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TitleDiscrimination based on Gly and Arg/Ser at position 673 betweendipeptidyl-peptidase (DPP) 7 and DPP11, widely distributed DPPs inpathogenic and environmental gram-negative bacteria

Author(s) Rouf, Shakh M.A.; Ohara-Nemoto, Yuko; Hoshino, Tomonori; Fujiwara,Taku; Ono, Toshio; Nemoto, Takayuki K.

Citation Biochimie, 95(4), pp.824-832; 2013

Issue Date 2012

URL http://hdl.handle.net/10069/30455

Right © 2012 Elsevier Masson SAS. All rights reserved.

NAOSITE: Nagasaki University's Academic Output SITE

http://naosite.lb.nagasaki-u.ac.jp

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Discrimination based on Gly and Arg/Ser at Position 673 between dipeptidyl-peptidase (DPP) 7

and DPP11, widely distributed DPPs in pathogenic and environmental Gram-negative bacteria

Shakh M. A. Roufa, Yuko Ohara-Nemotoa, Tomonori Hoshinob, Taku Fujiwarab,

Toshio Onoa, and Takayuki K. Nemotoa, *

aDepartment of Oral Molecular Biology, Course of Medical and Dental of Sciences, Nagasaki

University Graduate School of Biomedical Sciences, Nagasaki 852-8588, Japan bDepartment of Pediatric Dentistry, Course of Medical and Dental of Sciences, Nagasaki

University Graduate School of Biomedical Sciences, Nagasaki 852-8588, Japan

Running title: Discrimination between DPP7 and DPP11 based on residue at 673

*Corresponding author. Tel.: +81 95 819 7640. Fax: +81 95 819 7642.

E-mail address: tnemoto@nagasaki-u.ac.jp (T.K. Nemoto)

Department of Oral Molecular Biology, Course of Medical and Dental of Sciences, Nagasaki

University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki 852-8588, Japan

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ABSTRACT

Porphyromonas gingivalis, an asaccharolytic gram-negative rod-shaped bacterium,

expresses the novel Asp/Glu-specific dipeptidyl-peptidase (DPP) 11 (Ohara-Nemoto, Y. et al.,

(2011) J. Biol. Chem. 286, 38115-38127), which has been categorized as a member of the

S46/DPP7 family that is preferential for hydrophobic residues at the P1 position. From that

finding, 129 gene products constituting five clusters from the phylum Bacteroidetes have been

newly annotated to either DPP7 or DPP11, whereas the remaining 135 members, mainly from

the largest phylum Proteobacteria, have yet to be assigned. In this study, the substrate

specificities of the five clusters and an unassigned group were determined with recombinant

DPPs from typical species, i.e., P. gingivalis, Capnocytophaga gingivalis, Flavobacterium

psychrophilum, Bacteroides fragilis, Bacteroides vulgatus, and Shewanella putrefaciens.

Consequently, clusters 1, 3, and 5 were found to be DPP7 with rather broad substrate specificity,

and clusters 2 and 4 were DPP11. An unassigned S. putrefaciens DPP carrying Ser673 exhibited

Asp/Glu-specificity more preferable to Glu, in contrast to the Asp preference of DPP11 with

Arg673 from Bacteroidetes species. Mutagenesis experiments revealed that Arg673/Ser673 were

indispensable for the Asp/Glu-specificity of DPP11, and that the broad specificity of DPP7 was

mediated by Gly673. Taken together with the distribution of the two genes, all 264 members of

the S46 family could be attributed to either DPP7 or DPP11 by an amino acid at position 673. A

more compelling phylogenic tree based on the conserved C-terminal region suggested two gene

duplication events in the phylum Bacteroidetes, one causing the development of DPP7 and

DPP11 with altered substrate specificities, and the other producing an additional DPP7 in the

genus Bacteroides.

Keywords: dipeptidyl peptidase, S46 peptidase, substrate specificity, Porphyromonas gingivalis,

Bacteroidetes, Proteobacteria.

Abbreviations: DPP, dipeptidyl-peptidase; PgDPP7, DPP7 from P. gingivalis; PgDPP11 and

PeDPP11, DPP11 from P. gingivalis and P. endodontalis, respectively; Arg673- and

Ser673-DPP11, DPP11 carrying Arg673 and Ser673, respectively; GluV8, glutamyl endopeptidase

from Staphylococcus aureus; ac, acetyl; Z, benzyloxycarbonyl; MCA,

4-methycoumaryl-7-amide; NJ, neighbor-joining.

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1. Introduction

Porphyromonas gingivalis is a major causative organism of aggressive forms of chronic

periodontitis [1,2] leading to loss of permanent teeth [3-5], and also detected from extraoral

sources especially occluded arteries [6,7] and appendicitis [8]. This bacterium grows exclusively

under an anaerobic condition, forms biofilm [9], and does not utilize sugar, whereas it requires

proteinaceous substrates as carbon and energy sources [10,11]. Hence, P. gingivalis is considered

to be a model organism for studying bacterial pathogens possessing a metabolic network that

does not require sugar and oxygen.

Cell-associated peptidases in P. gingivalis, such as dipeptidyl-peptidases (DPPs), prolyl

tripeptidyl-peptidase A, as well as lysyl and arginyl gingipains, are responsible for the initial step

of its energy metabolism, because the organisms predominantly incorporate amino acids as di-

and tri-peptides from the surrounding environment [12, 13]. To date, three DPPs in P. gingivalis

have been well characterized. DPPIV specifically cleaves a C-terminal carbon-nitrogen bond of

Pro at the penultimate position (P1 position) from the N-terminus of substrates [14] and DPP7 is

preferential for hydrophobic residues at the P1 position [15], while we recently reported

Asp/Glu-specific DPP11, which is also expressed in Porphyromonas endodontalis, an important

pathogen in periapical lesions [16]. The discovery of P. gingivalis DPP11 (PgDPP11) solved a

long-lasting puzzle related to findings that peptidases responsible for the degradation of

polypeptides carrying Asp and Glu were apparently absent, while Glx (Glu and Gln) and Asx

(Asp and Asn) were the most abundantly utilized amino acids in P. gingivalis [12,13,17]. In

addition to these DPPs, genome information suggests existence of the DPPIII gene (PGN1645)

[11], which is considered to be expressed solely in eukaryotic cells [18-20]. When Pro is located

at the third position from the N-terminus, prolyl tripeptidyl-peptidase A specifically cleaves an

NH2-X-X-Pro-|-X bond [21, 22].

PgDPP11 encoded by the PG1283 gene was previously classified as an unassigned member

of DPP7 (PG0491, PgDPP7) in the S46 family (MEROPS peptidase database, Release 9.5) [23]

or considered to be an isoform of DPP7 [15]. As a consequence of the discovery of PgDPP11,

129 homologues in the family from the phylum Bacteroidetes, previously known as the

Cytophaga-Flavobacterium-Bacteroides (CFB) group, are currently annotated to either DPP7

(S46.001) or DPP11 (S46.002). Intriguingly, though many species possess a pair of DPP7 and

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DPP11 genes, the genus Bacteroides in this phylum exceptionally contains three S46-family

genes. Hence, it is of great interest to determine whether the third gene, which is currently

annotated to DPP11, truly possesses the substrate specificity of DPP11 or exhibits a novel

specificity. Furthermore, unassigned DPP7/11 members are widely distributed to the largest

phylum Proteobacteria, which comprises the majority of known gram-negative bacteria with

medical, industrial, and agricultural significance [24]. These include the genus Ferrimonas,

which is chemoorganotrophic [25], Colwellia, which adapts to cold marine environments [26],

and Xanthomonas, which causes plant diseases [27]. Therefore, validation of the hydrolytic

activities and specificities of these family members is important to elucidate their amino acid

metabolism.

The specificity of P. endodontalis DPP11 (PeDPP11) is primarily determined by the

interaction of Arg673 with the carboxyl group at the P1-position Asp/Glu of a substrate peptide

[16]. The amino acid at the corresponding position is Gly673 in PgDPP7, and exclusively either

Arg or Gly in all DPP7 and DPP11 members from the phylum Bacteroidetes. Gly673 in DPP7

seems to be compatible for its hydrophobic residue preference, however this notion has not been

directly tested because of the unavailability of recombinant DPP7 to date. In addition to the P1

position, there is also evidence that the hydrophobicity of the P2-position residue is important for

the activity of DPP11 [16]. In contrast, DPP7 and DPPIV seem to have no preference for other

residues except that Pro is unacceptable at the third position (P1’ position) [14, 23], although few

peptides were tested with DPP7 [15, 28].

In the present study, we determined the specificities of five DPP7/11 clusters from the

phylum Bacteroidetes and an unassigned DPP7/11 group from the phylum Proteobacteria in the

latest phylogenic tree of the S46 family. Interestingly, the species of the genus Bacteroides

carried one DPP11 and two DPP7 genes, one of which is currently annotated to DPP11 by

sequence homology. In addition, the specificity of an unassigned Shewanella DPP that carries

Ser673 was determined to be DPP11 subtype with the preference to Glu in contrast to

Arg673-DPP11. Based on these biochemical findings, we propose that DPP7 and DPP11 of all

264 S46-family members should be unambiguously attributed to either DPP7 or DPP11.

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2. Materials and methods

2.1. Materials

The expression and cloning vectors used were pTrcHisTOPO from Invitrogen and pQE60

from Qiagen. Low-molecular-weight markers were obtained from GE Healthcare. Restriction

enzymes and DNA-modifying enzymes came from Takara Bio and New England Biolabs,

respectively, while KOD Plus DNA polymerase came from Toyobo (Tokyo, Japan).

Met-Leu-MCA was obtained from Bachem (Bubendorf, Switzerland), and Leu-Asp-, Leu-Glu-,

acetyl (ac)-Leu-Asp-, and benzyloxycarbonyl (Z)-Leu-Leu-Gln-MCA were synthesized by

Thermo Fisher Scientific (Ulm, Germany) and TORAY (Tokyo, Japan). Other MCA peptides

were from the Peptide Institute Inc. (Osaka, Japan). Oligonucleotide primers came from

FASMAC (Atsugi, Japan). Thermolysin from Bacillus thermoproteolyticus rokko and trypsin

from bovine pancreas were obtained from Sigma-Aldrich. Recombinant Glu-specific

endopeptidase from Staphylococcus aureus (GluV8) was expressed and the subsequent

conversion to a mature form was performed as previously reported [29].

2.2. Bacterial strains

Capnocytophaga gingivalis JCM12953 and Flavobacterium psychrophilum JCM8519 were

provided by RIKEN BRC through the National Bio-Resource Project of MEXT, Japan. P.

gingivalis ATCC33277 was obtained from American Type Culture Collection. Each was

cultured in ABCM (anaerobic bacteria culture medium) broth (Eiken Chemical, Tokyo, Japan) in

the presence of 1 µg/ml of menadione (Sigma-Aldrich) at 35˚C under an anaerobic condition

(90% N2/5% H2/5% CO2).

2.3 Construction of expression plasmids

We prepared genomic DNA from P. gingivalis, C. gingivalis, and F. psychrophilum as

previously reported [30]. Genomic DNA from Bacteroides fragilis strain Onslow JGD06283,

Bacteroides vulgatus JGD06281, and Shewanella putrefaciens JCM20190T were provided by

RIKEN BRC. DNA fragments encoding cluster-1 and -2 DPPs of F. psychrophilum (locus tag:

FP1355 and FP0382, respectively) and C. gingivalis (CAPGI0001_0817 and CAPGI0001_1068,

respectively), cluster-5 DPPs from B. fragilis (BF_3010) and B. vulgatus (BVU_2253), DPP

6

from S. putrefaciens (Sputcn32_0757), and P. gingivalis DPPIV (PGN_1469) were amplified by

PCR with genomic DNA as templates and the primers listed in supplemental Table S1. PCR

fragments of cluster-2 DPP from C. gingivalis and cluster-1 and -2 DPPs F. psychrophilum were

directly inserted into pTrcHisTOPO. The PCR fragments of P. gingivalis DPPIV and PgDPP11

(PGN_0607), the cluster-1 DPP fragment from C. gingivalis, and the fragments from B. fragilis

and S. putrefaciens were digested with BamHI, and cloned into the BamHI site of pQE60. PCR

fragments encoding PgDPP7 (PGN_1479) and cluster-5 DPP from B. vulgatus were digested

with BglII, and cloned into the BamHI site of pQE60. Among them, since cluster-1 DPP from F.

psychrophilum was poorly expressed in this E. coli expression system, further biochemical

analysis was not performed.

2.4. Amino acid numbering and in vitro mutagenesis

To specify the positions of amino acid residues involved in the specificity of the DPPs, the

numbering was unified to that of PgDPP11 throughout this study. In vitro mutagenesis was

performed using a PCR technique with appropriate primers (supplemental Table S1) to introduce

an amino acid substitution of Gly673 in PgDPP7 to Arg, Ala, or Asp. Arg673 in PgDPP11 was

substituted to Ser. Ser673 and Lys680 in the S. putrefaciens DPP were substituted to Arg and Gly,

respectively, or simultaneously. All substitutions were confirmed by nucleotide sequencing.

2.5. Expression and purification of DPPs

Recombinant DPPs were expressed in Escherichia coli XL1-blue by induction with 0.2 mM

isopropyl-thiogalactopyranoside at 30 ˚C for 4 h. Recombinant proteins were purified by Talon

affinity chromatography, as previously reported [29].

2.6. Measurement of hydrolyzing activity

Reaction was started by addition of DPPs to reaction solution (200 µL) contained 50 mM

sodium phosphate (pH 7.0), 5 mM EDTA, and 20 µM MCA-substrate. After 1 h incubation at 37

˚C, fluorescence intensity was measured with excitation at 380 nm and emission at 460 nm.

Several dipeptidyl-MCA substrates were prepared from oligopeptidyl-MCA (0.4 mM) as

previously reported [16]. In practical terms, boc-Ile-Glu-Gly-Arg- and boc-Glu-Lys-Lys-MCA

were cleaved with GluV8 to produce Gly-Arg- and Lys-Lys-MCA, respectively, and

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Z-Leu-Arg-Gly-Gly-MCA with trypsin to produce Gly-Gly-MCA, while

ac-Lys-Thr-Lys-Gln-Leu-Arg-, Z-Val-Val-Arg-, and boc-Val-Leu-Lys-MCA were pretreated

with thermolysin to produce Leu-Arg-, Val-Arg-, and Leu-Lys-MCA, respectively.

2.7. Construction of phylogenic trees of the S46 family

PeDPP11, which was cloned recently in our laboratory [16], and 263 members of the S46

family in the MEROPS database (Release 9.6) were used for construction of phylogenic trees.

ClustalX software [31] (http://www.ebi.ac.uk) was used to align the sequences. The full-length

amino acid sequences and sequences of the conserved regions were used for construction of the

phylogenic trees. Phylogenic analysis with the neighbor-joining (NJ) algorithm [32] was

conducted using MEGA version 5 [33].

3．Results

3.1. Peptidase activities of DPPs belonging to clusters 1-5 from the phylum Bacteroidetes

In the S46 family listed in the previous MEROPS database (Release 9.5), 71 members

derived from the genus Bacteroides constituted only a single group, S46/DPP7, with 3 members

left unassigned [16]. In the version (Release 9.6), revised after the finding of DPP11, the number

of S46 family members reached 263, which were newly classified into S46.001/DPP7,

S46.002/DPP11, and unassigned members (supplemental Fig. S1). Among them, 129

homologues from the phylum Bacteroidetes constitute five clusters, in which cluster 3 includes

PgDPP7 and cluster 4 includes PgDPP11. Members of clusters 2 and 5 are newly annotated to

DPP11, whereas it has not been decided where cluster 1 should reside in either of the two DPPs.

Although there are several members with ambiguous locations in the branch, those are currently

annotated to DPP11 (supplemental Fig. S1, asterisks 1-4). The remaining 135 members, mainly

from the largest phylum Proteobacteria, could be classified into three groups, of which only

group 3 is annotated to DPP7, while groups 1 and 2 remain unannotated (supplemental Fig. S1).

Thus, since the S46 family members, except for PgDPP7 (PG_0491/PGN_1479), PgDPP11

(PG_12831/PGN_0607), and PeDPP11, have not been biochemically characterized, it is

worthwhile to determine directly the enzymatic activities of all five clusters. We chose DPPs

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from typical bacterial species as follows; cluster-1 and -2 DPPs from C. gingivalis and F.

psychrophilum, cluster-5 DPPs from B. fragilis and B. vulgatus, as well as PgDPP7 in cluster 3,

and PgDPP11 in cluster 4. Furthermore, P. gingivalis DPPIV (PG_1361/PGN_1469), which

belongs to the S9 peptidase family, was also examined as a representative of non-S46 family. All

DPPs were successfully expressed by use of an E. coli expression system and purified as

70-80-kDa species, except for cluster-1 DPP from F. psychrophilum (supplemental Fig. S2).

The substrate specificity of these recombinant DPPs was characterized using 11 dipeptidyl

MCA substrates (Fig. 1). In accord with a previous report [14], recombinant DPPIV most

preferentially hydrolyzed Gly-Pro-MCA, while that of Lys-Ala-MCA was less potent.

Hydrolysis of Met-Leu-MCA was nearly negligible as compared to these two substrates. These

results indicate that recombinant DPPIV faithfully reproduced the property of DPPIV expressed

in P. gingivalis. DPP7 from P. gingivalis has been reported to preferentially cleave a peptide

bond next to a hydrophobic amino acid and Ala at the P1 position [15]. This property was

reproduced with recombinant DPP7, which most potently cleaved Met-Leu-MCA and

Lys-Ala-MCA to a lesser extent. In addition, DPP7 hydrolyzed dipeptidyl MCA carrying

charged residues at the P1 position, such as Leu-Arg-, Leu-Asp-, Leu-Glu-, Val-Arg-, and

Leu-Lys-MCA, to some extent. An apparent characteristic of these substrates was the presence

of hydrophobic residues at the N-terminus (P2 position). PgDPP11 solely cleaved Leu-Asp- and

Leu-Glu-MCA, as previously reported [16].

The substrate specificity of C. gingivalis DPP in cluster 1, which is annotated as DPP11 in

the latest classification (Release 9.7), was similar to that of PgDPP7, with the most potency

toward Met-Leu-MCA, followed by Lys-Ala-, Leu-Arg- > Leu-Asp-, Leu-Glu-, > Leu-Lys-, and

>Val-Arg-MCA, while this DPP did not hydrolyze Gly-Arg-, Gly-Gly-, Lys-Lys-, or

Gly-Pro-MCA (Fig. 1). Therefore, this C. gingivalis DPP could be classified to DPP7. C.

gingivalis and F. psychrophilum DPPs in cluster 2 exhibited the specificity as that of PgDPP11

in cluster 4. Finally, B. fragilis and B. vulgatus cluster-5 DPPs demonstrated a substrate

preference similar to that of cluster-1 and -3 DPP7, although they have been annotated to DPP11.

This finding indicates that the discrimination between DPP7 and DPP11 is not simply achieved

by the comparison of full-length amino acid sequences, and thus, we suspect that the

discrimination might be achieved by the amino acid sequence region critical for the substrate

specificity of DPP7 and DPP11.

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3.2. Amino acid residue at position 673 critical to define substrate specificity of DPP7 and

DPP11

The alignment of the DPP sequences of 129 members of clusters 1-5 shows that three

conserved regions, each of which includes one of the essential residues of His85, Asp198, and

Ser655 of serine proteases (Fig. 2). Forty-three amino acid residues are conserved in more than

95% of the members. These highly conserved residues may be indispensable for functions

common to both DPP7 and DPP11, whereas residues specific for DPP7 or DPP11 may be

involved in each DPP-specific role. In this respect, the amino acid residue at position 673 was

unique, and Arg673 was entirely conserved in members of clusters 2 and 4 (DPP11), whereas the

position was exclusively Gly in clusters 1, 3, and 5 (DPP7).

We previously demonstrated an ionic interaction between Arg673 of DPP11 and the carboxyl

group of the P1-position Asp/Glu of a peptide substrate [16]. By analogy, it was postulated that

the tiny and hydrophobic residual group (hydrogen) of Gly673 is a prerequisite for DPP7 activity

to accept hydrophobic and bulky residues of a substrate at the P1 position. Hence, this hypothesis

was examined by use of a single amino acid substitution of Gly673 of PgDPP7 to Asp, Ala, and

Arg (Fig. 3). The hydrolyzing activity of PgDPP7 toward Met-Leu-MCA was abolished by the

Gly673Asp substitution (1.7%) and reduced to 22% by the Gly673Arg substitution, indicating

the need for a non-charged residue at this position. In the Gly673Ala substitution, 61% of the

Met-Leu-MCA hydrolyzing activity was maintained. These results strongly suggest that the tiny

hydrogen group (-H) of Gly673 is more suitable than a methyl group (-CH3) of Ala, presumably

because of acceptance of the bulky hydrophobic residue characteristic of DPP7. Furthermore, it

is of interest to note that the Gly673Arg substitution to mimic Arg673 of DPP11 could acquire the

activity toward Leu-Asp- and Leu-Glu-MCA. Thus, the residue at position 673 is critical for both

hydrolyzing activity and the substrate specificity of DPP7 as well as DPP11. Although we also

tested whether PgDPP7 Gly673Asp hydrolyzed the peptide carrying basic residue at the P1

position, it failed to hydrolyze Leu-Arg-MCA (data not shown), indicating that the residue at 673

is not the unique determinant of the substrate specificity.

Based on these biochemical findings together with the sequence homology, we considered

reasonably proposing that newly discovered DPP7/11 homologues from the phylum

Bacteroidetes can be readily assigned based on the amino acid residue at position 673.

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3.3. DPP from the phylum Proteobacteria and expression of Shewanella putrefaciens DPP

carrying Ser673

The S46 family contains 139 unassigned members mainly from the phylum Proteobacteria,

which constitutes three major groups (supplemental Fig. S1, Table S1). Among them, most

members in groups 1 and 3 carry Gly at position 673, thus they should be DPP7. On the other

hand, 30 members constituting group 2 were shown to possess neither Gly nor Arg, but rather

Ser at the position. Since most members of group 2 were from the genus Shewanella, we

expressed DPP (MEROPS ID: MER096825) from S. putrefaciens to determine the peptidase

activity of these members carrying Ser673. S. putrefaciens is a typical species of the genus that

has been isolated mostly from fish, poultry, and meats, but is also associated with a broad range

of human infections [34, 35]. It was found that S. putrefaciens DPP specifically cleaved

Leu-Asp- and Leu-Glu-MCA, demonstrating its entity as DPP11 (Fig. 4). This conclusion was

compatible with the fact that 18 of 19 Shewanella species possess a pair of genes encoding DPP7

with Gly673 and DPP11 with Ser673 (supplemental Table S2).

Interestingly, S. putrefaciens Ser673-DPP11 preferentially cleaved Leu-Glu-MCA as

compared to Leu-Asp-MCA, in contrast to the Leu-Asp-MCA preference of cluster-2 and -4

DPP11 (Figs. 1 and 5). In order to investigate the relationship between the amino acid at position

673 and Asp- and Glu-preferences, an amino acid swapping experiment was performed. In the

Arg673Ser mutant of PgDPP11, the Asp/Glu preference was inverted, i.e., peptidase activity

toward Leu-Asp-MCA nearly disappeared, whereas that to Leu-Glu-MCA partially remained and

vice versa for S. putrefaciens DPP11 (Fig. 5B).

These results of specificity described above strongly suggest that the amino acid residue at

position 673 is critical not only for discrimination between DPP7 and DPP11, but also for the

preference of either Asp or Glu in DPP11. Ser673 was specifically located in DPP11 of group 2

distributed in the genera Shewanella, Ferrimonas, Pseudoalteromonas, Stenotrophomonas, and

Pseudoxanthomonas, while a few members carrying Ser673 were exceptionally observed in group

1 from the genera Rhodothermus, Salinibacter, and Brevundimonas (supplemental Fig. S1,

asterisks 5 and 7; supplemental Table S2).

As for the interaction with the Asp/Glu residue at the P1 position of substrates, a specific

basic residue was surmised in Ser673-DPP11. Based on the alignment, we found that Lys680 is

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conserved in 21 Ser673-DPP11 members of all Shewanella species, and two other Ser673-DPP11

from Ferrimonas balearica and Pseudoalteromonas tunicate (supplemental Table S2), and that

most Arg673-DPP11 carry Gly680 (Fig. 2). In fact, substitution experiment findings demonstrated

that Ser673-DPP11-Lys680Gly abrogated that activity (Fig. 5). These results suggested that Lys680,

but not Ser673, could manage interactions with carboxyl group of substrates. Furthermore,

Lys680Gly substitution disrupted the acidic residue specificity, as it more efficiently degraded

Ala-Asn- and Leu-Gln-MCA (insert in Fig. 5). Finally, the double mutations (Ser673Arg and

Lys680Gly) that mimicked the amino acids of PgDPP11 induced a partial restoration of activity

for Leu-Asp-MCA. Therefore, these results indicate the importance of Lys680 in the enzymatic

activity of Ser673-DPP11.

3.4. A new phylogenic tree of S46 family

We constructed phylogenic trees based on each or total of the three conserved regions (see

Fig. 2). With region 1, cluster-4 members of the phylum Bacteroidetes were split into three

distinct locations: For instance, PgDPP11 was separated from PeDPP11 (data not shown). With

region 2, each cluster was distributed same as that classified with the full-length sequences (data

not shown). In contrast, the tree constructed using region 3 (Pro571/Ser571-Leu700) that carries an

essential Ser655 and specificity determining Gly/Arg/Ser673 (Fig. 6) may unify Bacteroidetes

DPP7 members into a single group comprising clusters 1, 3, and 5, and further accumulates

cluster-2 and -4 DPP11 at adjacent locations, as compared to the tree based on full-length

sequences (supplemental Fig. S1). In addition, the phylogenic tree obtained from the

combination of three regions (supplemental Fig. S3) was basically identical to that of region 3.

Accordingly, the phylogenic tree based on region 3 as well as that of the combination of

three regions enabled us to propose a simple and systematic evolutionary route of DPP7 and

DPP11 in the phylum Bacteroidetes. That is, the single ancestor gene of the S46 family was

initially duplicated into DPP7 and DPP11 genes in the phylum Bacteroidetes. Thereafter, a

second gene duplication may occur only in the DPP7 gene, which produced two DPP7 molecules

belonging to clusters 3 and 5 (Fig. 6).

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4. Discussion

In order to determine the substrate specificities of cluster-1 to -5 DPPs from the phylum

Bacteroidetes of the S46 family, we expressed at least two members from each of cluster 1, 2,

and 5, while cluster-1 DPP from Flavobacterium psychrophilum was not successfully expressed.

Moreover, we confirmed that the substrate specificities of Bacteroides fragilis DPP7 in cluster 3

(T. K. Nemoto and H. Nishimata, unpublished observation) and P. endodontalis DPP11 in

cluster 4 [16] were identical to those from P. gingivalis. Taken together with the sequence

similarity within each cluster, it was considered reasonable to conclude that members of one

cluster share an unique substrate specificity with either DPP7 or DPP11. In addition, the finding

that most species possess a set of DPP7 and DPP11 genes, except for the genus Bacteroides,

confirmed the validity of the classification.

The present study demonstrated that the amino acid at position 673 is involved in enzyme

activity together with the catalytic triad His85, Asp198, and Ser655, and is tightly associated with

the substrate specificities of DPP7 and DPP11 (Figs. 3 and 5). The substrate preference of DPP7

has been reported to be aliphatic or aromatic amino acid residues at the P1 position [15].

However, recombinant PgDPP7 cleaved Leu-Arg-MCA at an efficiency of 55% as compared to

that of Met-Leu-MCA (Fig. 1). DPP7 from Capnocytophaga gingivalis (cluster 1), Bacteroides

fragilis (cluster 5), and Bacteroides vulgatus (cluster 5) also cleaved Leu-Arg-MCA, thus DPP7

probably possesses a broader specificity, even for negatively and positively charged substrates at

the P1 position, than previously reported. An extensive analysis with various dipeptidyl MCA

substrates indicated the hydrophobic preference of DPP7 at the P2 position as well as the P1

position, which partly overcame the inaccessibility to the P1 position residue (S.M.A. Rouf, Y.

Ohara-Nemoto, and T. K. Nemoto, unpublished observation). These results may be compatible

with a previous observation [15], in which native DPP7 produced dipeptides from oligopeptides

with hydrophobic residues at the P2 position.

Our study also demonstrated that Ser673-DPP11 is capable of Asp/Glu-specific hydrolysis

and that Ser673-DPP11 exhibits a Glu preference in contrast to the Asp preference of authentic

Arg673-DPP11 (Fig. 5). We previously reported that the size of Arg673 defined the preference to

Asp, because the Asp preference was gradually shifted to Glu by the substitution of Arg673 to the

smaller amino acid residues His, Gln, and Gly, in that order [16]. By analogy, the Glu preference

13

of Ser673-DPP11 would be explained by the conversion from Arg673 to Ser. In addition, based on

the amino acid sequence alignment, the ε-amino group of Lys680 in Ser673-DPP11 is suspected to

locate at the position of the guanidinium group of Arg673 of PgDPP11 in a steric manner. As

expected, we found that substitution of Lys680 to Gly, which mimicked the amino acid

conversion from Ser673-DPP11 to Arg673-PgDPP11, abrogated the activity, suggesting that Lys680

is involved in substrate specificity and enzymatic activity (Fig. 5).

Among three conserved regions of the S46 family members, C-terminal conserved region 3

composed of 230 residues seems to be most important for activity and specificity, since the

alkoxide of catalytic Ser655 is directly associated with the carbonyl carbon of Asp/Glu in a

substrate and initiates the cleavage of peptide bond [40]. Moreover, the complete sequence

homology around Ser655, i.e., Thr650-Thr-Gly-Gly-Asn-Ser655-Gly-Ser-Pro-Val659, between

PgDPP11 and GluV8 indicates the importance of this sequence in enzymatic activity [36]. In

particular, using the analogy of Gly193 and Ser195 of trypsin [37, 38], the backbone amide

hydrogen atoms of Gly653 and Ser655 may serve to stabilize the developing negative charge on the

carbonyl oxygen atom of the cleaved amides. Furthermore, Gly673 and Arg673/Ser673, critical for

specificity, were localized in the same region (Fig. 2). The residue at position 673 may

equivalent to Asp185 of trypsin located in the catalytic pocket responsible for attracting and

stabilizing the charged P1-position residue of a substrate. This assumption may be also true for

Lys680 of Ser673-DPP11.

Hoshino et al. [39] reported the evolution of the cariogenic characteristics of Streptococcus

mutans using glucosyltransferase. In that study, a middle catalytic domain (residues 400-900)

instead of the 1500-amino acid full-length sequence of glucosyltransferase was used for the

alignment, because the rest of the sequence varied enormously among species. In accord with

that study, the present phylogenic tree based on the conserved region 3 as well as the

combination of three conserved regions indicates an evolutionary route of DPP7 and DPP11,

which could more reasonably explain the relationships among the five clusters of the S46 family

compared to the tree based on the full-length sequence. Proteins generally accumulate amino

acid substitutions at variable rates based on the requirements of the sequences. Hence, for

multi-domain proteins, specificity-determining restricted regions may be preferable for study of

their classification. By use of the C-terminal conserved region 3 for comparison, we could figure

a simple evolutionary route of DPP7 and DPP11 genes in phylum Bacteroidetes (Fig. 6). We

14

further examined whether classification based on the conserved region 3 could be successfully

expanded to DPPs from phyla other than Bacteroidetes. They were tentatively separated into

three groups, of which groups 2 and 3 were exclusively composed of Ser673-DPP11 and

Gly673-DPP7, respectively (Fig. 6).

Acknowledgements

We greatly acknowledge Mariko Naito (Nagasaki University) for valuable discussion and

Takeshi Kobayakawa for technical assistance. This work was supported in grants-in-aid for

scientific research from the Ministry of Education, Science, Sports, and Culture of Japan (to Y.

O.-N., S. K. and T. K. N.) and from the Institute for Fermentation, Osaka (to T. K. N.). S. M. A.

Rouf is a graduate student supported by Japanese Government (MEXT) Scholarship.

15

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20

Fig. 1. Substrate specificity of DPPIV and DPP7/11 belonging to the five clusters of the S46

family. The hydrolyzing activities of P. gingivalis (A) DPPIV, (B) DPP7 in cluster 3, and (C)

DPP11 in cluster 4 toward dipeptidyl-MCA substrates were determined. (D) Cluster-1 DPP from

C. gingivalis, (E) cluster-2 DPPs from C. gingivalis and F. psychrophilum, and (F) cluster-5

DPPs from B. fragilis and B. vulgatus. Values are shown as the mean ± S.D. (n=3).

FIGURE 1

A

0.01

0.1

1

10

100cluster 5

B. fragilis B. vulgatus

C

0.01

0.1

1

10

100

1000

10000cluster 2

C. gingivalis F. psychrophilum

cluster 3

B

0.001

0.01

0.1

1

10

100cluster 1 C. gingivalis

MetLeu

LeuA

rgLy

sAla

LeuA

sp

LeuG

lu

ValArg

LeuL

ys

GlyArg

GlyGly

LysL

ys

GlyPro

Spe

cific

act

ivity

(pm

ol/m

in/µ

g)

D FE

0.01

0.1

1

10

100

1000DPPIV

Spe

cific

act

ivity

(pm

ol/m

in/µ

g)

0.01

0.1

1

10

100

1000

PgDPP7

0.01

0.1

1

10

100

1000cluster 4 PgDPP11

MetLeu

LeuA

rgLy

sAla

LeuA

sp

LeuG

lu

ValArg

LeuL

ys

GlyArg

GlyGly

LysL

ys

GlyPro

MetLeu

LeuA

rgLy

sAla

LeuA

sp

LeuG

lu

ValArg

LeuL

ys

GlyArg

GlyGly

LysL

ys

GlyPro

MetLeu

LeuA

rgLy

sAla

LeuA

sp

LeuG

lu

ValArg

LeuL

ys

GlyArg

GlyGly

LysL

ys

GlyPro

MetLeu

LeuA

rgLy

sAla

LeuA

sp

LeuG

lu

ValArg

LeuL

ys

GlyArg

GlyGly

LysL

ys

GlyPro

MetLeu

LeuA

rgLy

sAla

LeuA

sp

LeuG

lu

ValArg

LeuL

ys

GlyArg

GlyGly

LysL

ys

GlyPro

21

FIGURE 2-1

1 10 20 30 40 50 60 70 80 90Cluster1_CgDPP -MRKLIFSLVTSFFLLLPS-VIRADEGMWFLMFIKRLN-ERDMQKKGLQLTAEEIYSINNNSLKNAIVQFNGGCTASIISPDGLVITNHHCGYGAIAGLSCluster2_CgDPP MKRFFKMALFLGVSALYG-----QQGGMWIPSLLEGMN-AKEMKTLGMKMTVADIYSVNKSSLKDAAPHFNGGCSSEVISDKGLLLTNHHCGYGQIQAHSCluster2_FpDPP -MKYLKLFLLLFIIQTQA-----QQGGMWIPSLLSGMN-ETEMKNLGMKISADDIYSVNHSSLKDAVPHFNGGCTSEVISPKGLILTNHHCGFDAIQNHSCluster3_PgDPP7 MQMKLKSILLGAALLLGASGVAKADKGMWLLNELNQEN-LDRMRELGFTLPLDSLYSFDKPSIANAVVIFGGGCTGITVSDQGLIFTNHHCGYGAIQSQSCluster4_PgDPP11 MKKRLLLPLFAALCLSQI---AHADEGMWLMQQLGRKY--AQMKERGLKMKEYDLYNPNGTSLKDAVVLFDGGCTGEVVSDRGLVLTNHHCGYDMIQAHSCluster5_BfDPP -MNRLKLYLLALTALAVCS--AKADEGMWLLQLMQQQHSIDMMKKQGLKLEAQDLYNPNGVSLKDAVGIFGGGCTGEIISPEGLILTNHHCGYASIQQHSCluster5_BvDPP -MKKFKLLLLALMCVAFLP--SKADEGMWLLQLMQEQHLADRMKAQGLLLEADDIYNPNRVSLKDAVGIFGGGCTGEIISPDGLILTNHHCGYGAIQQHSGroup2_SpDPP ----MRIALVATLVLTSG--IANADEGQWQPYQMPSIA--DKLSERGIDIPAEKLADLTSYPMNAVVGLG--YCTASFVSPQGLVVTNHHCAYKAIQYNTConsensus GmW G a GC S GL TNHHC S

100 110 120 130 140 150 160 170 180 190Cluster1_CgDPP TPEHNYLKDGYWAKDRSQELPP-KSLYVRFFVRMDNVTDRMLSVVNSSMSEKERQDALNREMEKIQKENSEGGKY------VVSVRPFFQGNEYYYFVYQCluster2_CgDPP TLQNDYLANGFWAKSLAEELPN-KNLKVTFMVRIDDVTKQVLKGTESITDETEKAKLIEKNIAEVIKTAPKEAWQ------ENSVKAFYDGNQYILFVTECluster2_FpDPP SVDHDYLTNGFWAMKMEDELPN-ENLVVTFIVSINDVTAQVLDGVASITSETEKQNKIQENITKVTASFAKEAWQ------ENKVRTFFEGNQYILFVTECluster3_PgDPP7 TVDHDYLRDGFVSRTMGEELPI-PGLSVKYLRKIVKVTDKVEGQLKGITDEMERLRKAQEVCQELAKKENADENQ------LCIVEPFYSNNEYFLIVYDCluster4_PgDPP11 TLEHNYLENGFWAMREADELPN-KDISVVFIDKIEDVTDYVKKELKAIKDPNSMDYLSPKYLQKLADKKAGKNFSAKNPGLSVEIKAFYGGNLYLMFTKKCluster5_BfDPP SVEHDYLTDGFWATSRDKELPT-PGLKFTFIERIEDITDIVNLRIAAK-EITESESFSSTFLNKLAKELFEKSDLKGKKGIVPQALPFYAGNKFYMFYKKCluster5_BvDPP SVEHDYLTDGFWAKSRKEELPT-PGLKFKFVERIVDVTDKVNNKVKSG-EVKEEETFEYDFLKKLADEELKASDLNGKAGISAQALPFYAGNKFYLIYLKGroup2_SpDPP KQEHNYLEQGFLATSMDKEPSAGPNERLYITEAVTDVTKDVTKELSQD-PLTRYEEIQNNSKALIKNCEVDDNYR------C-NVRSFHNGLEYYLIKQLConsensus G A E T G l

200210 220 230 240 250 260 270 280 290

Cluster1_CgDPP DFKDVRFVGTPPENVGKFGGDTDNWEWPRHTGDFSVFRVYTDKDGNPAPYSPNNIPMKAKKYLNVTLKGVQENDFAMILGYPGRTNRWVSSHWVDQQVKYCluster2_CgDPP TFKDVRLVGAPPSSIGKFGSDTDNWVWPRHTGDFSLFRIYADKNNRPAEYSKDNVPYKPKHFFPISLKGVKEGDFVLVFGYPGTTQEYLPSAAVAQIENVCluster2_FpDPP VFKDVRLVGAPPSLIGKFGSDTDNWVWPRHTGDFSMFRVYANKNNHPAAYSKDNVPYIPKHFLPVSLDGVQEDDFTMVMGYPGKTQEYLPSFAVAQIVNECluster3_PgDPP7 VFKDVRMVFAPPSSVGKFGGDTDNWMWPRHTGDFSVFRVYAGADNRPAEYSKDNKPYKPVYFAAVSMQGYKADDYAMTIGFPGSTDRYLTSWGVEDRIENCluster4_PgDPP11 TYTDVRLVGAPPSSIGKFGADTDNWIWPRHTGDFSIFRIYADKNGNPAPYSEDNVPLKPKRFFNISLGGVQENDYAMIMGFPGTTHRYFTASEVDEWKSICluster5_BfDPP VYPDVRMVAAPPSSIGKFGGETDNWMWPRHTGDFSMFRIYADANGEPAEYSASNVPLKTKKHLNISIKGLKEGDYAMIMGFPGSTSRYLTVSEVKERMEACluster5_BvDPP TYSDVRMVAAPPSSIGKFGGETDNWMWPRHTCDFSVFRIYADANGEPAEYNENNVPLKAKKHLAISLKGINEGDYAMIMGFPGSTNRYLTQSEVKQRMHSGroup2_SpDPP MIRDVRLVYAPPESVGGYGGDIDNYEYPRHSGDFAFLRAYVGKDGKPAAYAEDNIPYKPKSYLKVNADGVKAGDGVFVAGYPGTTSRYNLTSELKFASDWConsensus D R V aP G Gg DN PR t D R Y PA n P G g G PG T R

300 310 320 330 340 350 360 370 380 390Cluster1_CgDPP GYPAWVEASKTAMDAMKAHMDKDKAVRLKYASRYASLANYWKNRQGMIDALTAHKTADLKRAAEKKFAVWANKPENKAEYGNVLSDLATYFEKTNQEAANCluster2_CgDPP INPARIGIRDIVLKVQDSYMRKDQGIKIKYAAKYARVANYWKKWMGETKGLKKSGAVALKQQQEAKFQQAIQKANKQAQYGNLLSDFNRLYKEIEPYTLACluster2_FpDPP TNPAKIEIREAALKVQDGFMRKDNAIKIQYASKYAGVANYWKKWIGESQGLKKSNAIGLKQNFEKDFQQKVIAAGKQNEYGNLLADFQKYYTEITPYAVSCluster3_PgDPP7 ENNPRIEVRGIKQGIWKEAMSADQATRIKYASKYAQSANYWKNSIGMNRGLARLDVIGRKRAEERAFADWIRKNGKSAVYGDVLSSLEKAYKEGAKANRECluster4_PgDPP11 DNDIRIRMRDIRQGVMLREMLADPQIKIMYSAKYAASQNAYKRAIGANWAIKTRGLRQNKQAMQDRLIAWGAKQG-TPRYEEAVHEIDATVAKRADLRRRCluster5_BfDPP SNAPRIRIRGTRQDVLKEAMNASDKVRIQYANKYAGSSNYWKNSIGMNKAIIDNNVLGTKAEQEAKFAKFAKEKN-NTDYMNVVAKIDEAVAKTSPIKYQCluster5_BvDPP TNEPRIRIRGVRQDVLKKEMAASDKVRIQYASKYAGSSNYWKNSIGMNKAIIDNKVLETKAEQEAKFAAFAKAKG-NTDYEKVVSEIDAAIEKSNPILYNGroup2_SpDPP LYPTQAKRYQLQIDTINAMGQEDADIAIKYAGNMASMANRMKKLNGLLAGFKATDIIGIKQSREDNFLAWLKQNP--KLNQNLIAELEVLLAEQQQVFQSConsensus N K G

Region 1

Region 2

*

FIGURE 2-2

400 410 420 430 440 450 460 470 480 490Cluster1_CgDPP HNYLLLFFRASRIVPQANGY-VKQLNTYLNSSSDQEKQQIRERIAKELDAYY-SESYLPAEIDLFADNLKLYADKATD--IPQEIA-QIKSQYNGDFRKFCluster2_CgDPP ANLNSEFIFRNIDLLSNGSR-LLQLEKALEDKGEQSFNDRKKNLLNTFKEIF-KDNDKQVDKDVFEKVVVFYAANMPKNLLINSLK-------NFDAKKLCluster2_FpDPP RDYFNEVVVKNTELLSLGYK-LYQLEQVFITKGEQAFNDRKENLIKSQADFF-KDFNATVDEKVFEQLVALYATKAPKEFLPISLL-------NVEYKKFCluster3_PgDPP7 MTYLSETLFGGTEVVR-----FAQFANALATN-PDAHAGILKSLDDKY-----KDYLPSLDRKVLPAMLDIVRRRIPADKLPDIFKNVIDKKFKGDTKKYCluster4_PgDPP11 YWMIEEGIIRGIEFARSPIP-TEDETKALQGNDASARKEAIDKIRTRYSKFANKDYSAEVDKKVAVAMLTEYLKEIPYENLPLHLR-LVKDRFAGDVQAYCluster5_BfDPP QTCLTETFFGGIEFGSPFMV-MDKLKEALEQKNDSSIEANIKVLKEVFNDIHNKDYDHEVDRKVAKALLPLYAEMIPAGQRPAIYD-VIEKEYKGDYNAYCluster5_BvDPP YTCFREVFQGGIEFGTPYLI-LDKLKDAIKNKDKEAINKNIETLKKVYADIHNKDYDHEVDRKVAKALLPLYAEMVPADALPAFYT-TIQKDFKGNYDAYGroup2_SpDPP NYYFTNAQSSTLLTAANSLYRLAKEKQKSDAEREIGYQERDLAMFSSRLKRIDSSFHVDVDKTLWQQDLRAYLAQPNR--IAALDDALDLNNKETNLEAK

500 510 520 530 540 550 560 570 580Cluster1_CgDPP AAEVFARSIFTTKENFENFMNNPS----SDALQSDPIAQIARVMIDKYYNSQSEALK---DGYEKAFRKYVKGMRDSK--VSLILYPDANSTLRLTYGSVCluster2_CgDPP ADNLYNNSFLTSLSGVESVLNLSAA-EFKERMKNDVGIQFVRELKEMNDTQVFPVYDRLNTQIHALQRTYMKAILEFSK-PSDRIFPDANGTLRVTYGKVCluster2_FpDPP APSIYSKSKLVDYANFKALLSGDAK-AVLKKISLDKGYAFVKSLADNYSKNIAPRYDEINLKINALQRIYMKAQLELY--PNSRIFPDANSTLRVTYGKVCluster3_PgDPP7 ADFVFDKSVVPYSDKFHAMLKSMDKEKFAKAIEKDPAVELSKSVIAAARAIQADAMANA-YAIEKGKRLFFAGLREMY--PGRALPSDANFTMRMSYGSICluster4_PgDPP11 VDDIFARSVFGSEAQFDAFAAVPS----VEKLAEDPMVLFASSVFDEYRKLYNELRPYD-DPILRAQRTYIAGLLEMD--GDQDQFPDANLTLRFTYGQVCluster5_BfDPP VDAMYDTSILANQANFDKFIKKPT----VKAIEKDIATQYSRAKFDKYTNLAEQMGKLP-EELALLHKTYIRGLGEMK--LPVPSYPDANFTIRLTYGNVCluster5_BvDPP VDHCYDNSIFSNEANFNKFIKKPT----VKAIEKDPMTAYVRAKYDLMDKLGNELAESM-KGMDLLHKTYVRGLCEMY--SPEPKAPDANFTIRLTYGNVGroup2_SpDPP LDGLYSLTTLTDQAQRLAWMDADTT---TFETSTDPFIRLAVALYDTNMAQEKAEKILD-GKLSTARPDYMAAVIEYYKANNWPVYPDANGTLRISYGMV

PDAN T R G

590 600 610 620 630 640 650Cluster1_CgDPP KSLPKDKRNHDVKRNYYTTFKTMLEKYKPGDAEFDMPKKFVEMYEKKDFGRYLDK----------------------DGTMHVCFLTNNDITGGNSGSPVCluster2_CgDPP AGFSP--- ADAVTYSAHTTLDGVMEKYVPGDYEFDVPQHLRDLQAKKDFGRYGDK----------------------NGKMPLCFLSTCHTTGGNSGSPACluster2_FpDPP KGYSP---KDAIYYNPTTYLDGAIEKYIPGDYEFDVPKKLIDLYNNKDYGQYGE-----------------------NGKLPVCFIGTNHTTGGNSGSPACluster3_PgDPP7 KGYEP---QDGAWYNYHTTGKGVLEKQDPKSDEFAVQENILDLFRTKNYGRYAE-----------------------NGQLHIAFLSNNDITGGNSGSPVCluster4_PgDPP11 KGYSP---RDNVYYGHQTTLDGVMEKEDPDNWEFVVDPKLKAVYERKDFGRYADR----------------------SGRMPVAFCATTHTTGGNSGSPVCluster5_BfDPP KPYSP---KDGVYYKYYTTTDGILEKENPEDREFVVPAKLKELIEKKDFGRYALP----------------------NGEMPVCFLSTNDITGGNSGSPVCluster5_BvDPP KSYNP---KDGVHYKYYTTLKGVMEKEDPTNPEFVVPAKLKELYEAKDFGRYALP----------------------NGDMPACFLTTNDITGGNSGSPVGroup2_SpDPP DGYQS---RDALYKQPFTRLDGIVAKHTGAEP-YNAPQKLLDAISEQRFGDHLVKSVYQDPRGWICRLFSCLDKPEEFNSVPVNFLSSVDTTGGNSGSPV

Tt K P F D tGGNSGSP

660 670 680 690 700 710 720Cluster1_CgDPP MNGKGELIGLAFDGNIEAMAGDVIFDKKLQRTIVVDIRYVLWCIDTFGGAKHIVDEMTIIQ----Cluster2_CgDPP IDANGNLIGLNFDRVWEGTMSDIHYDPKLCRNIMVDIRYVLFVIDKYAGAGHLVNEMKLIK----Cluster2_FpDPP VDAQGNLIGLNFDRVWEGTMSDIHYDPSICRNVMVDMRYVLFIVDKFAGAKHLINEMKLVHPKKKCluster3_PgDPP7 FDKNGRLIGLAFDGNWEAMSGDIEFEPDLQRTISVDIRYVLFMIDKWGQCPRLIQELKLI-----Cluster4_PgDPP11 MNANGELIGLNFDRNWEGVGGDIQYLADYQRSIIVDIRYVLLVIDKVGGCQRLLDEMNIVP----Cluster5_BfDPP LNENGELIGCAFDGNWESLSGDINFDNNLQRCINLDIRYVLFILEKLGGCGHLINEMTIVE----Cluster5_BvDPP INGNGELIGAAFDGNWESLSGDINFDNNLQRCIAVDIRYVLFIIDKLGGCKHLIDEMTIVE----Group2_SpDPP FNGKGELVGLNFDSTYEAITKDWFFNPTITRAVHVDIRYILWMMDKVDHADNLIKELDLVRN---Consensus R VD Ry L L E

Consensus

Consensus

ConsensusRegion 3

G l G FD

22

Fig. 2. Alignments of amino acid sequences of expressed DPPs and conserved amino acid

residues in S46 family members. Amino acid sequences of recombinant DPPs expressed in this

study were shown. Amino acid number is represented as that of PgDPP11. Conserved residues,

highly similar, and weakly similar residues among 8 DPPs are shown in red, green, and blue,

respectively. As consensus residues, identical amino acids in 264 members of the S46 family in

more than 95%, 80%, and 70% are indicated by capital, capital-italic, and small letters,

respectively. Conserved regions 1 (Ala61-Glu114), 2 (Asp198-Arg281), and 3 (Pro571/Asp571-Leu700)

are boxed. Arrowheads indicate essential amino acids forming the catalytic triad of the serine

protease family. Entirely conserved Gly673 in clusters-1, -3, and -5 DPP7 members, or Arg673 in

cluster-2 and -4 DPP11 members, but was converted to Ser673 in Group-2 DPP are boxed. An

asterisk indicates Gly/Ser680, which is substituted by Lys680 in S. putrefaciens DPP (see text for

details). Cluster-1 CgDPP (C. gingivalis, MEROPS ID: MER217135), Cluster-2 CgDPP (C.

gingivalis, MER217541), Cluster-2 FpDPP (F. psychrophilum, MER109496), Cluster-3 PgDPP7

(P. gingivalis, MER14366), Cluster-4 PgDPP11 (P. gingivalis, MER34628), Cluster-5 BfDPP (B.

fragilis, MER039993), Cluster-5 BvDPP (B. vulgatus, MER61211), Group-2 SpDPP (S.

putrefaciens, MER096825).

Fig. 3. Role of Gly673 in enzyme activity of DPP7. Hydrolyzing activities of PgDPP7 wild-type

Gly673, and single amino acid substitution mutants Gly673Asp, Gly673Ala, and Gly673Arg were

determined with Met-Leu-, Leu-Asp , and Leu-Glu-MCA. Values are shown as the mean ± S.D.

(n=3).

FIGURE 3

0

20

40

60

80

Gly673 Gly673Asp Gly673Ala Gly673Arg

Met-Leu Leu-Asp Leu-Glu

Spe

cific

act

ivity

(pm

ol/m

in/µ

g)

23

Fig. 4. Alterations of Asp/Glu preference by substitution of Arg673/Ser673 of PgDPP11 and

SpDPP11 to Ser673/Arg673 respectively. (A) The substrate specificity of S. putrefaciens DPP

(MER096825) was determined with dipeptidyl MCA substrates. (B) The hydrolyzing activities

of PgDPP11 wild-type Arg673, substitution mutant Arg673Ser, S. putrefaciens DPP11 wild-type

Ser673, and substitution mutant Ser673Arg were determined. Values are shown as the mean ± S.D.

(n=3).

FIGURE 4

0.01

0.1

1

10

100

1000

Spe

cific

act

ivity

(pm

ol/m

in/µ

g)

SpDPP11

A B

MetLeu

LeuA

rgLy

sAla

LeuA

sp

LeuG

lu

ValArg

LeuL

ys

GlyArg

GlyGly

LysL

ys

GlyPro

PgDPP11

Arg673Ser Ser673Arg

Leu-AspLeu-Glu

0.01

0.1

1

10

100

1000

Spe

cific

act

ivity

(pm

ol/m

in/µ

g)

Arg673 Ser673

FIGURE 5

0

100

200

300

400

500

Leu-Asp Leu-Glu Ala-Asn Leu-Gln

Lys680Gly Ser673Arg/Lys680Gly

Spe

cific

act

ivity

(pm

ol/m

in/µ

g)

wt

Lys6

80Gly

Ser673

Arg/

Lys6

80Gly

0.01

0.1

1

10

100

1000

wt

24

Fig. 5. Effect of Lys680Gly substitution on hydrolyzing activities of S. putrefaciens

Ser673-DPP11. The hydrolyzing activity of S. putrefaciens DPP11 wild-type (wt), substitution

mutants Lys680Gly and Ser673Arg/Lys680Gly were determined with four dipeptidyl MCA

substrates. Inserts are represented as logarithmic scales. Values are shown as the mean ± S.D.

(n=3).

7GM

ER

2749

997G

MER2799

53

7GMER02

8076

7GM

ER27

9871

6 4

7GM

ER21

7280

7GM

ER

2172

817G

ME

R10

9141

51

58

7GM

ER

1091

05

7GM

ER27

9432

7GM

ER27

9564

7GM

ER03

9992

80

75

84

7GM

ER21

7344

7GM

ER11

8379

7GM

ER28

0092

7GMER18

0324

7GMER23

5044

7GMER280025

7GMER109068 9 3

88

5 8

6 7

96

7GM

ER21

7223

7GM

ER06

1297

7GMER18

0211 9 9

62

7GMER22

7864

11GMER12

4742

11GMER27

9265

11GMER279340

9 7

9 3

7GMER27

8904

7GMER24

5884

7GMER275386

9 9

7GMER217453

7GMER1089626 9

7GMER2753027GMER275178 7 07GMER2791777GMER2174277GMER0956546 6

9 97 6

5 0 5 67 6

uGMER242678

uGMER129379uGMER239692

8 0

7GMER248763uGMER217568uGMER277162

uGMER217313uGMER2400007 5uGMER043795uGMER064694

uGMER1726968 4

uGMER118610

uGMER109666

uGMER109689

uGMER282040

uGMER1096419 0

6 0 7 1

11GMER02571311RMER136394

11RMER217693

11RMER1094016 0

11RMER2176737 5

11RMER217594

11RMER118465 6

95 1

11RM

ER235126

11RM

ER217655

11RM

ER280330

11RM

ER

0399919

811R

ME

R171742

11RM

ER

10966511R

ME

R028074

11RM

ER

217550

94

11RM

ER217599

11RM

ER217597

11RM

ER109366

11RM

ER217542

11RM

ER217538

90

53

65

93

11RM

ER163085

11RM

ER176769

72

11RM

ER136307

11RM

ER240287

11RM

ER217674

11RM

ER061213

60

11RMER217658

11RM

ER217656

11RMER217657

6 8

9 8

95

98

11RM

ER217400

11RMER109716

99

11RMER095694

53

11RMER217397

11RMER245871

9 9

5 6

2 8

99

11R

MER

0650

4011

RMER

2175

91

93

53

11RM

ER28

2246

11RMER

1094

25

8 1

11RMER24

0104

11RMER10

9624

11RMER11

8534

5 2

11RMER21

7666

11RMER217518

9 8

8 6

11RMER242837

7 311RMER136480

11RMER22996211RMER276280

7 8

9 7

7 6

uRMER217093

uRMER109241

9 9

11RMER129448

11RMER109854

9 7

11RMER229538

8 8

11RMER239699

9 9

11RMER109737

11RMER127480

11RMER109578

11RMER109476

11RMER068033

9 4

6 4

5 3

9 1

uSMER109783

uSMER178462

9 9uSMER234647

9 9

uSMER22

4590

uSMER109388

6 4

uSMER07

5899

uSMER107797

uSMER071508

uSMER113776

uSMER118823

9 4 uSMER089732

uSMER070845

uSMER125033

uSMER203080

uSMER145137

uSMER026110

5 2 uSMER076127uSMER057559uSMER076258

uSMER074416uSMER074442

uSMER076243

6 2

5 55 79 9

7GMER13

6575

7GMER082302

7GMER1099147GMER089729

7GMER2032237GMER096969

5 9

7GMER1453477GMER1186647GMER113592

6 5

5 8

5 2

7GMER071511

7GMER096834

7GMER076118

7GMER108928

7GMER075078

7GMER074443

7GMER074413

6 7

5 97 5

7GM

ER026109

7GM

ER075894

6 1

7GM

ER109021

7GMER224475

6 3

7GMER068120

7GMER057818

7 6

8 2

6 18

4

7GMER040699

7GMER109044

7GMER064377

7GMER108988

7GMER109386

7GM

ER108887

7GM

ER109096

74

7GM

ER116742

7GM

ER114456

7GM

ER1095437GM

ER180722

7GM

ER197194

7GM

ER014367

7GM

ER113819

94

7GM

ER171948

7GM

ER110037

95

7GM

ER

234615

7GM

ER

1088967G

ME

R229450

82

99

51

uGMER108856

uGMER025898

7 4

uGMER108900

5 0

uGM

ER221965

uGM

ER217444

83

85

uGM

ER230407

73

uRMER217683

uSMER137074

99

uSMER130666

uSMER058364

90

uGM

ER130667

80

uGM

ER125327

uGM

ER136893

53

uGM

ER030572

uGM

ER235742

uGM

ER065521uR

ME

R217415

uGM

ER

153993uG

MER

0659929

9

uGM

ER

067962uG

ME

R109603

uGM

ER

255525uG

ME

R036977

uGM

ER

064297uG

ME

R068085

uGM

ER109687

98

uGM

ER

134491uG

MER

090845

75

0.05NJ

Cluster

5

Clu

ster

1

Cluster

3

Cluster 4

Cluster 2

Group 2

Group 3

Group 1

9 8

7GMER2172677GMER217181

6 1

7GMER203644

7GMER179133

7GMER280392

7GMER280542

7GMER242888

7GMER221301

6 7

11GMER180464

11GMER217065

11GMER217067 9 8

11GMER217170

11GMER144886

11GMER2402857 7

7 6

9 0

11GMER124817

11GMER276858

7 1

11GMER109273

5 2

11GMER235125

8 1

11GMER21725411G

MER180879

11GM

ER109454

11GM

ER163087

11GM

ER109242

11GMER217252

11GMER217241

11GMER217239

11GMER028075

11GMER217225

88 9

16

8

8 4

11GM

ER039993

11GMER061211

uGMER217135

7GMER014366

11RM

ER10

9496

11RMER217541

11RMER034628PeDPP11

uSMER096825

*7

*5

*6

*3

*4

*2

*1

FIGURE 6

25

Fig. 6. Phylogenic tree of S46 family based on sequences of conserved region 3. A phylogenic

tree was constructed using an NJ method [32] with ClustalX software [31] based on conserved

region 3 (Pro/Ser571-Leu700). Basic information for DPPs is included in the names, as follows:

first 7 (DPP7), 11 (DPP11), or u (unassigned) in the MEROPS database (Release 9.6), followed

by G (Gly673), R (Arg673), or S (Ser673), then finally the MEROPS codes. For example, PgDPP7

and PgDPP11 were written as 7GMER014366 and 11RMER034628, respectively. As an

exception, PeDPP11 is simply expressed the same as that cloned in the laboratory [16] and is not

yet registered in the database. DPPs expressed in this study are boxed. The phylum Bacteroidetes

consisted of five clusters and others mainly comprising the phylum Proteobacteria consisted of

three groups. See text and supplemental Fig. S1 for members marked by asterisks. Basic

information and the names of the DPPs are described in supplemental Table S2.

26

SUPPLEMENTAL DATA

Supplemental Table S1 Primers used for expressions of DPPIV, DPP7, and DPP11 from P. gingivalis, and DPPs from clusters 1, 2, and 5 of the phylum Bacteroidetes. Restriction sites are indicated by italics.

Primer DPP Sequence (5’-3’)

5PgDPPIVD23Bam PgDPPIV GGCTCAGGGATCCGACAAACCCGTAGATTT

3PgDPPIVL723Bam

PgDPPIV AGAGATACTGGATCCAAGATTGTCGAACAA

5PgDPP7Q2Bgl PgDPP7 GACTGCTAGATCTCAAATGAAATTAAAAAGTATTC

3PgDPP7I712Bgl PgDPP7 GGCGAATAGATCTGATCAACTTCAGCTC

5PgDPP7Q2 PgDPP7 deletion CAAATGAAATTAAAAAGTATTCTTCTCG

pQE60-M1Rev PgDPP7 deletion CATGGTTAATTTCTCCTCTTTAATGA

5PgDPP11K2Bam PgDPP11 GTAGGATCCAAAAAAGACTATTGCTCCCCCTC

3PgDPP11P720Bam PgDPP11 ATAGGATTCGGGAACGATATTCATTTCATCCAAC

5CgDPPG1R2Bam C. gingivalis cluster-1 TAATTGGGATCCAGAAAACTTATTTTTTC

3CgDPPG1Q715Bam

C. gingivalis cluster-1 AATAGGGATCCCTGGATAATAGTCATTTC

5FpDPPC1D22 F. psychrophilum cluster-1

GATGAAGGAATGTGGTTTTTAATGT

3FpDPPC1K716 F. psychrophilum cluster-1

CTATTATTTAACTAAAGTCATTTCA

5CgDPPC2L16 C. gingivalis cluster-2 CTCTATGGACAGCAAGGCGGGAT

3CgDPPC2K712 C. gingivalis cluster-2 CTATTACTTGATGAGTTTCATTT

5FpDPPC2Q18 F. psychrophilum cluster-2

CAACAAGGGGGCATGTGGATTCC

3FpDPPC2K713 F. psychrophilum cluster-2

CTATTATTTTTTCTTAGGGTGAA

5BfDPPC5N2Bam B. fragilis cluster-5 ATATAGGATCCAACAGACTCAAACTTTAC

3BfDPPC5E721Bam

B. fragilis cluster-5 TCATAGGATCCTTCAACAATCGTCATTTC

5Bv DPPC5K2Bgl B. vulgatus cluster-5 TTATGAGATCTAAAAAATTCAAACTTTTATTG

27

3BvDPPC5E721Bgl B. vulgatus cluster-5 TACATAGATCTTTCAACAATAGTCATTTC

5SpDPP SR2Bam S. putrefaciens TAATCAGGATCCCGTATTGCACTGGTTGCGAC

3SpDPP SN732Bam S. putrefaciens ACTATAGGATCCATTTCTCACTAAATCCAGTTC

28

Supplemental Table S2 Classification of DPP7 and DPP11 based on amino acid residue at position 673. DPPs in the S46 family are classified as DPP7 carrying Gly673, and DPP11 carrying either Asp673 or Ser673. DPPs are represented by the MEROPS codes and numbers 1-263 are identical to those of the MEROPS database (Release 9.6). Annotation in the database is indicated by italics (unassigned), bold (DPP11), and underlining (DPP7). MEROPS codes in grey backgrounds are DPPs with substrate specificity directly identified in the present and previous [refs. 14-16] studies. aNos. 61 and 62 were 93.44% identical, presumably due to the different strains of a single organism. bNos. 98 and 231 were 25.81% identical. cComposed of 1200 residues, of which residues 1-626 were homologous to the S46 family members.

Species and strains DPP7 DPP11

Gly673 Arg673 Ser673 Shewanella sediminis 1 (MER109914) 36 (MER070845) Shewanella benthica 2 (MER136575) 37 (MER125033) Shewanella woodyi 3 (MER082302) 35 (MER076258) Shewanella violacea 4 (MER203223) 38 (MER203080) Shewanella loihica 5 (MER089729) 34 (MER089732) Shewanella putrefaciens 6 (MER096834) 44 (MER096825) Shewanella sp. W3-18-1 7 (MER076118) 45 (MER076127) Shewanella baltica 8 (MER108928) 46 (MER057559) Shewanella sp. ANA-3 9 (MER075078) 41 (MER076243) Shewanella sp. MR-7 10 (MER074443) 40 (MER074442) Shewanella sp. MR-4 11 (MER074413) 39 (MER074416) Shewanella oneidensis 12 (MER026109) 42 (MER026110) Shewanella halifaxensis 13 (MER118664) 32 (MER118823) Shewanella pealeana 14 (MER113592) 31 (MER113776) Shewanella piezotolerans 15 (MER145347) 33 (MER145137) Shewanella denitrificans 16 (MER071511) 47 (MER071508) Shewanella frigidimarina 17 (MER096969) 49 (MER107797) Shewanella amazonensis 18 (MER075894) 48 (MER075899) Shewanella sp. HN-41 43 (MER276432) Ferrimonas balearica 19 (MER224475) 50 (MER224590) Pseudoalteromonas tunicata 20 (MER109021) 52 (MER109388) Kangiella koreensis 21 (MER068120) Rheinheimera sp. A13L 22 (MER276692) 51 (MER276752) Colwellia psychrerythraea 23 (MER057818) Glaciecola nitratireducens 24 (MER281964) gamma proteobacterium 25 (MER276352)

29

IMCC3088 Erythrobacter sp. NAP1 26 (MER109386) Erythrobacter sp. SD-21 27 (MER108887) Parvularcula bermudensis 28 (MER108988) Alteromonas macleodii 29 (MER109096) Idiomarina loihiensis 30 (MER040699) Burkholderia sp. JV3 53 (MER272800) Stenotrophomonas sp. SKA14 54 (MER171948) 64 (MER178462) Stenotrophomonas maltophilia 55 (MER110037) 65 (MER109783) Pseudoxanthomonas suwonensis 56 (MER234615) 66 (MER234647) Xanthomonas albilineans 57 (MER197194) Xanthomonas oryzae 58 (MER108896) Xanthomonas alfalfae 59 (MER274065) Xanthomonas fuscans 60 (MER229450) Xylella fastidiosa

61 (MER014367)a 62 (MER113819)a

Burkholderia oklahomensis 63 (MER180722) Haliangium ochraceum 67 (MER116742) 72 (MER127480) Sorangium cellulosum 68 (MER114456) Plesiocystis pacifica 69 (MER109543) 73 (MER109737) Solibacter usitatus 70 (MER109044) 76 (MER109578) Anaeromyxobacter sp. Fw109-5 71 (MER064377) Myxococcus xanthus 249 (MER068085) 74 (MER068033) Stigmatella aurantiaca 250 (MER109687) 75 (MER109476) Candidatus Chloracidobacterium thermophilum

77 (MER274999)

Pedobacter sp. BAL39 78 (MER109854) Pedobacter heparinus 213 (MER129379) 79 (MER129448) Sphingobacterium spiritivorum 80 (MER229538) Pedobacter saltans 212 (MER239692) 81 (MER239699) Aureococcus anophagefferens 82 (MER243787) Alistipes shahii 83 (MER279340) 162 (MER229962) Alistipes sp. HGB5 84 (MER279265) Alistipes putredinis 85 (MER124742) 163 (MER136480) Cytophaga hutchinsonii 86 (MER025713) Bacteroides coprophilus 135 (MER217170) 87 (MER176769) Bacteroides plebeius 134 (MER136226) 88 (MER163085) Bacteroides salanitronis 132 (MER240285) 89 (MER240287)

30

Bacteroides coprocola 133 (MER144886) 90 (MER136307)

Bacteroides sp. 9_1_42

137 (MER217067) 184 (MER217223)

91 (MER217658)

Bacteroides dorei

139 (MER180464) 185 (MER180211)

92 (MER217657)

Bacteroides sp. 3_1_33 93 (MER217656) Bacteroides vulgatus

136 (MER061211) 186 (MER061297)

94 (MER061213)

Bacteroides sp. 4_3_47 138 (MER217065) 95 (MER217674) Bacteroides sp. D2 126 (MER217252) 96 (MER217599) Bacteroides sp. 2_2_4 165 (MER217280) 97 (MER217597) Bacteroides ovatus

125 (MER109242) 166 (MER109141)

98 (MER109366)b 231 (MER109241)b

Bacteroides sp. D1

128 (MER217239) 167 (MER217281)

99 (MER217538)

Bacteroides sp. 2_1_22 127 (MER217241) 100 (MER217542) Bacteroides finegoldii 124 (MER163087) 101 (MER171742) Bacteroides caccae

123 (MER109454) 168 (MER109105)

102 (MER109665)

Bacteroides sp. 1_1_6

129 (MER217225) 171 (MER279953)

103 (MER217550)

Bacteroides thetaiotaomicron

130 (MER028075) 170 (MER028076)

104 (MER028074)

Bacteroides sp. 2_1_56FAA 175 (MER279492) 105 (MER280467) Bacteroides sp. 3_2_5 174 (MER279564) 106 (MER217655) Bacteroides fragilis

131 (MER039993) 173 (MER039992)

107 (MER039991)

Bacteroides sp. 2_1_16 172 (MER279432) 108 (MER280330) Bacteroides stercoris

120 (MER124817) 180 (MER118379)

109 (MER118465)

Bacteroides eggerthii

119 (MER276858) 181 (MER217344)

110 (MER217594)

Bacteroides uniformis

117 (MER109273) 177 (MER109068)

111 (MER109401)

Bacteroides sp. D20 178 (MER280025) 112 (MER217693) Bacteroides cellulosilyticus

122 (MER217254) 183 (MER280092)

113 (MER217673)

Bacteroides intestinalis 121 (MER180879) 114 (MER136394)

31

182 (MER180324) Bacteroides helcogenes

116 (MER235125) 179 (MER235044)

115 (MER235126)

Bacteroides clarus 118 (MER277010) Bacteroides sp. 1_1_14 169 (MER279871) Bacteroides sp. 4_1_36 176 (MER279717) Bacteroides sp. 20_3 194 (MER275178) Bacteroides sp. 3_1_19 195 (MER275302) Bacteroides sp. 2_1_33B 198 (MER217427) Parabacteroides sp. D13 196 (MER279177) Bacteroides sp. D2 232 (MER217093) Parabacteroides merdae 192 (MER108962) 140 (MER109716) Parabacteroides johnsonii 193 (MER217453) 141 (MER217400) Parabacteroides distasonis 197 (MER095654) 142 (MER095694) Porphyromonas gingivalis 189 (MER014366) 143 (MER034628) Porphyromonas asaccharolytica 191 (MER245884) 144 (MER245871) Porphyromonas uenonis 190 (MER275386) 145 (MER217397) Odoribacter splanchnicus 146 (MER275825) Bacteroidetes oral taxon 274 147 (MER277162) Flavobacteria bacterium BAL38 218 (MER109689) 148 (MER109425) Flavobacterium columnare 219 (MER282040) 149 (MER282246) Flavobacterium psychrophilum 217 (MER109641) 150 (MER109496) Capnocytophaga gingivalis 229 (MER217135) 151 (MER217541) Capnocytophaga canimorsus 228 (MER255367) 152 (MER255206) Capnocytophaga sputigena 226 (MER172696) 153 (MER217591) Capnocytophaga ochracea 227 (MER064694) 154 (MER065040) Flavobacteriales bacterium ALC-1 155 (MER118534) Lacinutrix sp. 5H-3-7-4 156 (MER251262) unidentified eubacterium SCB49 157 (MER109624) Weeksella virosa 158 (MER240104) Flavobacteriaceae bacterium 3519-10

230 (MER217568) 159 (MER217518)

Chryseobacterium gleum 160 (MER217666) Alistipes sp. HGB5 161 (MER276280) Fluviicola taffensis 164 (MER242837) Paraprevotella xylaniphila 187 (MER277088) Porphyromonas endodontalis 188 (MER278904) 264 PeDPP11

32

Dysgonomonas mossii 199 (MER275543) Dysgonomonas gadei 200 (MER275470) Paludibacter propionicigenes 201 (MER227864) Prevotella timonensis 202 (MER280542) Prevotella oralis 203 (MER280616) Prevotella salivae 204 (MER280687) Prevotella copri 205 (MER179133) Prevotella sp. oral taxon 472 206 (MER217181) Prevotella sp. oral taxon 317 207 (MER217267) Prevotella ruminicola 208 (MER203644) Prevotella melaninogenica 209 (MER221301) Prevotella denticola 210 (MER242888) Prevotella bivia 211 (MER280392) Runella slithyformis 214 (MER252347) Fluviicola taffensis 215 (MER242678) Haliscomenobacter hydrossis 216 (MER248763) Flavobacteria bacterium BAL38 218 (MER109689) Flavobacterium columnare 219 (MER282040) Flavobacterium branchiophilum 220 (MER274656) Flavobacterium johnsoniae 221 (MER043795) unidentified eubacterium SCB49 222 (MER109666) Kordia algicida 223 (MER118610) Weeksella virosa 224 (MER240000) Sphingobacterium spiritivorum 225 (MER217313) Capnocytophaga ochracea 227 (MER064694) Rhodothermus marinus 235 (MER130667) 233 (MER130666) Salinibacter ruber 234 (MER058364) Spirosoma linguale 236 (MER090845) Opitutus terrae 237 (MER125327) bacterium Ellin514 238 (MER136893) Isosphaera pallida 239 (MER235742) Rhodopirellula baltica 240 (MER030572) Gemmata obscuriglobus 241 (MER217415) Acidobacteria bacterium 242 (MER065521) Anaeromyxobacter dehalogenans 243 (MER153993) Anaeromyxobacter sp. K 244 (MER125498)c Anaeromyxobacter dehalogenans 245 (MER065992)

33

Anaeromyxobacter sp. Fw109-5 246 (MER064297) Gloeobacter violaceus 247 (MER036977) Myxococcus fulvus 248 (MER251918) Collimonas fungivorans 251 (MER255525) Myxococcus fulvus 252 (MER251542) Myxococcus xanthus 253 (MER067962) Rhodospirillum centenum 254 (MER134491) uncultured bacterium 255 (MER109603) Brevundimonas subvibrioides 256 (MER221965) 263 (MER217683) Brevundimonas sp. BAL3 257 (MER217444) 262 (MER137074) Asticcacaulis excentricus 258 (MER230407) Sphingomonas sp. SKA58 259 (MER108856) Novosphingobium aromaticivorans 260 (MER025898) Sphingopyxis alaskensis 261 (MER108900)

34

SUPPLEMENTAL FIGURE LEGENDS

Supplemental FIGURE S1

11R

MER

2173

9711

RM

ER

2458

719

9

78

61

11R

ME

R09

5694

11R

ME

R21

7400

11R

MER

1097

169

99

9

99

11R

MER

2176

5611

RM

ER21

7657

11R

ME

R21

7658

99

11R

ME

R21

7674

11R

ME

R06

1213

92 9

9

11R

ME

R13

6307

11R

MER

2402

87

84

11R

MER

1630

85

11R

MER

1767

69

79

99

99

11R

ME

R13

6394

11R

MER

2176

739

9

11R

MER

2175

94

11R

MER

1184

659

9

11RM

ER23

5126

11RM

ER21

7693

11RM

ER10

9401

9 9

96

11RMER28

0330

11RMER03

9991

11RMER21

7655

9 9

11RMER10

9665

11RMER171742

5 5

11RMER028074

11RMER217550 9 9

11RMER21754211RMER2175389 9

11RMER10936611RMER217597

11RMER2175997 7

9 98 8

9 9

8 39 9

9 9

56

11G

MER

2172

39

11G

MER

2172

41 9 2

11GMER217252

11GMER109242

5 3

9 9

11GMER1630876 3

11GMER1094549 5

11GMER02807511GMER217225 9 9 9 911GMER21725411GMER180879 9 9

6 39 811GMER12481711GMER276858 9 9

11GMER10927311GMER235125 9 8 9 9

9 2

11GMER144886

11GMER2402859 9

11GMER217170

9 9

11GMER180464

11GMER217065

11GMER2170679 09 9

8 2 9 9

9 4

11GMER124742

11GMER279340

11GMER279265

9 99 9

7 4

11GMER025713

7GMER2791777GMER095654 6 8

7GMER217427

6 9

7GMER275178

7GMER2753026 3

9 9

7GMER108962

7GMER2174539 9

9 9

7 5

7GMER278904

7GMER275386

7GMER245884

9 9

7 6

9 9

7GMER227864

6 8

99

99

76

90

7GM

ER280542

7GM

ER179133

7GM

ER217181

7GM

ER217267

7GM

ER203644

7GM

ER280392

7GM

ER

2213017G

ME

R242888

98

99

7GMER217223

7GMER1802119 8

7GMER0612979 9

7GMER109068

7GMER280025 9 9

7GMER235044

7 6

7GMER180324

7GMER280092

9 9

5 2

7GMER118379

7GMER217344

9 9

9 9

7GMER039992

7GMER279564

7 5

7GMER279432

9 9

7GMER279871

7GM

ER2799537

7

7GM

ER0280769

9

7GM

ER1091057G

MER

2172817G

MER

2172807G

MER

109141

99

81

62

99

98

99

9 9

78

9 9

11GM

ER

277162uG

ME

R239692

uGM

ER

129379

99

7GM

ER

248763

75

uGM

ER

242678uG

ME

R217568

uGM

ER

172696uG

ME

R064694

99

99

uGM

ER

240000uG

ME

R217313

77

uGM

ER109666

uGM

ER118610

65

uGM

ER043795

uGM

ER109689

uGM

ER109641

uGM

ER282040

59

99

92

73

96

99

98

69

71

68

9 9

uRM

ER10

9241

uRM

ER21

7093

99

11R

MER

2762

8011

RM

ER22

9962

99

11R

MER

1364

80

99

11R

MER

2428

37

11R

MER

2175

1811

RM

ER21

7666

99

11RM

ER24

0104

80

11RM

ER11

8534

11RM

ER10

9624

99

11RM

ER10

9425

11RM

ER28

2246

94

99

11RMER

2175

91

11RMER06

5040

9 9

9 7

9 4

9 4

99

82

72

9 211

RMER

1098

54

11RM

ER12

9448

9 9

11RMER22

9538

8 9

11RMER23

9699

9 9

11RM

ER06

8033

11RMER10

9476

9 9

11RMER10

9578

9 9

11RMER12

7480

9 9

9 8

11RMER10

9737

99

uSMER178462

uSMER

109783

uSMER234647

99

7 47 9

8 98 0

7GM

ER274999

uSMER130666

uSMER058364

99

uGM

ER130667

93

uGM

ER090845

99

uGM

ER125327

uGM

ER136893

98

uGM

ER235742

55

uGM

ER030572

66

uRM

ER217415

59

uGM

ER065521

89

uGM

ER221965

uGM

ER217444

99

uGM

ER230407

83

uGM

ER108900

uGM

ER108856

uGM

ER

025898

64

99

uSM

ER

137074uR

ME

R217683

99

98

uGM

ER

109603uG

ME

R134491

uGM

ER

067962uG

ME

R255525

uGM

ER

036977uG

ME

R068085

uGM

ER109687

99

uGM

ER064297

uGM

ER153993

uGM

ER065992

99

64

93

67

67

80

99

99

0.05NJ

Cluster 2

Cluster 4

Clu

ster

5

Cluster 1

Cluster

3

Group 1

9 9

9 9

6 9

9 8

5 28 9

9 2

7 3

7GMER19

7194

7GMER10

8896

7GMER23

4615

7GMER229450

7GMER171948

7GMER110037

9 9

7GMER014367

7GMER113819

9 9

7GMER180722

7GMER109044

7GMER064377

7GMER109543

7GMER116742

7GMER114456

9 9

9 5

5 1

6 3

9 9

9 8

9 9

uSMER089732uSMER070845uSMER125033uSMER203080

9 9

uSMER076258

uSMER145137

uSMER113776

uSMER118823

9 9

9 6

uSMER076127

9 9

uSMER057559

uSMER026110

uSMER076243

uSMER

074416

uSMER

074442

97

91

96

57

9 9

uSMER

107797

uSMER

071508

uSMER

075899

uSMER

224590

uSMER

109388

8 2

6 79 2

9 999

76

9 9

8 3

7GMER109021

7GMER224475

7GMER075894

7GMER071511

7GMER118664

7GMER1135929 8

9 9

9 88 7

7GMER040699

7GMER109386

7GMER108887

9 9

7GMER108988

9 7

7GMER109096

9 9

7GMER068120

7GMER057818

6 7

9 99 9

7 26 49 99 4

7GMER1453477GMER096969

7GMER1099147GMER1365757GMER0823027GMER089729

7GMER2032237GMER026109

7GMER096834

7GMER076118

7GMER108928

7GMER075078

7GM

ER074443

7GM

ER074413

Group 3

Group 2

11RM

ER10

9496

11RMER21

7541

uGM

ER

217135

11GMER039993

11GMER061211

11R

ME

R03

4628

uSMER096825

PeD

PP

11

7GMER014366

*2*1

*3

*4

*7

*6*5

35

Supplemental Fig. S1. Phylogenic tree of S46 family based on full-length amino acid sequences.

The phylogenic tree of the S46 family was constructed using an NJ method by comparisons of

the full-length sequences. Basic information for the DPPs is included in the names, as follows:

first, 7 (DPP7), 11 (DPP11), or u (unassigned) in the MEROPS database (Release 9.6), followed

by G (Gly673), R (Arg673), or S (Ser673), and finally the MEROPS codes. For example, PgDPP7

and PgDPP11 are expressed as 7GMER014366 (DPP7-Gly673-MER014366) and

11RMER034628 (DPP11-Arg673-MER034628), respectively. DPPs expressed in this study are

boxed. Members from phylum Bacteroidetes were separated into five clusters and others were

classified into three groups. Members with asterisks 1-4 were not categorized into any clusters or

groups. Members with asterisks 5-7 were categorized into group 1 and possessed Arg673 or

Ser673.

Supplemental Fig. S2. SDS-PAGE of recombinant DPPs. E. coli-expressed purified samples

(0.5 µg) of PgDPPIV (lane 1), PgDPP7 (lane 2), PgDPP11 (lane 3), cluster-1 DPP from C.

gingivalis (lane 4), cluster-2 DPPs from C. gingivalis (lane 5) and F. psychrophilum (lane 6), and

cluster-5 DPPs from B. fragilis (lane 7) and B. vulgatus (lane 8) were separated using

SDS-PAGE on 10% polyacrylamide gels. Separated proteins were stained with

Coomassie-brilliant blue. Lane M, low-molecular-weight marker.

Supplemental FIGURE S2

kDa

97

66

45

M 1 2 3 4 5 6 7 8

36

Supplemental FIGURE S3

Cluster 3

Clus

ter 5

*2*1

*3

*4

*7

*6

*5

Group 1

Group 3

Cluster 2

Cluster 4

Clu

ster

1

Group 2

7GM

ER21

7280

7GM

ER10

9141

53

7GM

ER21

7281

7GM

ER27

9871

7GM

ER02

8076

7GM

ER27

9953

6678

7GM

ER

1091

05

62

7GM

ER

2794

327G

MER

0399

92

7GM

ER27

9564

99

77

7GM

ER23

5044

7GM

ER11

8379

7GM

ER21

7344

72

7GM

ER10

9068

7GM

ER28

0025

99

7GMER

1803

24

7GMER28

0092

92

54 76

99

7GM

ER21

7223

7GM

ER18

0211

7GMER

0612

97 100

98

7GM

ER20

3644

7GM

ER21

7181

7GM

ER21

7267

98

7GMER

2805

42

7GMER17

9133

50

7GMER28

0392

7GMER22

1301

7GMER242888

87

81

100

70

7GMER

2753

86

7GMER24

5884

100

uGMER27

8904

7GMER01

4366

7GMER10

8962

7GMER21745398

7GMER275302

7GMER2751787GMER2791777GMER0956547GMER217427

6799

96

94

99

7GMER22786411GMER12474211GMER27934011GMER279265

94

98

11GMER02571311GMER277162uGMER242678

uGMER239692uGMER12937995

7GMER248763

51

uGMER217568uGMER240000uGMER217313 83

uGMER109641uGMER282040

58

uGMER10968991

uGMER043795

uGMER109666

uGMER118610

uGMER217135

uGMER172696

uGMER06469498

5977

71

11GMER21706711GMER217065

11GMER06121171

11GMER180464100

11GMER217170

11GMER240285

11GMER14488692

95

11GMER235125

11GMER109273

84

11GMER276858

11GMER12481779

93

11GMER180879

11GMER217254

82

11GMER217225

11GMER028075

92

11GMER109454

11GMER163087

11GM

ER03999311G

MER

10924211G

ME

R217252

11GM

ER217241

11GM

ER217239

59 5393

6292

100

11RMER217657

11RMER217656

11RMER217658

80

11RMER061213

51

11RMER217674

99

11RMER176769

11RMER163085

97

11RM

ER240287

11RM

ER136307

8351

97

11RM

ER217538

11RM

ER217542

82

11RM

ER217550

11RM

ER

028074

7553

11RM

ER

21759911R

ME

R217597

11RM

ER

10936670

84

11RM

ER

10966511R

ME

R171742

83

11RM

ER

23512611R

ME

R039991

11RM

ER280330

11RM

ER217655

99

11RMER118465

11RMER217594

98

11RMER109401

11RMER217693

94

11RMER217673

11RMER136394

95

6155

98

1007G

ME

R274999

11R

MER

2458

7111

RM

ER21

7397

100

PeD

PP11

85

11R

MER

0346

2811

RM

ER09

5694

11R

MER

1097

16

11RM

ER21

7400

100

87

7899

11R

MER

2762

80

11R

MER

2299

62

64

11RM

ER13

6480

100

11RR

MER

2428

3711

RM

ER21

7518

11RM

ER21

7666

100

11RM

ER24

0104

61

11RM

ER11

8534

11RM

ER10

9624

97

11RM

ER21

7541

11RM

ER10

9425

11RMER28

2246

98

11RMER10

9496

11RMER21

7591

11RMER065040

100

98

57

86

uRMER

1092

41

uRMER21

7093

100 11

RMER06

8033

11RMER10

9476

99

11RMER10

9578

89

11RMER12

7480

11RMER10

9737

63

97

11RMER23

9699

11RMER22

9538

11RMER109854

11RMER129448

99

76

100

55

uSM

ER12

5033

uSM

ER20

3080

51

uSM

ER08

9732

uSMER

0708

45

uSMER07

6258

uSMER14

5137

uSMER113776

uSMER118823

9299 uSMER057559

uSMER074416

uSMER074442

81 uSMER07624354 uSMER026110uSMER096825uSMER076127

81545281

11SMER075899uSMER071508uSMER107797

93

uSMER224590

82

uSMER109388

99

uSMER234647uSMER178462uSMER109783100

100

50

7GMER1719487GMER11003796

7GMER1088967GMER22945074

7GMER2346157GMER0143677GMER113819

97

7GMER197194

100

7GMER1807227GMER116742

7GMER114456

70

7GMER109543

7GMER109044

7GMER064377

7GMER040699

7GMER1093867GMER108887

7GMER108988

7GMER109096

99

uSMER276352

7GMER068120

7GMER057818

71

7GMER2244757GMER109021

7GMER075894

7GMER071511

7GMER096834

7GMER076118

84

7GMER108928

7GMER075078

7GM

ER074443

7GM

ER074413

80

64

7GM

ER026109

7GM

ER118664

7GM

ER113592

1007G

MER

145347

7GM

ER203223

7GM

ER096969

7GM

ER089729

7GM

ER109914

7GM

ER

1365757G

MER

082302

5354

64

86

96

50

53

52

uSMER

130666

uSMER

058364

80

uGM

ER130667

95

uGM

ER090845

84

uGM

ER125327

uGM

ER235742

55

uGM

ER136893

uGM

ER065521

uGM

ER030572

uRM

ER217415uG

MER

025898uG

MER

108900

52uG

ME

R108856

65uG

ME

R221965

uGM

ER

217444

99

85

uGM

ER

230407

74

uSM

ER

137074uR

MER

217683

100

uGM

ER109603

uGM

ER

134491uG

MER

067962uG

MER

036977uG

MER

255525uG

MER

153993uG

MER

065992

100

uGM

ER064297

uGM

ER068085

uGM

ER109687

99

6484

64

100

0.05NJ

37

Supplemental Fig. S3. Phylogenic tree of S46 family based on the three conserved regions.

Three conserved regions (Ala61-Glu114, Asp198-Arg281, and Pro571/Asp571-Leu700) were used for

calculation. Other notations are same as those in supplemental Fig. S1.