Reversible Nε-Lysine Acetylation Regulates the Activity of Acyl-CoA Synthetases Involved in Anaerobic Benzoate Catabolism inRhodopseudomonas palustris

Heidi A. Crosby1, Erin K. Heiniger2, Caroline S. Harwood2, and Jorge C. Escalante-Semerena*,1

1Department of Bacteriology of the University of Wisconsin, Madison, WI 53706 USA2Department of Microbiology of the University of Washington, Seattle, WA 98195 USA

AbstractRhodopseudomonas palustris grows photoheterotrophically on aromatic compounds available inaquatic environments rich in plant-derived lignin. Benzoate degradation is regulated at thetranscriptional level in R. palustris in response to anoxia and the presence of benzoate and/orbenzoyl-CoA (Bz-CoA). Here, we report evidence that anaerobic benzoate catabolism in thisbacterium is also regulated at the posttranslational level. In this pathway, benzoate is activated toBz-CoA by the AMP-forming Bz-CoA synthetase (BadA) enzyme. Mass spectrometry andmutational analysis data indicate that residue Lys512 is critical to BadA activity. Acetylation ofLys512 inactivated BadA; deacetylation reactivated BadA. Likewise, 4-hydroxybenzoyl-CoA(HbaA) and cyclohexanecarboxyl-CoA (AliA) synthetases were also reversibly acetylated. Weidentified one acetyltransferase that modified BadA, Hba, and AliA in vitro. The acetyltransferaseenzyme is homologous to the protein acetyltransferase (Pat) enzyme of Salmonella enterica svTyphimurium LT2, thus we refer to it as RpPat. RpPat also modified acetyl-CoA (Ac-CoA)synthetase (Acs) from R. palustris. In vivo data indicate that at least two deacetylases reactivateBadAAc. One is SrtN (encoded by srtN, formerly rpa2524), a sirtuin-type NAD+-dependentdeacetylase (O-acetyl-ADP-ribose-forming); the other deacetylase is LdaA (encoded by ldaA, forlysine deacetylase A; formerly rpa0954), an acetate-forming protein deacetylase. LdaAreactivated HbaAc and AliAAc in vitro.

INTRODUCTIONAromatic compounds are widespread in the environment, where they are primarily derivedfrom plant material such as lignin (Dagley, 1981). In the last century, human contaminationwith aromatic compounds such as polychlorinated biphenyls, polyaromatic hydrocarbons,benzene, toluene, xylene and others has become an environmental concern. Thebiodegradation of aromatic compounds presents unique biochemical challenges because ofthe high resonance energy that stabilizes benzene rings. However, many microorganismspossess the metabolic capabilities necessary for the use of these recalcitrant compounds ascarbon and energy sources under aerobic and anaerobic conditions (Diaz, 2004, Fuchs,2008, Gibson & Harwood, 2002, Vaillancourt et al., 2006). Although microbes use differentpathways depending on oxygen availability, all aromatic compound degradation pathwaysrequire activation of the stable aromatic ring and then subsequent ring cleavage. Underanoxic conditions,, aromatic rings tend to be activated by coenzyme A (CoA)

*Corresponding author: Department of Bacteriology, University of Wisconsin, 6478 Microbial Sciences Building, 1550 Linden Drive,Madison, WI 53706. Tel: 608-262-7379; Fax: 608-265-7909; escalante@bact.wisc.edu.

NIH Public AccessAuthor ManuscriptMol Microbiol. Author manuscript; available in PMC 2011 May 1.

Published in final edited form as:Mol Microbiol. 2010 May ; 76(4): 874–888. doi:10.1111/j.1365-2958.2010.07127.x.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

thioesterification prior to ring modification and ring reduction steps leading to ring cleavage(Carmona et al., 2009, Fuchs, 2008). Under oxic conditions, dioxygenases destabilize thearomatic ring by hydroxylation, before they catalyze ring cleavage (Vaillancourt et al,2006). More recently, hybrid pathways have been described that involve formation of CoAderivatives of aromatic acids, followed by ring hydroxylation by dioxygenases (Zaar et al.,2001, Zaar et al., 2004, Fuchs, 2008).

The photoheterotrophic, purple non-sulfur α-proteobacterium Rhodopseudomonas palustrishas one of the best-studied systems for anaerobic aromatic compound degradation (Harwoodet al., 1999). When growing photosynthetically in the absence of oxygen, R. palustrisconverts aromatic compounds to Ac-CoA, which is then used as carbon and energy source.

The first step in the degradation of benzoate, and the related compounds 4-hydroxybenzoate(4-HBA) and cyclohexanecarboxylate (CHC), is the activation of the acid to thecorresponding CoA thioester by an AMP-forming acyl-CoA synthetase (Fig. 1a). R.palustris has at least three acyl-CoA synthetases with overlapping specificity; BadAprimarily activates benzoate to Bz-CoA, and HbaA and AliA activate 4-HBA and CHC,respectively, to their CoA thioesters (Geissler et al., 1988,Gibson et al., 1994,Samanta &Harwood, 2005,Egland et al., 1995). In the central benzoate pathway, the aromatic ring ofBz-CoA is reduced, hydrated, and cleaved, yielding the linear compound pimeloyl-CoA,which is degraded via β-oxidation yielding Ac-CoA and CO2 (Egland et al., 1997,Harrison& Harwood, 2005). The products of HbaA and AliA (4-hydroxybenzoyl-CoA andcyclohexanecarboxyl-CoA, respectively), feed into the central benzoate degradationpathway (Fig. 1a).

Three transcriptional regulators control benzoate degradation in R. palustris by modulatingexpression of the badDEFGAB operon that encodes the O2-sensitive 4-component Bz-CoAreductase (BadDEFG), Bz-CoA synthetase (BadA) and a ferredoxin (BadB). AadR, (ahomolog of E. coli Fnr), activates the transcription of the badDEFGAB operon in responseto anoxia (Dispensa et al., 1992). In addition, BadR (an activator), and BadM (a repressor),respond to benzoate or Bz-CoA (Egland & Harwood, 1999, Peres & Harwood, 2006).

To date, a role for posttranslational regulation of the enzymes involved in anaerobicaromatic compound degradation has not been reported. Several groups have shown that theactivity of members of the AMP-forming family of acyl-CoA synthetases in bacteria andeukaryotes is regulated by reversible acylation of the epsilon amino group (Nε) of aconserved lysine residue (Starai et al., 2002, Garrity et al., 2007, Gardner et al., 2006,Hallows et al., 2006, Schwer et al., 2006). Hence we considered the possibility that theactivity of aryl-CoA synthetases BadA and HbaA, and that of the alicyclic acyl-CoAsynthetase AliA, could be controlled by reversible Nε-Lys acylation.

In Salmonella enterica, the protein acyltransferase (SePat) enzyme uses Ac-CoA orpropionyl-CoA (Pr-CoA) to acetylate Ac-CoA synthetase (Acs) or propionylate Pr-CoAsynthetase (PrpE) (Starai & Escalante-Semerena, 2004, Garrity et al., 2007); in both cases,acylation inactivates the enzyme. The lysine residues that are acylated (K609 in Acs, K592in PrpE) are located within the active site and are required for catalysis of the first half-reaction, in which the fatty acid and ATP react to form an acyl-AMP intermediate (Reger etal., 2007; Horswill and Escalante, 2002). Acetylated Acs (AcsAc) and propionylated PrpE(PrpEPr) are reactivated by deacylation, a reaction catalyzed by the NAD+-dependent CobBsirtuin enzyme (Starai et al., 2002, Starai et al., 2003, Garrity et al., 2007, Hoff et al., 2006).

The SePat protein is a member of the yeast Gcn-5 family of acyltransferases (a.k.a. GNATs,pfam 00583) (Vetting et al., 2005a). SePat is an unusual GNAT in that it contains twodistinct domains. The N-terminal domain (~791 aa) is similar to an ADP-forming acyl-CoA

Crosby et al. Page 2

Mol Microbiol. Author manuscript; available in PMC 2011 May 1.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

synthetase, but lacks the catalytic histidine residue critical for the activity of these enzymes.The C-terminal domain of the SePat enzyme (~95 aa) is homologous to the catalytic domainof GNATs (Starai & Escalante-Semerena, 2004, Vetting et al., 2005b).

In the Gram-positive bacterium Bacillus subtilis, Ac-CoA synthetase (BsAcsA) is alsoregulated by acetylation (Gardner et al., 2006). While acetylation of BsAcsA has the sameeffect as in S. enterica, the posttranslational modification system in B. subtilis is somewhatdifferent. In the latter, BsAcsA is acetylated by BsAcuA, another GNAT-like protein that ismuch smaller than SePat, and contains only the yGcn-5 acetyltransferase domain (Gardner etal., 2006, Gardner & Escalante-Semerena, 2008). B. subtilis has two different deacetylasescapable of reactivating BsAcsA. One of these, BsAcuC, uses a Zn(II) ion to hydrolyze theacetyl moiety, generating free acetate (Gardner et al., 2006, Nielsen et al., 2005). The otherdeacetylase, SrtN, is a sirtuin-type deacetylase similar to CobB in S. enterica (Gardner &Escalante-Semerena, 2009, Denu, 2005).

There are many AMP-forming acyl-CoA synthetases found in all domains of life, includingat least 40 in R. palustris, which activate a wide range of fatty and aromatic acids forcatabolic and biosynthetic purposes (Larimer et al., 2004).

In this paper, we report in vivo and in vitro evidence that the enzymatic activity of RpBadA,RpHbaA and RpAliA is negatively affected by acetylation, that a single acetylation eventrenders the enzymes inactive, that the site of acetylation is a catalytically important,conserved Lys residue in this family of enzymes, and that reactivation of the modifiedenzyme is achieved upon deacetylation. Thus, in R. palustris, the degradation of aromaticcompounds is regulated at the posttranslational and transcriptional level. Our findings raisethe possibility that Nε-Lys acetylation is also important in other anaerobic aromaticdegrading microbes.

RESULTSAMP-forming acyl-CoA synthetases involved in anaerobic benzoate catabolism in R.palustris have a conserved lysine residue, which in other proteins is the site of acylation

An alignment of the protein sequences of the R. palustris acyl-CoA synthetases BadA,HbaA and AliA with S. enterica Acs and PrpE, shows that all three R. palustris acyl-CoAsynthetases contain the catalytically important lysine residue that is modified in SeAcs andSePrpE (Fig. 1b). From the crystal structure of the Bz-CoA synthetase of Burkholderiaxenovorans LB400 (61% identical to BadA, Protein Data Base code 2V7B), we inferred thatresidue K512 in BadA (K520 in B. xenovorans Bz-CoA synthetase) lies within the C-terminal domain of the protein (Bains & Boulanger, 2007), and hypothesized it could bemodified by acetylation.

The crystal structure of B. xenovorans Bz-CoA synthetase shows that residue K520 is part ofthe lid for the benzoate-binding pocket, and that this residue forms two hydrogen bonds withthe carboxylate group of benzoate (Fig. 2a) (Bains & Boulanger, 2007). We modeled anacetylated lysine residue at position 520 using the Pymol molecular graphics softwarepackage (http://www.pymol.org), and, as shown in figure 2b, the presence of the acetylmoiety would block the interactions between the ε-amino group of K520 and the carboxylategroup of benzoate (Fig. 2b).

Genes of R. palustris encoding a reversible Nε-Lys acetylation systemIn R. palustris, the genes encoding the BadA, HbaA and AliA acyl-CoA synthetases arelocated within a 24-gene cluster dedicated to aromatic compound transport and degradation(Egland et al., 1997). This gene cluster contains badL, whose product is homologous to

Crosby et al. Page 3

Mol Microbiol. Author manuscript; available in PMC 2011 May 1.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

members of the yGcn-5 acetyltransferase family of enzymes (Vetting, et al., 2005a). Weinvestigated the possibility that BadL could modify BadA, Hba, and AliA. For this purpose,we isolated recombinant BadL protein to homogeneity from an E. coli overexpression strain,and used it in our in vitro protein acetylation assay. BadL did not modify BadA, HbaA orAliA under the conditions of our assay (data not shown).Additional bioinformatics analysisof the R. palustris genome identified a homolog of the S. enterica pat gene (Table 1), but didnot identify a homolog of the acuA gene of B. subtilis. We did find, however, a genehomologous to the S. enterica cobB gene, which encodes a NAD+-dependent deacetylasesirtuin (Tsang & Escalante-Semerena, 1998), and a Zn-dependent lysine deacetylasehomologous to the B. subtilis acuC gene. Hereafter we refer to the putative sirtuin-encodingR. palustris gene as srtN, and the putative Zn-dependent deacetylase as ldaA (for lysinedeacetylase protein A, Table 1). We investigated the possibility that the putative R. palustrisPat, LdaA and SrtN proteins were involved in anaerobic benzoate catabolism in R. palustris.

In vivo evidence that RpPat, RpLdaA, and RpSrtN modulate BadA activityTo determine whether the logic of reversible Nε-Lys acetylation systems found in otherbacteria also applied to R. palustris, we constructed in-frame deletions of ldaA, srtN, and patin this bacterium. We predicted that inactivation of the deacetylase genes would result inirreversible acetylation, thus inactivation, of the acyl-CoA synthetases we were testing.Consequently R. palustris protein deacetylase-deficient strains would not grow on aromaticsubstrates such as benzoate. Although we did not observe significant growth defects onbenzoate when either ldaA or srtN was deleted individually (not shown), there was a growthdefect when both putative deacetylase genes were inactivated (Fig. 3). Growth on benzoatewas restored when either ldaA or srtN was supplied in trans, suggesting that bothdeacetylases affect growth on benzoate in vivo (Fig. 3a). This growth defect was specific togrowth on aromatic and alicyclic compounds, as we saw no defect on other carbon sourcessuch as malonate (Fig. S1). If the observed phenotypes were caused by modification of thesynthetases, and RpPat modified these enzymes, we reasoned that deletion of the putativeRpPat acetyltransferase would restore anaerobic benzoate catabolism in the ldaA srtN patstrain. Growth of the ldaA srtN pat strain was reproducibly better than the ldaA srtN strain(Fig. 3b), although on some occasions the ldaA srtN pat strain exhibited a slight lagcompared to the wild type.

To determine the extent of acetylation of BadA, we partially purified an H10-tagged copy ofBadA from each strain (wild type, ldaA srtN, and ldA srtN pat) grown to full densityphotosynthetically on benzoate. The activity of BadA was measured after incubation withjust NAD+, as a control to determine baseline activity, and after deacetylation with the CobBdeacetylase from S. enterica; as seen in Table 2, SeCobB efficiently deacetylated. BadApurified from the wild type strain was equally active before and after incubation withSeCobB and NAD+, suggesting that the extent of acetylation of BadA was negligible. In theldaA srtN strain, however, the activity of BadA increased 12-fold after incubation withSeCobB and NAD+, indicating that the majority of BadA was acetylated in this strain.Similarly, BadA purified from the ldaA srtN pat strain was 8-fold more active afterincubation with SeCobB and NAD+. Taken together, these results demonstrate that thegrowth defect observed in the ldaA srtN strain on benzoate was most likely due to nearlycomplete acetylation of BadA. Deletion of pat in the ldaA srtN background improvedgrowth on benzoate, but, at least during stationary phase, it appeared that there might beanother acetyltransferase in R. palustris that could acetylate BadA.

In vivo, BadA is acetylated at residue K512 during photoheterotrophic growth on benzoateTo determine the location of the site of modification in BadA, the latter was isolated from anldaA srtN / pRpBADA4 (H10-BadA) strain grown photosynthetically on benzoate. We used

Crosby et al. Page 4

Mol Microbiol. Author manuscript; available in PMC 2011 May 1.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

nickel affinity chromatography to isolate H10-BadA, which was subjected to trypsindigestion, and the resulting peptides were analyzed by tandem mass spectrometry. Individualsequence determination of these peptides using mass spectrometric techniques revealed thatresidue K512 was acetylated (Fig. 4).

Is BadA benzoylated or acetylated in vivo?As mentioned above, the activity of Ac-CoA synthetase is controlled by acetylation (Staraiet al., 2002), while the activity of Pr-CoA synthetase is controlled by propionylation (Garrityet al., 2007). In light of this information, we investigated the possibility that BadA, whichgenerates Bz-CoA, was modified by benzoylation. To increase the likelihood of detectingbenzoylation we increased the intracellular Bz-CoA concentration by inactivation of thebadF gene, which encodes one subunit of Bz-CoA reductase (Egland et al., 1997). For thispurpose, we constructed strain JE12146 [badF ldaA srtN / pRpBADA4 (H10-BadA)], whichwe grew on PM medium containing succinate (10 mM) and benzoate (3 mM). Massspectrometric analysis of H10-BadA isolated from strain JE12146 resulted in a nearlyidentical fragmentation pattern as the one shown in Fig. 4, indicating that, in vivo, BadA wasacetylated, not benzoylated.

In vitro, Rp Pat acetylates and inactivates acyl-CoA synthetases BadA, HbaA, and AliAWe confirmed that RpPat directly acetylated acyl-CoA synthetases by incubating purifiedPat with [14C-1]-Ac-CoA and either BadA, HbaA, AliA or Acs from R. palustris (Fig. 5).Based on data obtained in other systems, we expected RpAcs to serve as positive control forRpPat activity. In each case, the labeled acetyl group was transferred to the CoA synthetaseonly when RpPat was present in the reaction mixture. RpPat did not acetylate a variant ofBadA with a K512A substitution (BadAK512A) (Fig. 5), indicating that BadA was modifiedonce, and that residue K512 was the site of acetylation. Similarly, variants HbaAK503A andAcsK606A were not acetylated by RpPat, which suggested that these lysine residues were theonly sites of acetylation in HbaA and Acs, respectively (Fig. 5).

Unlike other acyl-CoA synthetases we have investigated, AliA contains a second lysineresidue at position K535 that is three residues away from the presumed acetylation site(K532) (Fig. 1b). We tested acetylation of AliAK532A and AliAK535A variants, and foundthat the AliAK532A protein was not acetylated, demonstrating that residue K532 was thesingle site of acetylation (Fig. 5). These acetylated lysine residues are homologous to thepreviously reported sites of acylation in S. enterica Acs and PrpE proteins (indicated with anarrow in Fig. 1b) (Starai et al., 2002,Garrity et al., 2007).

Acetylation dramatically affected the activity of all acyl-CoA synthetases tested. Each acyl-CoA synthetase was pre-incubated with RpPat and either CoA, as a negative control, or Ac-CoA, and the activity of the acyl-CoA synthetase was measured spectrophotometrically.Upon acetylation, the activity of BadAAc decreased to <1% of that of BadAWT, and wascomparable to that of the BadAK512A variant (Table 3). The activities of HbaA, AliA, andAcs all decreased below the limit of detection after incubation with RpPat and Ac-CoA. K-to-A substitutions at the acetylation sites of HbaA, AliA and Acs resulted in a decrease ofactivity to <3% of wild-type activity (Table 3), demonstrating that these lysine residues werecrucial for activity.

LdaA deacetylates and reactivates acyl-CoA synthetases BadA, HbaA, and AliAAttempts to purify LdaA were met with limited success due to poor solubility of the protein.However, enough protein was present in cell-free extracts enriched for LdaA to demonstrateprotein deacetylase activity. This information was obtained after the incubation of cell-freeextracts of strain JE11959 (E. coli MG1655 ΔcobB / pRpLDAA1 ldaA+) with [14C]-acetyl-

Crosby et al. Page 5

Mol Microbiol. Author manuscript; available in PMC 2011 May 1.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

BadA. Within five minutes of incubation, the levels of acetylation of BadA were below thelimit of detection (>90% deacetylation), compared with no substantial deacetylation usingcell-free extracts from a strain containing the empty cloning vector (JE11958) (Fig. 6a). Wealso asked if deacetylation of acyl-CoA synthetases would restore their activity. Weincubated BadA, HbaA and AliA with RpPat and unlabeled acetyl-CoA. After incubation,the reaction mixtures were incubated with cell-free extracts from strain JE11959 (enrichedfor LdaA) or JE11958 (devoid of LdaA). The activity of BadA and AliA was restored tolevels observed before acetylation, and HbaA activity was restored to 65% of itsunacetylated activity under the conditions of our experiment. These results demonstratedthat deacetylation by LdaA reactivated BadA, HbaA and AliA.

Purified RpSrtN did not deacetylate any of the acetylated acyl-CoA synthetases, even withthe addition of the S. enterica nicotinamidase enzyme PncA, which has been used todiminish inhibition by the byproduct nicotinamide (Garrity et al., 2007). However, the S.enterica CobB sirtuin deacetylated [14C]-acetyl-BadA in vitro and restored enzyme activity.We also tested whether E. coli cell lysates enriched in RpSrtN would deacetylate BadA; wedid not observe any deacetylation. At present, it is unclear why RpSrtN was inactive in vitro.

DISCUSSIONAnaerobic benzoate catabolism in R. palustris is under the control of reversible Nε-Lysacetylation

We have reported here the first evidence of reversible Nε-Lys acetylation control of acyl-CoA synthetases in an α-proteobacterium, and, to our knowledge, the first posttranslationalregulation of aryl-CoA synthetases. We identified one protein acetyltransferase enzyme in R.palustris (RpPat) that effectively modifies three AMP-forming acyl-CoA synthetasesinvolved in aromatic and alicylic acid activation. The fact that a single modification of aconserved Lys residue close to the C terminus of the protein is sufficient to render all three>95% inactive, reflects the importance of the Lys residue on the function of BadA, HbaA,and AliA.

Two cognate protein deacetylases (i.e. RpLdaA, RpSrtN) appear to work in concert withRpPat to modulate the activity of BadA, HbaA, and AliA. It is unclear, how RpLdaA andRpSrtN specifically contribute to aromatic acid degradation in this bacterium. However, theabsence of both deacetylases has a clear impact on growth on benzoate, and, as shown intable 2, when BadA is purified from a ldaA srtN strain, it is mostly in the acetylated state.

The lack of activity of RpSrtN was unexpected, since sirtuins from other sources (e.g.SeCobB) are active under the same conditions we tested. An explanation for this observationrequires further investigation.

Are other protein acetyltransferases involved?Our data clearly show that RpPat can efficiently inactivate BadA, HbaA and AliA (andacetyl-CoA synthetase) in vitro, and that deletion of pat in an ldaA srtN strain restoresgrowth on benzoate to near wild type levels. However, BadA is still partially acetylated in apat strain, strongly suggesting that one or more yet-to-be-identified acetyltransferases withPat-like activity exist in R. palustris.

A model for the posttranslational control of acyl-CoA synthetases in R. palustrisThe scheme in figure 7 summarizes what we know about the control of Bz-CoA synthetase(BadA) activity by Nε-Lys acetylation system of this bacterium. Based on our data, weextrapolate that Hba and AliA activities are controlled in a similar manner. This work has

Crosby et al. Page 6

Mol Microbiol. Author manuscript; available in PMC 2011 May 1.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

unveiled what is likely to be a complex system for metabolic control in R. palustris. Wesuspect there are many other proteins under reversible Nε-Lys acetylation control in thisbacterium.

Physiological role of acyl-CoA synthetase regulationBadA, HbaA, and AliA are members of the AMP-forming acyl-CoA synthetase family ofenzymes, and each of these enzymes catalyzes the first step in benzoate, 4-HBA, and CHCdegradation, respectively. Each enzyme consumes ATP and CoA to convert the substrate tothe corresponding CoA thioester. The next step in benzoate degradation is catalyzed by Bz-CoA reductase, which, in the denitrifying bacterium Thauera aromatica, uses 2 ATP todrive the transfer of electrons from ferredoxin to Bz-CoA (Boll et al., 1997, Boll & Fuchs,1995). Since the first two steps of aromatic acid degradation are energetically expensive, wehypothesize that Nε-Lys acetylation is a mechanism that prevents uncontrolled Ac-CoAsynthesis, which, if it happened, would consume large amounts of ATP and free CoA, andmight lead to growth arrest. The fact that srtN+ provided in trans restored growth of an ldaAsrtN strain on aromatic compounds, suggests a link between the reactivation of acetylatedacyl-CoA synthetases and a physiological state where NAD+ is high. Such a condition maysignal a need to catabolize benzoate to generate carbon and reducing power for growth.Thus, control of protein function by acetylation may be an important system for maintainingCoA and redox homeostasis with a direct link to the carbon and energy status of the cell.

Implications of the acetylation of acyl-CoA synthetasesIt has been suggested that BadR, a transcriptional activator of the badDEFGAB operon, maysense Bz-CoA, rather than benzoate (Egland & Harwood, 1999). Indeed, in the denitrifyingbacterium Azoarcus sp. strain CIB, the genes encoding enzymes involved in the anaerobicdegradation of benzoate are organized in a single operon that is regulated by the BzdRrepressor, which responds to Bz-CoA (Barragan et al., 2005). Our data showing that Bz-CoA synthetase (BadA) activity is regulated by acetylation, suggests that thisposttranslational modification is important for modulating the concentration of Bz-CoA incells. This may, in turn, affect transcriptional regulation, providing additional inputs into theregulatory network.

Extent of CoA synthetase regulation by reversible Nε-Lys acylationPrevious work has shown that AMP-forming acyl-CoA synthetases that activate short chainfatty acids, such as acetate and propionate, are regulated by reversible Nε-Lys acetylationand propionylation, respectively, in bacteria and eukaryotes (Starai et al., 2002, Garrity etal., 2007, Gardner et al., 2006, Schwer et al., 2006, Hallows et al., 2006). The AMP-formingacyl-CoA synthetases are a large and diverse group of enzymes that can activate diversesubstrates, including short-, medium-, and long-chain fatty acids, dicarboxylic acids,aromatic acids, and substituted aromatic acids such as 4-chlorobenzoate. R. palustris, whichhas unusually broad metabolic capabilities, has at least 40 genes annotated to encodeputative AMP-forming acyl-CoA synthetases (Larimer et al., 2004). Many of these enzymescontain the conserved Lys residue that is acetylated in Acs (Lys606 in RpAcs), which is notsurprising since it appears to play a role in substrate binding (Reger et al., 2007, Conti et al.,1997, May et al., 2002, Nakatsu et al., 2006). It will be interesting to determine the extent ofregulation of acyl-CoA synthetases by Nε-Lys acetylation.

In summary, the results presented here demonstrate that, in the photosynthetic α-proteobacterium Rhodopseudomonas palustris, the activities of acetyl-CoA synthetase, twoaryl-CoA synthetases, and one alicyclic-CoA synthetase are regulated at theposttranslational level by reversible acetylation of the epsilon amino group of a conservedLys residue. We have identified three enzymes (one acetyltransferase, and two deacetylases)

Crosby et al. Page 7

Mol Microbiol. Author manuscript; available in PMC 2011 May 1.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

that orchestrate the control of the above-mentioned synthetases, and we hypothesize thatthere is at least one more acetyltransferase involved. The modifying/demodifying enzymesare likely a regulatory system that is finely tuned to CoA (carbon status) and redox (energystatus) homeostasis in this bacterium. In view of these results and the widespreadparticipation of AMP-forming acyl-CoA synthetases in aromatic acid degradation it seemspossible that posttranslational modification by Nε-Lys acetylation constitutes an commonand previously overlooked mechanism for regulating these pathways.

EXPERIMENTAL PROCEDURESBacterial strains, growth conditions, chemicals

All strains used in this study are listed in Table S1, and a list of plasmids can be found inTable S2. Escherichia coli strains were grown at 37°C in lysogeny broth (LB, Difco)(Bertani, 1951,Bertani, 2004). All R. palustris strains are derivatives of R. palustrisCGA009 (Larimer et al., 2004) and were cultured at 30 °C in YP rich medium (20 g yeastextract L−1, 20 g peptone L−1), or defined basal photosynthetic medium [PM, (Kim &Harwood, 1991)] supplemented with NaHCO3 (10 mM) and either succinate (10 mM),malonate (10 mM), benzoate (3 mM), 4-hydroxybenzoate (3 mM) orcyclohexanecarboxylate (3 mM). For growth curves, three- or four-days old, aerobicallygrown cultures of R. palustris were grown in YP medium supplemented with kanamycinwhere appropriate. Cultures were diluted (1:10) in triplicate into 5 ml of anoxic PMsupplemented with the appropriate carbon source and kanamycin; all manipulations wereperformed in Balch tubes (Balch & Wolfe, 1976). Tubes were incubated at 30 °C in lightwithout shaking. Cell density was monitored at 660 nm using a Manostat Spec20D; eachgrowth curve was repeated at least three times. When used, ampicillin was at 100 µg ml−1

unless otherwise indicated, chloramphenicol was at 20 µg ml−1, gentamicin was at 100 µgml−1, and kanamycin was at 50 µg ml−1 (E. coli) or 75 µg ml−1 (R. palustris). Radiolabeled[14C-1]-Ac-CoA (54 mCi/mmol) was purchased from Moravek, and all other chemicalswere obtained from Sigma.

Molecular techniquesDNA manipulations were performed using standard techniques (Ausubel, 1989). Restrictionendonucleases were purchased from Fermentas. DNA was amplified using PfuUltra IIFusion DNA polymerase (Stratagene), and site-directed mutagenesis was performed usingthe QuikChange kit from Stratagene unless otherwise noted. Plasmid DNA was purifiedusing the Wizard Plus SV Miniprep kit (Promega) and PCR products were purified using theQiaQuick PCR Purification kit (Qiagen). DNA sequencing was performed using BigDye(ABI PRISM) protocols, and sequencing reactions were resolved and analyzed at theUniversity of Wisconsin-Madison Biotechnology Center. Oligonucleotide primer sequencesare listed in Table S3 (Supplementary Material).

Construction of gene deletions in R. palustrisIn-frame deletions of R. palustris pat and badF were generated using described protocols(Schafer et al., 1994). A DNA fragment was amplified using overlap extension PCR (Hortonet al., 1993) that contained 1 kb of upstream DNA fused in-frame to 1 kb of downstreamDNA. The PCR product was cut with EcoRI and HindIII enzymes, ligated into plasmidpK18mobsacB (Schafer et al., 1994), and transformed into E. coli strain DH5α. Theresulting plasmid was purified and electroporated into R. palustris to generate a strain inwhich the plasmid had integrated into the chromosome. A kanamycin resistant colony wasused to inoculate 5 ml of YP + kanamycin medium, grown aerobically at 30 °C, andtransferred to 50 ml of anaerobic PM + succinate (10 mM) + sucrose (10%, w/v), withoutantibiotic, to select for loss of the integrated plasmid. This culture was diluted and plated on

Crosby et al. Page 8

Mol Microbiol. Author manuscript; available in PMC 2011 May 1.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

PM + succinate (10 mM). Kanamycin sensitive colonies were screened by PCR for deletionof the gene; the presence of the deletion was established by DNA sequencing.

Deletions of srtN and ldaA were constructed similarly, except that plasmid pJQ200SK,which encodes gentamicin resistance (Quandt & Hynes, 1993), was used instead of plasmidpK18mobsacB. To delete the R. palustris srtN gene, a fusion of 1 kb of upstream DNA and1 kb of downstream DNA was ligated into plasmid pJQ200SK using restriction enzymesSacI and BamHI; SacI and NotI were used for the ldaA deletion plasmid. The resultingplasmids were electroporated into R. palustris, and gentamicin resistant colonies werestreaked on PM + succinate (10 mM) plates containing sucrose (10%, w/v). Gentamicin-sensitive colonies arising on these plates were screened by PCR for the deletion, which wasconfirmed by DNA sequencing.

Plasmids used for protein overproductionThe badA, hbaA and aliA genes were amplified from R. palustris genomic DNA using theprimers listed in Table S2 (Supplementary Material), and were cloned into the NdeI andBamHI sites of plasmid pET-16b. Each protein was fused to an N-terminal H10 tag. ThebadA gene was also cloned into the NdeI and EcoRI sites of plasmid pTEV5 (Rocco et al.,2008), which directed the synthesis of BadA with an N-terminal H6 tag, cleavable by TEVprotease. The acs gene (formerly rpa0211) was cloned into the NheI and EcoRI sites ofpTEV5. Plasmids directing the synthesis of variants BadAK512A and AliAK532A weregenerated from the pET-16b vectors using site-directed mutagenesis. Alleles encoding theremaining variants (AcsK606A, HbaAK503A, and AliAK535A) were constructed byamplification using overlap extension PCR with the mutagenesis and cloning primers, andthe resulting products were ligated into plasmids pTEV5 (AcsK606A) or pET-16b(HbaAK503A and AliAK535A). The presence of mutations was confirmed by DNAsequencing. R. palustris pat was amplified from genomic DNA, and was cloned into theKpnI and HindIII sites of plasmid pKLD66 (Rocco et al., 2008). The fifth codon of pat is arelatively rare arginine codon in E. coli (CGA), thus it was changed to the more commoncodon CGT using site-directed mutagenesis. The resulting plasmid, pRpPAT2, expressedRpPat with sequential H6 and MBP tags at the N-terminus, both of which were cleavable byTEV protease. The srtN gene was amplified from R. palustris genomic DNA and cloned intothe NdeI and XhoI sites of pET-24b, which expressed RpSrtN with a C-terminal H6 tag.

Plasmids for expression in R. palustrisThe R. palustris ldaA+ gene was amplified and cloned into the HindIII and EcoRI sites ofplasmid pBBR1MCS-2 (Kovach et al., 1995) to generate plasmid pRpLDAA4. The R.palustris srtN gene was amplified with an optimized ribosome-binding site for E. coli, andwas cloned into the KpnI and HindIII sites of plasmid pBBR1MCS-2 to generate plasmidpRpSRTN4. The R. palustris badA+ gene was amplified from plasmid pRpBADA1, andcloned into the HindIII and EcoRI sites of plasmid pBBR1MCS-2 to generate plasmidpRpBADA4; the latter directed the synthesis of H10-BadA.

Plasmid for expression in E. coliThe R. palustris ldaA+ gene was amplified with an optimized ribosome-binding site for E.coli, and was cloned into the XbaI and HindIII sites of plasmid pBAD30 (Guzman et al.,1995) to generate plasmid pRpLDAA1, which expressed ldaA+ under the control of anarabinose-inducible promoter. The R. palustris srtN+ gene was cloned in a similar manner,except that the gene was cloned into the KpnI and HindIII sites of pBAD30.

Crosby et al. Page 9

Mol Microbiol. Author manuscript; available in PMC 2011 May 1.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Protein purificationpET-16b vectors containing badA+, hbaA+, or aliA+ were transformed into a Δpat derivativeof strain of E. coli C41λ(DE3) (Miroux & Walker, 1996)to prevent acetylation prior topurification. The resulting strains were grown until early stationary phase and sub-cultured(1:100) into two liters of LB supplemented with ampicillin (100 µg ml−1). Cultures weregrown with shaking to an OD600 ~0.6, and protein expression was induced with isopropyl-1-thio-β-D-galactopyranoside (IPTG, 0.5 mM). Cultures were grown overnight at 16 °C, cellswere harvested by centrifugation at 10,500 × g for 12 min in a Beckman Coulter Avanti J-20XOI refrigerated centrifuge with a JLA-8.1000 rotor. Cell pellets were re-suspended in 30ml of buffer ABadA [sodium phosphate buffer (50 mM, pH 8.0), NaCl (300 mM), imidazole(5 mM)] containing 1 mg ml−1 lysozyme and 1 mg ml−1 DNaseI. Cells were disrupted usinga French pressure cell (Spectronic instruments) at 1.26 kPa (2–3 times) and debris wasremoved by centrifugation at 39,000 × g for 30 min. Proteins were purified by Ni-affinitypurification using an ÄKTA FPLC Purifier system (Amersham Biosciences) equipped witha 1-ml HisTrap HP column. The column was equilibrated with buffer A before loading thefiltered cell-free extract, and then washed with 10 ml of Buffer ABadA, followed by 15 ml of4% buffer BBadA [sodium phosphate buffer (50 mM, pH 8.0), NaCl (300 mM), imidazole(250 mM)]. Proteins were desorbed from the resin using a 15-ml linear gradient to 100% B.Each protein was dialyzed ≥3 h against buffer 1 [Tris-Cl (50 mM, pH 7.5, at 4 °C), NaCl(100 mM), EDTA (1 mM), glycerol (20%, v/v)] and then buffer 2 [Tris-Cl (50 mM, pH 7.5,at 4 °C), NaCl (100 mM), glycerol (20%, v/v)], before drop freezing into liquid N2. Proteinconcentrations were determined by measuring the absorbance at 280 nm. The molarextinction coefficients used to calculate protein concentrations were 134,420 M-1 cm-1 forRpAcs, 59,600 M-1 cm-1 for AliA, 65,430 M-1 cm−1 for BadA, 69,840 M−1 cm−1 for HbaA,and 68,560 for RpPat.

R. palustris Ac-CoA synthetase (RpAcs) and untagged BadApTEV5 vectors expressing RpH6-Acs and H6-BadA were transformed into a Δpat strain ofE. coli C41λ(DE3) to prevent acetylation prior to purification. Conditions used for theoverproduction and purification of RpH6-Acs and H6-BadA were similar to those describedabove. The H6 tag was removed using H6-TEV protease (Blommel & Fox, 2007) asdescribed (Garrity et al., 2007), and RpAcs and BadA were purified away from the tag andH6-TEV protease by passage over a 1 ml HisTrap HP column.

R. palustris protein acety transferase (RpPat)Plasmid pRpPAT2 was transformed into E. coli BL21λ(DE3) / pLysS and expressed in fourliters of LB + ampicillin (200 µg ml−1) + chloramphenicol (20 µg ml−1). After inductionwith IPTG (0.5 mM), the temperature was reduced to 28 °C and the cultures were allowed togrow overnight with shaking. RpH6-MBP-Pat was purified as described above, with minoradjustments, and the His6-MBP tag was removed with H6-TEV protease. For the firstpurification, buffer APat contained sodium phosphate (50 mM, pH 7.5), NaCl (500 mM),imidazole (20 mM) and tris(2-carboxyethyl)phosphine (TCEP, 0.5 mM), and buffer BPatwas the same as buffer APat except that the imidazole concentration was increased to 500mM. For the second purification step (removal of the tag and H6-TEV protease) the samebuffers were used, except imidazole was omitted from buffer APat. Purified Pat was dialyzedthree times against successively lower NaCl concentrations, into storage bufferPat [4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES, 50 mM, pH 7.5), NaCl (150 mM),glycerol (20%, v/v)] and drop frozen into liquid N2.

Crosby et al. Page 10

Mol Microbiol. Author manuscript; available in PMC 2011 May 1.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

R. palustris sirtuin deacetylase ( Rp SrtN)Plasmid pRpSRTN6 was transformed into E. coli C41λ(DE3) and expressed in 4 L of LB +kanamycin, and ZnSO4 (50 µM). After induction with IPTG (0.5 mM), the temperature wasreduced to 28 °C and the cultures were allowed to grow overnight with shaking. RpSrtN waspurified using Ni-affinity chromotography as described above for BadA, HbaA and Alia,except that 0.5 mM TCEP was added to the purification buffers, and 5 mM DTT wasincluded in the storage buffers.

S. enterica CobB sirtuin deacetylase and PncA nicotinamidasePlasmid pCOBB33, expressing S. enterica CobB fused to an N-terminal chitin bindingdomain tag, was transformed into E. coli C41λ(DE3) and expressed in 4 L of LB +ampicillin, and ZnSO4 (50 µM). S. enterica CobB was purified as described (Garrity et al.,2007). Expression and purification of S. enterica PncA nicotinamidase were as described(Garrity et al., 2007).

In vitro protein acetylation assayProtein acetylation was observed using radiolabeled Ac-CoA as described (Starai &Escalante-Semerena, 2004). Reactions contained HEPES buffer (50 mM, pH 7.0), TCEP (1mM), KCl (20 mM), [1-14C]Ac-CoA (19 µM), acyl-CoA synthetase (3 µM), and RpPat(0.06 µM). Reactions (25 µl total volume) were incubated for 60 min at 30°C. Samples (5 µleach) were resolved using SDS-PAGE (Laemmli, 1970), and proteins were visualized byCoomassie Blue staining (Sasse, 1991). Gels were dried and exposed overnight to aMultiPurpose Phosphor Screen (Packard). Radioactivity was detected using a CycloneStorage Phosphor System (Packard) equipped with OptiQuant v 04.00 software (Packard).

In vitro CoA synthetase activity assayCoA synthetases (3 µM each) were individually incubated with Pat (1 µM) and 50 µM Ac-CoA or CoASH (non-acetylated control) for 2h at 30 °C using the same buffer systemdescribed above for 14C acetylation assays. Acetylation reactions were stopped by bufferexchange into HEPES (50 mM, pH 7.5) containing KCl (20 mM). Acyl-CoA synthetaseactivity was measured using an NADH-consuming assay (Reger et al., 2007). Reactionscontained HEPES (50 mM, pH 7.5), TCEP (1 mM), ATP (2.5 mM), CoASH (1 mM),MgCl2 (5 mM), phosphoenolpyruvate (3 mM), NADH (0.1 mM), pyruvate kinase (1 U),myokinase (5 U), lactate dehydrogenase (1.5 U), and either benzoate, 4-hydroxybenzoate,cyclohexanecarboxylate, or acetate (0.2 mM). Reactions were started by the addition ofacyl-CoA synthetase (30 nM), and the absorbance at 340 nm was monitored for 10 minusing a PerkinElmer Lambda 40 UV-visible spectrophotometer. Enzyme activities werecalculated as described (Garrity et al., 2007).

Protein deacetylation assaysThe R. palustris ldaA+ gene was expressed in strain JE11691, a CobB (sirtuin) deacetylase-deficient strain of E. coli K-12 MG1655. The R. palustris srtN+ strain was expressedsimilarly, using strain JE11960. Cultures (100 ml each) were grown overnight in LB +ampicillin (100 µg ml−1) + arabinose (1 mM). Cells were harvested by centrifugation, andre-suspended in 1 ml of deacetylase buffer containing HEPES (50 mM, pH 7.5), NaCl (300mM), dithiothreitol (1 mM), and ZnCl2 (25 µM). Cells were lysed with BugBuster® reagent(Novagen) supplemented with protease inhibitor cocktail (Sigma). Cell debris was removedby centrifugation, and the supernatant was dialyzed for 1 h against deacetylase buffer in amicrodialyzer (Pierce). AliA and untagged BadA proteins were acetylated with 20 µM[1-14C]-Ac-CoA or unlabeled Ac-CoA and 0.06 µM RpPat as described above, and theacetylation reaction was stopped by buffer exchange into HEPES (50 mM, pH 7.5), TCEP (1

Crosby et al. Page 11

Mol Microbiol. Author manuscript; available in PMC 2011 May 1.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

mM), and KCl (20 mM) using Microcon YM-30 centrifugal filter units. HbaA wasacetylated similarly, except that KCl was omitted from the reaction, and 50 µM acetyl-CoAand 0.5 µM Pat were used to achieve greater extents of acetylation. A sample of dialyzed,clarified cell-free extracts containing 80 µg of protein was added to the reaction mixture (50µl final volume) containing acetylated BadA, and 10-µl samples were removed over timeand quenched with SDS-PAGE gel-loading buffer. BadA protein was visualized using SDS-PAGE, and radioactivity was quantified using phosphorimaging as described above. Todetermine CoA synthetase activity, 100 µl of the acetylation reaction was incubated withcell lysates (250 µg of protein) from strains expressing ldaA or the empty vector. Thedeacetylation reactions were incubated for 30 min at 30 °C, and the rate of AMP generationwas measured using the coupled spectrophotometric assay described above.

We tested whether RpSrtN or SeCobB could deacetylate BadAAc. For this purpose weincubated [14C-Ac]-BadA (6 µM) with RpSrtN or SeCobB (6 µM) and NAD+ (1 mM) inHEPES buffer (50 mM, pH 7.5) containing TCEP (1 mM). Proteins were resolved usingSDS-PAGE and radioactivity was quantified by phosphorimaging. Addition of PncAnicotinamidase (~4 µM) to the reaction mixture did not enhance deacetylation.

Purification of BadA from R. palustrisFive hundred-ml cultures of strains JE13049 (CGA009 / pRpBADA4), JE11639 (CGA009ldaA srtN / pRpBADA4), and JE13050 (CGA009 ldaA srtN pat / pRpBADA4) were grownuntil they reached stationary phase (four to seven days) anaerobically in the light on PM +benzoate (3 mM). Similarly, an 800-ml culture of strain JE12146 (CGA009 ldaA srtNbadF / pRpBADA4) was grown on PM + succinate (10 mM) + benzoate (3 mM) for eightdays in the light. Cells were harvested by centrifugation, and re-suspended in 10 ml of bufferABadA plus 1 mg ml−1 of lysozyme. Cells were lysed by sonication and cell debris wasremoved by centrifugation. H10-BadA protein was purified using a His-Bind Quick 900cartridge (Novagen) with the same buffer system for BadA as described above. Forsubsequent deacetylation reactions, purified BadA was dialyzed overnight into HEPES (50mM, pH 7.5) buffer containing NaCl (150 mM).

The synthetase activity of BadA was then assessed. To do this, BadA (1 µM) purified fromstrains JE11639, JE13049, and JE13050 (see table 2) was deacetylated with SeCobB (or nodeacetylase control to estimate the in vivo activity of BadA) as described in the abovesection for 2 h at 37 °C. A sample of the deacetylation reaction mixtures was used to assessBadA activity. The concentration of BadA in the synthetase activity assays was 40 nM.

Identification of acetylation sites by mass spectrometryTo determine the site of acetylation, purified BadA protein was incubated with RpPat and 50µM Ac-CoA as described above, and compared to a non-acetylated control. To determinethe identity of the in vivo modification of BadA, H10-BadA purified from R. palustris wasresolved by SDS-PAGE and the band corresponding to BadA was excised. In-gel trypsindigestion and mass spectrometric analysis of the peptides was performed at the University ofWisconsin-Madison Mass Spectrometry Facility. Tryptic digestion and peptide recovery wasperformed as outlined on website:http://www.biotech.wisc.edu/ServicesResearch/MassSpec/ingel.htm.

Peptides were analyzed by nanoLC-MS/MS using the Agilent 1100 nanoflow systemconnected to a hybrid linear ion trap-orbitrap mass spectrometer LTQ-Orbitrap (ThermoFisher Scientific) equipped with a nanoelectrospray ion source. Capillary HPLC wasperformed using an in-house fabricated column with integrated electrospray emitter (11)except that 360 µm × 75 µm fused silica tubing was used. The column was packed with 5

Crosby et al. Page 12

Mol Microbiol. Author manuscript; available in PMC 2011 May 1.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

µm C18 particles (Column Engineering) to ~12 cm. Sample loading (8 µl) and desaltingwere achieved using a trapping column (Zorbax 300SB-C18, 5 µM, 5 × 0.3 mm, Agilent) inline with the autosampler. HPLC solvents were as follows: Loading: acetonitrile (1%, v/v),acetic acid (0.1 M); A: acetic acid in water (0.1 M), and B: acetonitrile (95%, v/v), aceticacid in water (0.1 M). Sample loading and desalting were done at 10 µl/min with the loadingsolvent delivered from an isocratic pump. Gradient elution was performed at 200 nL/minand increasing %B in A of 0 to 40 in 100 min, 40 to 60 in 15 min, and 60 to 100 in 5 min.The LTQ-Orbitrap was set to acquire MS/MS spectra in data-dependent mode as follows:MS survey scans from charge-to-mass ratio (m/z) from 300 to 2000 were collected in profilemode at a resolving power of 100,000. MS/MS spectra were collected on the three most-abundant signals in each survey scan. Dynamic exclusion was employed to increasedynamic range and maximize peptide identifications. This feature excluded precursors up to0.55 m/z below and 1.05 m/z above previously selected precursors. Precursors remained onthe exclusion list for 15 s. Singly charged ions and ions for which the charge state could notbe assigned were rejected from consideration for MS/MS. Raw MS/MS data were convertedto mgf file format using Trans Proteomic Pipeline (Seattle Proteome Center, Seattle, WA).Resulting mgf files were searched against NCBI non-redundant proteobacteria amino acidsequence database using in-house Mascot search engine (Matrix Science, London, UK) withmethionine oxidation and lysine acetylation as variable modifications.

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

AcknowledgmentsThis work was supported by PHS grant R01-GM62203 from the National Institute of General Medical Sciences (toJ.C.E.-S.), and DOE grant DE-FG02-08ER15707 to C.S.H. H.A.C. was supported in part by PHS BiotechnologyTraining Grant T32 GM08349, and an NSF graduate research fellowship. We are grateful to Grzegorz Sabat at theUniversity of Wisconsin Biotechnology Center for assistance with mass spectrometry, and Ana Misic for help withmodeling in Pymol.

Abbreviations

4-HBA 4-hydroxybenzoic acid

CHC cyclohexanecarboxylate

HEPES 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid

TCEP tris(2-carboxyethyl)phosphine

IPTG isopropyl-1-thio-β-D-galactopyranoside

CoASH coenzyme A

Bz,CoA benzoyl-CoA

Ac-CoA acetyl-CoA

PPi pyrophosphate

O-AADPR O-acetyl-ADP-ribose

Nm nicotinamide

Crosby et al. Page 13

Mol Microbiol. Author manuscript; available in PMC 2011 May 1.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

REFERENCESBains J, Boulanger MJ. Biochemical and structural characterization of the paralogous benzoate CoA

ligases from Burkholderia xenovorans LB400: defining the entry point into the novel benzoateoxidation (box) pathway. J. Mol. Biol. 2007; 373:965–977. [PubMed: 17884091]

Balch WE, Wolfe RS. New approach to the cultivation of methanogenic bacteria: 2-mercaptoethanesulfonic acid (HS-CoM)-dependent growth of Methanobacterium ruminantium in apressurized atmosphere. Appl. Environ. Microbiol. 1976; 32:781–791. [PubMed: 827241]

Barragan MJ, Blazquez B, Zamarro MT, Mancheno JM, Garcia JL, Diaz E, Carmona M. BzdR, arepressor that controls the anaerobic catabolism of benzoate in Azoarcus sp. CIB, is the firstmember of a new subfamily of transcriptional regulators. J. Biol. Chem. 2005; 280:10683–10694.[PubMed: 15634675]

Bertani G. Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J.Bacteriol. 1951; 62:293–300. [PubMed: 14888646]

Bertani G. Lysogeny at mid-twentieth century: P1, P2, and other experimental systems. J. Bacteriol.2004; 186:595–600. [PubMed: 14729683]

Blommel PG, Fox BG. A combined approach to improving large-scale production of tobacco etchvirus protease. Protein Expr. Purif. 2007; 55:53–68. [PubMed: 17543538]

Boll M, Albracht SS, Fuchs G. Benzoyl-CoA reductase (dearomatizing), a key enzyme of anaerobicaromatic metabolism. A study of adenosinetriphosphatase activity, ATP stoichiometry of thereaction and EPR properties of the enzyme. Eur. J. Biochem. 1997; 244:840–851. [PubMed:9108255]

Boll M, Fuchs G. Benzoyl-coenzyme A reductase (dearomatizing), a key enzyme of anaerobicaromatic metabolism. ATP dependence of the reaction, purification and some properties of theenzyme from Thauera aromatica strain K172. Eur. J. Biochem. 1995; 234:921–933. [PubMed:8575453]

Carmona M, Zamarro MT, Blazquez B, Durante-Rodriguez G, Juarez JF, Valderrama JA, BarraganMJ, Garcia JL, Diaz E. Anaerobic catabolism of aromatic compounds: a genetic and genomic view.Microbiol. Mol. Biol. Rev. 2009; 73:71–133. [PubMed: 19258534]

Conti E, Stachelhaus T, Marahiel MA, Brick P. Structural basis for the activation of phenylalanine inthe non-ribosomal biosynthesis of gramicidin S. Embo J. 1997; 16:4174–4183. [PubMed:9250661]

Dagley, S. New perspectives in aromatic catabolism. In: Leisinger, T.; Cook, AM.; Hutter, R.; Nuesch,J., editors. Microbial degradation of xenobiotics and recalcitrant compounds. New York, N.Y:Academic Press; 1981. p. 181-186.

Denu JM. Vitamin B(3) and sirtuin function. Trends Biochem. Sci. 2005; 30:479–483. [PubMed:16039130]

Diaz E. Bacterial degradation of aromatic pollutants: a paradigm of metabolic versatility. Int.Microbiol. 2004; 7:173–180. [PubMed: 15492931]

Dispensa M, Thomas CT, Kim MK, Perrotta JA, Gibson J, Harwood CS. Anaerobic growth ofRhodopseudomonas palustris on 4-hydroxybenzoate is dependent on AadR, a member of thecyclic AMP receptor protein family of transcriptional regulators. J. Bacteriol. 1992; 174:5803–5813. [PubMed: 1522059]

Egland PG, Gibson J, Harwood CS. Benzoate-coenzyme A ligase, encoded by badA, is one of 3ligases able to catalyze benzoyl-coenzyme A formation during anaerobic growth ofRhodopseudomonas palustris on benzoate. J. Bacteriol. 1995; 177:6545–6551. [PubMed:7592432]

Egland PG, Harwood CS. BadR, a new MarR family member, regulates anaerobic benzoatedegradation by Rhodopseudomonas palustris in concert with AadR, an Fnr family member. J.Bacteriol. 1999; 181:2102–2109. [PubMed: 10094687]

Egland PG, Pelletier DA, Dispensa M, Gibson J, Harwood CS. A cluster of bacterial genes foranaerobic benzene ring biodegradation. Proc. Natl. Acad. Sci. U S A. 1997; 94:6484–6489.[PubMed: 9177244]

Crosby et al. Page 14

Mol Microbiol. Author manuscript; available in PMC 2011 May 1.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Fuchs G. Anaerobic metabolism of aromatic compounds. Ann. N. Y. Acad. Sci. 2008; 1125:82–99.[PubMed: 18378589]

Gardner JG, Escalante-Semerena JC. Biochemical and mutational analyses of AcuA, theacetyltransferase enzyme that controls the activity of the acetyl coenzyme a synthetase (AcsA) inBacillus subtilis. J. Bacteriol. 2008; 190:5132–5136. [PubMed: 18487328]

Gardner JG, Escalante-Semerena JC. In Bacillus subtilis, the sirtuin protein deacetylase encoded by thesrtN gene (formerly yhdZ), and functions encoded by the acuABC genes control the activity ofacetyl-CoA synthetase. J. Bacteriol. 2009; 191:1749–1755. [PubMed: 19136592]

Gardner JG, Grundy FJ, Henkin TM, Escalante-Semerena JC. Control of acetyl-coenzyme Asynthetase (AcsA) activity by acetylation/deacetylation without NAD(+) involvement in Bacillussubtilis. J. Bacteriol. 2006; 188:5460–5468. [PubMed: 16855235]

Garrity J, Gardner JG, Hawse W, Wolberger C, Escalante-Semerena JC. N-lysine propionylationcontrols the activity of propionyl-CoA synthetase. J. Biol. Chem. 2007; 282:30239–30245.[PubMed: 17684016]

Geissler JF, Harwood CS, Gibson J. Purification and properties of benzoate-coenzyme A ligase, aRhodopseudomonas palustris enzyme Involved in the anaerobic degradation of benzoate. J.Bacteriol. 1988; 170:1709–1714. [PubMed: 3350788]

Gibson J, Dispensa M, Fogg GC, Evans DT, Harwood CS. 4-Hydroxybenzoate-coenzymeA ligasefrom Rhodopseudomonas palustris - Purification, gene sequence, and role in anaerobicdegradation. J. Bacteriol. 1994; 176:634–641. [PubMed: 8300518]

Gibson J, Harwood C. Metabolic diversity in aromatic compound utilization by anaerobic microbes.Annu. Rev. Microbiol. 2002; 56:345–369. [PubMed: 12142480]

Gouet P, Courcelle E, Stuart DI, Metoz F. ESPript: multiple sequence alignments in PostScript.Bioinformatics. 1999; 15:305–308. [PubMed: 10320398]

Guzman LM, Belin D, Carson MJ, Beckwith J. Tight regulation, modulation, and high-levelexpression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 1995; 177:4121–4130. [PubMed: 7608087]

Hallows WC, Lee S, Denu JM. Sirtuins deacetylate and activate mammalian acetyl-CoA synthetases.Proc. Natl. Acad. Sci. U S A. 2006; 103:10230–10235. [PubMed: 16790548]

Harrison FH, Harwood CS. The pimFABCDE operon from Rhodopseudomonas palustris mediatesdicarboxylic acid degradation and participates in anaerobic benzoate degradation. Microbiology.2005; 151:727–736. [PubMed: 15758219]

Harwood CS, Burchhardt G, Herrmann H, Fuchs G. Anaerobic metabolism of aromatic compounds viathe benzoyl-CoA pathway. FEMS Microbiol. Rev. 1999; 22:439–458.

Hoff KG, Avalos JL, Sens K, Wolberger C. Insights into the sirtuin mechanism from ternarycomplexes containing NAD+ and acetylated peptide. Structure. 2006; 14:1231–1240. [PubMed:16905097]

Kim M-K, Harwood CS. Regulation of benzoate-CoA ligase in Rhodopseudomonas palustris. FEMSMicrobiol. Lett. 1991; 83:199–203.

Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RM 2nd, Peterson KM. Four newderivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene. 1995; 166:175–176. [PubMed: 8529885]

Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227:680–685. [PubMed: 5432063]

Larimer FW, Chain P, Hauser L, Lamerdin J, Malfatti S, Do L, Land ML, Pelletier DA, Beatty JT,Lang AS, Tabita FR, Gibson JL, Hanson TE, Bobst C, Torres JL, Peres C, Harrison FH, Gibson J,Harwood CS. Complete genome sequence of the metabolically versatile photosynthetic bacteriumRhodopseudomonas palustris. Nat. Biotechnol. 2004; 22:55–61. [PubMed: 14704707]

May JJ, Kessler N, Marahiel MA, Stubbs MT. Crystal structure of DhbE, an archetype for aryl acidactivating domains of modular nonribosomal peptide synthetases. Proc. Natl. Acad. Sci. U S A.2002; 99:12120–121205. [PubMed: 12221282]

Miroux B, Walker JE. Over-production of proteins in Escherichia coli: mutant hosts that allowsynthesis of some membrane proteins and globular proteins at high levels. J. Mol. Biol. 1996;260:289–298. [PubMed: 8757792]

Crosby et al. Page 15

Mol Microbiol. Author manuscript; available in PMC 2011 May 1.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Nakatsu T, Ichiyama S, Hiratake J, Saldanha A, Kobashi N, Sakata K, Kato H. Structural basis for thespectral difference in luciferase bioluminescence. Nature. 2006; 440:372–376. [PubMed:16541080]

Nielsen TK, Hildmann C, Dickmanns A, Schwienhorst A, Ficner R. Crystal structure of a bacterialclass 2 histone deacetylase homologue. J. Mol. Biol. 2005; 354:107–120. [PubMed: 16242151]

Peres CM, Harwood CS. BadM is a transcriptional repressor and one of three regulators that controlbenzoyl coenzyme A reductase gene expression in Rhodopseudomonas palustris. J. Bacteriol.2006; 188:8662–8665. [PubMed: 17041049]

Quandt J, Hynes MF. Versatile suicide vectors which allow direct selection for gene replacement inGram-negative bacteria. Gene. 1993; 127:15–21. [PubMed: 8486283]

Reger AS, Carney JM, Gulick AM. Biochemical and crystallographic analysis of substrate binding andconformational changes in acetyl-CoA synthetase. Biochemistry. 2007; 46:6536–6546. [PubMed:17497934]

Rocco CJ, Dennison KL, Klenchin VA, Rayment I, Escalante-Semerena JC. Construction and use ofnew cloning vectors for the rapid isolation of recombinant proteins from Escherichia coli.Plasmid. 2008; 59:231–237. [PubMed: 18295882]

Samanta SK, Harwood CS. Use of the Rhodopseudomonas palustris genome sequence to identify asingle amino acid that contributes to the activity of a coenzyme A ligase with chlorinatedsubstrates. Mol. Microbiol. 2005; 55:1151–1159. [PubMed: 15686561]

Sasse, J. Detection of proteins. In: Ausubel, FA.; Brent, R.; Kingston, RE.; Moore, DD.; Seidman, JG.;Smith, JA.; Struhl, K., editors. Current Protocols in Molecular Biology. New York: WileyInterscience; 1991. p. 10.16.11-10.16.18.

Schafer A, Tauch A, Jager W, Kalinowski J, Thierbach G, Puhler A. Small mobilizable multi-purposecloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defineddeletions in the chromosome of Corynebacterium glutamicum. Gene. 1994; 145:69–73. [PubMed:8045426]

Schwer B, Bunkenborg J, Verdin RO, Andersen JS, Verdin E. Reversible lysine acetylation controlsthe activity of the mitochondrial enzyme acetyl-CoA synthetase 2. Proc. Natl. Acad. Sci. U S A.2006; 103:10224–10229. [PubMed: 16788062]

Starai VJ, Celic I, Cole RN, Boeke JD, Escalante-Semerena JC. Sir2-dependent activation of acetyl-CoA synthetase by deacetylation of active lysine. Science. 2002; 298:2390–2392. [PubMed:12493915]

Starai VJ, Escalante-Semerena JC. Identification of the protein acetyltransferase (Pat) enzyme thatacetylates acetyl-CoA synthetase in Salmonella enterica. J. Mol. Biol. 2004; 340:1005–1012.[PubMed: 15236963]

Starai VJ, Takahashi H, Boeke JD, Escalante-Semerena JC. Short-chain fatty acid activation by acyl-coenzyme A synthetases requires SIR2 protein function in Salmonella enterica andSaccharomyces cerevisiae. Genetics. 2003; 163:545–555. [PubMed: 12618394]

Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W:improving the sensitivity of progressivemultiple sequence alignment through sequence weighting, position-specific gap penalties andweight matrix choice. Nucleic Acid Res. 1994; 22:4673–4680. [PubMed: 7984417]

Tsang AW, Escalante-Semerena JC. CobB, a new member of the SIR2 family of eucaryotic regulatoryproteins, is required to compensate for the lack of nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase activity in cobT mutants during cobalaminbiosynthesis in Salmonella typhimurium LT2. J. Biol. Chem. 1998; 273:31788–31794. [PubMed:9822644]

Vaillancourt FH, Bolin JT, Eltis LD. The ins and outs of ring-cleaving dioxygenases. Crit. Rev.Biochem. Mol. Biol. 2006; 41:241–267. [PubMed: 16849108]

Vetting MW, Carvalho LPSd, Yu M, Hegde SS, Magnet S, Roderick SL, Blanchard JS. Structure andfunctions of the GNAT superfamily of acetyltransferases. Arch. Biochem. Biophys. 2005a;433:212–226. [PubMed: 15581578]

Vetting MW, de Carvalho LP, Roderick SL, Blanchard JS. A novel dimeric structure of the RimLNalpha-acetyltransferase from Salmonella typhimurium. J. Biol. Chem. 2005b; 280:22108–22114.[PubMed: 15817456]

Crosby et al. Page 16

Mol Microbiol. Author manuscript; available in PMC 2011 May 1.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Zaar A, Eisenreich W, Bacher A, Fuchs G. A novel pathway of aerobic benzoate catabolism in thebacteria Azoarcus evansii and Bacillus stearothermophilus. J. Biol. Chem. 2001; 276:24997–25004. [PubMed: 11306574]

Zaar A, Gescher J, Eisenreich W, Bacher A, Fuchs G. New enzymes involved in aerobic benzoatemetabolism in Azoarcus evansii. Mol. Microbiol. 2004; 54:223–238. [PubMed: 15458418]

Crosby et al. Page 17

Mol Microbiol. Author manuscript; available in PMC 2011 May 1.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Figure 1. Anaerobic benzoate catabolism in R. palustrisA. Pathways for degradation of benzoate, 4-hydroxybenzoate, and cyclohexanecarboxylatein R. palustris. BadA, benzoyl-CoA synthetase; HbaA, 4-hydroxybenzoyl-CoA synthetase;AliA, cyclohexanecarboxyl-CoA synthetase. Adapted from (Egland & Harwood, 1999). B.Alignment of protein sequences near C-terminal end of acyl- and aryl-CoA synthetases(residues 504-523 of BadA). Arrow indicates lysine residue that is acylated in S. entericaAcs and PrpE. Rp, R. palustris; Se, S. enterica; Acs, Ac-CoA synthetase; PrpE, propionyl-CoA synthetase. Alignment generated with ClustalW (Thompson et al., 1994) and ESPript(Gouet et al., 1999).

Crosby et al. Page 18

Mol Microbiol. Author manuscript; available in PMC 2011 May 1.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Figure 2. Modeling the effect of Nε-Lys acetylation on the structure of benzoyl-CoA synthetasefrom B. xenovorans LB400A.The crystal structure of Bz-CoA synthetase from B. xenovorans LB400 (PDB 2V7B) wasreported with benzoate (shown in purple) in the active site (Bains & Boulanger, 2007). Aclose-up view of this region shows the hydrogen bonds formed between the oxygen atoms ofbenzoate (red) and the epsilon amino group of residue Lys520 (blue), with bond distancesindicated. B. An acetylated Lys residue was modeled in lieu of Lys520 using Pymol(http://www.pymol.org). Acetylation of Lys520 suggests the loss of hydrogen bondingbetween the epsilon amino group of Lys520 and the carboxylic acid oxygen atoms ofbenzoate.

Crosby et al. Page 19

Mol Microbiol. Author manuscript; available in PMC 2011 May 1.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Figure 3. Photoheterotrophic growth of R. palustris on benzoate(A) Growth behavior of the R. palustris ΔldaA ΔsrtN strain carrying a plasmid encoding awild-type allele of ldaA or srtN. Data points are averages of three replicates, and error barsrepresent standard deviations. Open triangles, wild type (CGA009) harboring plasmidpBBR1MCS-2 as a vector control; filled triangles, strain JE11636 (ldaA srtN /pBBR1MCS-2); circles, strain JE11637 (ldaA srtN / pRpLDAA4); squares, strain JE11638(ldaA srtN / pRpSRTN4). (B) Growth behavior of the R. palustris ΔldaA ΔsrtN Δpat strain(open circles) compared to the wild type (open triangles) and the ΔldaA ΔsrtN strain (filledtriangles). Optical density was monitored during photosynthetic growth on 3 mM benzoate

Crosby et al. Page 20

Mol Microbiol. Author manuscript; available in PMC 2011 May 1.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Figure 4. Residue K512 of BadA is the site of acetylationLC/MS-MS analysis of H10-BadA protein purified from R. palustris strain JE11639 (ldaAsrtN / pRpBADA4) grown photosynthetically on benzoate (3 mM). MS/MS spectrum of the915.52 amu tryptic peptide, where peaks in red indicate b series m/z (predicted fragment ionmasses of TATGKAcIQR with the charge on the NH2-terminal amino acid), and blue peaksindicate y series m/z (predicted fragment ion masses of TATGKAcIQR with the charge onthe COOH-terminal amino acid). These ion series confirm that the sequence of the 915.52amu peptide as TATGKAcIQR, which corresponds to the predicted acetylation site ofLys512.

Crosby et al. Page 21

Mol Microbiol. Author manuscript; available in PMC 2011 May 1.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Figure 5. Acetylation of BadA, HbaA, AliA, and Acs proteins using [14C-1]-Ac-CoAEither wild-type (WT) or Lys-to-Ala variants of CoA synthetases were incubated with[14C-1]-Ac-CoA with or without RpPat. Top panel shows SDS-PAGE of acetylationreactions; lower panel shows the phosphor image of the same gel.

Crosby et al. Page 22

Mol Microbiol. Author manuscript; available in PMC 2011 May 1.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Figure 6. Deacetylation of BadA, HbaA and AliA using LdaA in cell lysatesA.[14C]-BadAAc protein was incubated with cell lysates of E. coli harboring either plasmidpRpLDAA1 (triangles) or cloning vector (squares). Aliquots of the reaction were quenchedat each time point, resolved using SDS-PAGE, and quantified by phosphor imaging. Datarepresent averages and standard deviations of three reactions. B. BadA, HbaA and AliAactivity is restored upon deacetylation. Each acyl-CoA synthetase (or no-synthetase control)was pre-incubated with or without Pat and acetyl-CoA, and the acetylation reaction wasstopped by buffer exchange. Cell-free extracts of strains JE11958 (empty vector) or JE11959(pRpLDAA1) were added to the reaction, and acyl-CoA synthetase activity was measured intriplicate.

Crosby et al. Page 23

Mol Microbiol. Author manuscript; available in PMC 2011 May 1.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Figure 7. Proposed model for the posttranslational regulation of BadAIn this model, BadA protein is acetylated by RpPat (and possibly by other unknownacetyltransferases) at residue Lys512, rendering it inactive. BadA is reactivated by thedeacetylases LdaA and SrtN. LdaA uses water to hydrolyze the acetyl group, releasingacetate, whereas SrtN uses NAD+ as a substrate, generating O-acetyl-ADP-ribose (O-AADPR) and nicotinamide (Nm). Regulation of HbaA and AliA is predicted to be similar tothat for BadA. CoASH, coenzyme A; PPi, pyrophosphate.

Crosby et al. Page 24

Mol Microbiol. Author manuscript; available in PMC 2011 May 1.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Crosby et al. Page 25

Table 1

Similarity of R. palustris proteins to homologs in S. enterica LT2 and B. subtilis SMY

R. palustrisgene

Proteinname

Predictedfunction

Homology toS. entericaa

Homology toB. subtilis

rpa4240 Pat acyltransferase 37/55 NAb

rpa0954 LdaAc deacetylase NA 33/48

rpa2524 SrtNd deacetylase 35/49 39/49

aHomology listed as percent identity/percent similarity of protein sequences.

bNA, not applicable because there is not a homolog in this species.

cLdaA is homologous to AcuC in B. subtilis SMY.

dSrtN is homologous to CobB in S. enterica sv Typhimurium LT2.

Mol Microbiol. Author manuscript; available in PMC 2011 May 1.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Crosby et al. Page 26

Table 2

Activity of BadA partially purified fromR. palustris

Strain Genotype Pre-incubation with:

NAD+a SeCobB + NAD+

JE13049 WT / pRpBADA4b 1.0 ± 0.0 1.0 ± 0.0

JE11639 ldaA srtN / pRpBADA4 0.1 ± 0.0 1.2 ± 0.1

JE13050 ldaA srtN pat / pRpBADA4 0.1 ± 0.1 0.8 ± 0.1

Each measurement was performed in triplicate; the experiment was performed twice.

aEnzyme activity reported as µmol AMP min−1 mg(BadA)−1

bbadA+ cloned into pBBR1MCS-2

Mol Microbiol. Author manuscript; available in PMC 2011 May 1.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Crosby et al. Page 27

Table 3

CoA synthetase enzyme activity

Pre-incubation with:

R. palustris enzyme Substrate Pat + CoAa Pat + AcCoA

BadA benzoate 8.1 ± 0.3 0.06 ± 0.0

BadAK512A benzoate 0.05 ± 0.0 0.05 ± 0.0

HbaA 4-hydroxybenzoate 2.3 ± 0.1 BLDb

HbaAK503A 4-hydroxybenzoate BLD BLD

AliA cyclohexanecarboxylate 10.1 ± 0.5 BLD

AliAK532A cyclohexanecarboxylate 0.3 ± 0.03 0.3 ± 0.0

AliAK535A cyclohexanecarboxylate 9.6 ± 0.4 BLD

Acs acetate 12.7 ± 0.3 BLD

AcsK606A acetate BLD BLD

aEnzyme activity reported as µmol AMP min−1 mg−1.

bBLD: below the limit of detection.

Mol Microbiol. Author manuscript; available in PMC 2011 May 1.