Vol. 171, No. 6

Cloning, Sequencing, and Overexpression of mvaA, Which EncodesPseudomonas mevalonii 3-Hydroxy-3-Methylglutaryl

Coenzyme A ReductasetMICHAEL J. BEACHt AND VICTOR W. RODWELL*

Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907

Received 28 November 1988/Accepted 15 March 1989

We have cloned, determined the primary structure of, and overexpressed in Escherichia coli the gene mvaA,which is the 1,287-base structural gene for the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase[EC 1.1.1.88] ofPseudomonas mevalonii. The amino acid composition of HMG-CoA reductase agreed with thatpredicted from the nucleotide sequence of mvaA, and DNA-derived sequences were identical to all experimen-tally determined peptide sequences. Overexpression of mvaA in E. coli yielded quantities of HMG-CoAreductase over 1,500-fold higher than those present in control cultures. Comparison of the primary structureof the P. mevalonii enzyme with the DNA-derived primary structure for a mammalian HMG-CoA reductaserevealed two regions of similarity suggestive of functional relatedness. An open reading frame, ORF1, lies onthe 3' side of mvaA, and a potential ribosome-binding site for ORF1 overlaps the termination codon of mvaA.

Several microorganisms can utilize the isoprenoid precur-sor mevalonate as a sole carbon source (4, 13, 16, 49, 50, 52).In Pseudomonas mevalonii (previously referred to as Pseiu-domonas sp. M [16, 17]), growth on mevalonate is mediatedby an inducible active transport system (16). InternalizedR-mevalonate is then oxidatively deacylated to S-3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) in a reactioncatalyzed by HMG-CoA reductase [EC 1.1.1.88]. P. meva-lonii HMG-CoA reductase, which is coinduced with a meva-lonate transport system (16, 17), has been purified to homo-geneity and shown to be a tetramer of apparently identicalsubunits, each of approximate Mr 43,000 (17). In vertebrates,yeasts, and plant tissues, HMG-CoA reductase (EC 1.1.1.34)catalyzes the conversion of HMG-CoA to mevalonate in areaction that is rate limiting for isoprenoid biosynthesis (43).Although no method exists for purification of the membrane-bound holoenzyme, hamster (11, 46), human (30), and yeast(3) genes that encode HMG-CoA reductase have beencloned and characterized by complete (11, 30, 46) or partial(3) DNA sequencing. No plant or procaryotic HMG-CoAreductase gene has been similarly examined.We report here a partial protein sequence for P. inevalonii

HMG-CoA reductase, the complete DNA sequence of thecorresponding structural gene mvaA, and overexpression ofthe gene in Escherichia coli. This new information, togetherwith the availability of large quantities of the recombinantenzyme, makes the P. mevalonii enzyme attractive foranalysis of the active site and catalytic mechanism of HMG-CoA reductase via chemical modification, site-specific mu-tagenesis, and eventual crystallization. This work will alsofacilitate investigation of the regulation of mevalonate catab-olism in Pseudomonas spp.

(This research was carried out by Michael J. Beach inpartial fulfillment of the requirements for the Ph.D. degreefrom Purdue University, West Lafayette, Ind.)

* Corresponding author.t Journal paper 11,971 from the Purdue University Agricultural

Experiment Station.t Present address: Hepatitis Branch, Centers for Disease Control,

Atlanta, GA 30333.

MATERIALS AND METHODS

Bacterial strains, plasmids, bacteriophage, and culture con-ditions. Plasmid cloning and M13 propagation were con-ducted in E. coli JM103 (36), using M9 medium, L broth (34),B broth, or 2x YT medium (36). Phage lambda was propa-gated in E. coli Q359 (25) on NZYM medium (34). PlasmidspUC18 and pUC19, phage derivatives M13mpl8 andM13mpl9, and lambda EMBL4 have been previously de-scribed (14, 54). Plasmid pKK177-3, a shortened version ofpKK223-3 (9) in which the remainder of the tet gene betweenthe BamHI and PvuII sites has been deleted (J. Brosius,personal communication), was obtained from Scott Buckel,Carnegie-Mellon University, Pittsburgh, Pa.

Purification and assay of P. mevalonii HMG-CoA reductase.HMG-CoA reductase activity was assayed and the enzymewas purified to homogeneity as described by Gill et al. (17).

Reduction, alkylation, and cleavage of protein with cyano-gen bromide. Purified HMG-CoA reductase (450 nmol) wasdialyzed against 9% (vol/vol) formic acid, lyophilized, andsuspended in reduction-alkylation buffer (130 mM Tris [pH8.0], 6 M guanidine hydrochloride, 1 mg of EDTA per ml)that contained dithioerythritol in a fourfold molar excessover the predicted sulfhydryl content. The solution wasincubated at room temperature for 4 h and then dialyzedagainst reduction-alkylation buffer. Cysteine residues werealkylated by treatment first with 2 pCi of [1-14C]iodoace-tamide (specific activity, 53 Ci/mol) and then with nonradio-active iodoacetamide. (Use of [14C]iodoacetamide was in-tended to facilitate identification of cysteine-containingpeptides. However, no peptide yielded enough sequence toascertain the positions of the two cysteine residues.) After 30min, excess dithioerythritol was added to neutralize unre-acted iodoacetamide. The reduced, alkylated protein wasthen dialyzed three times against 9% (vol/vol) formic acid,lyophilized, and suspended in 70% (vol/vol) formic acid. Acrystal of cyanogen bromide was added, and cleavage wasallowed to proceed for 20 h at room temperature in the dark.

Peptide isolation. Cyanogen bromide-cleaved peptideswere initially fractionated on Sephadex G-50SF equilibratedin 9% (vol/vol) formic acid. This fractionation led to sevenmajor peaks, which were pooled and lyophilized. Lyophi-

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PSEUDOMONAS HMG-CoA REDUCTASE GENE STRUCTURE

lized peptide pools CB-I through CB-VII were further puri-fied by reverse-phase high-performance liquid chromatogra-phy (HPLC) (23, 31, 32, 42), using a model 5000 liquidchromatograph (Varian, Palo Alto, Calif.) equipped witheither a Varichrome or a UV-50 detector and a 250-by-4.1-mm C18 reverse-phase Synchrom Synchropak RP-Pcolumn. Portions of selected peptide pools were dissolvedeither in 9% (vol/vol) formic acid (CB-III and CB-IV) or in 6M guanidine hydrochloride (CB-V) and injected into thechromatograph. Fractions were eluted by using a binarygradient of 0.1% (vol/vol) trifluoroacetic acid in water as theprimary mobile phase. The secondary phase was either1-propanol-0.1% trifluoroacetic acid or acetonitrile-0.1%trifluoroacetic acid. Flow rates were approximately 0.7ml/min. The percentage of organic solvent in the gradientwas increased with time (1%/min in most cases) from 0 to60%. The detector was set at 230 nm for analytical runs andincreased to 240 nm for preparative work. All peptides wereinitially purified with 1-propanol as the secondary phase.Peptides from pool CB-V were rechromatographed, usingacetonitrile as the secondary phase.

Sequence analysis. Purified peptides were sequenced on amodel 890C automated sequencer (Beckman Instruments,Inc., Fullerton, Calif.) (22, 24). The sequencer-derived prod-ucts were treated as described previously (32, 40). Phenyl-thiohydantoin derivatives were analyzed by HPLC (40, 55).Phenylthiohydantoin-arginine was identified by HPLC or bya spot test (33). Phenylthiohydantoin-histidine was identifiedby the Pauly test (39).

Oligonucleotides. Oligonucleotide probe mixtures weresynthesized on an Applied Biosystems synthesizer by ShuJin Chan (University of Chicago, Chicago, Ill.).DNA isolation. Plasmids and M13 replicative form were

purified by the method of Birnboim (7). Chromosomal DNAwas prepared generally as described by Saito and Miura (44)but with the phenol extraction performed at room tempera-ture and TE substituted for saline-citrate. No further purifi-cation was performed after the spooled DNA was washedwith ethanol. Small quantities of lambda DNA were pre-pared by the method of Benson and Taylor (5). High-titerliquid lysates of lambda were prepared as described bySilhavy et al. (51). Phage DNA was prepared by using aglycerol step gradient as described by Maniatis et al. (34).M13 template for sequencing was purified as describedpreviously (1).

Transformations. Transformations using E. coli JM103 anda 2-min heat shock were done by the method of Hanahan(20). SOB medium (20) contained twice the quantity of Mg2+ions recommended for E. coli DH1 cells.

Oligonucleotide labeling. Oligonucleotide probe mixtureswere 5' end labeled with [_y-32P]ATP by using T4 polynucle-otide kinase. The radioactive probe was purified on Sepha-dex G-50SF equilibrated with 50 mM triethylamine (pH 8.0)and routinely had a specific activity of greater than 108cpm4Lg.

Construction and screening of a Pseudomonas pUC19 li-brary. A 0.7- to 1.3-kilobase-pair (kbp) fraction of a Sall-HindIl digest of P. mevalonii DNA was ligated into simi-larly cleaved pUC19 and transformed into JM103. Whitecolonies were screened with combined 17-mer and 14-merprobes by the colony hybridization method of Grunstein andHogness (18) as modified by Woods (53). Hybridization with2.5 x 106 cpm per filter was conducted at 37°C, and the finalwash was at 42°C.

Construction and screening of a genomic library in EMBL4.P. mevalonii chromosomal DNA was partially digested with

Sau3AI to obtain fragments of approximately 20 kbp (34).The DNA was ligated into SalI-BamHI-cleaved EMBL4 andpackaged by using an in vitro packaging kit (AmershamCorp., Arlington Heights, Ill.). Infection and amplification(34) always gave titers in excess of 1010 PFU/ml. TheEMBL4 library was screened with an 840-bp SalI-HindIllfragment that had been labeled by using an Amersham nicktranslation kit. The labeled probe, which was purified asdescribed above for oligonucleotide probes, routinely had aspecific activity of greater than 107 cpm/,ug. Plaques werereplica plated onto nitrocellulose, amplified in situ (34), andscreened by the method of Benton and Davis (6), using 1.5 x106 cpm of probe per filter. Hybridization and washing wereconducted at 68°C.

Construction of nested deletions for sequencing. Two meth-ods were used to generate nested deletions in the HMG-CoAreductase gene, present as an insert in the polylinker regionof M13mpl8 and M13mpl9. Deletions were made in theSall-HindIIl fragment by using exonuclease III essentiallyas described by Bartlett and Matsumura (2) and Henikoff(21). Nested deletions in the KpnI-PstI mvaA fragment werecarried out by using a Cyclone kit (International Biotechnol-ogies, Inc., New Haven, Conn.) according to the specifica-tions of the manufacturer.DNA sequencing. M13 template was prepared as described

previously (1). Dideoxy sequencing was done by the methodof Sanger et al. (45), using the deoxynucleotide triphosphatesolutions described by Amersham (1). Compressions in thesequencing gel caused by the high G+C content (65%) of P.mevalonii DNA were resolved by sequencing the oppositestrand or by electrophoresis on 40% formamide gels (35).

Construction of the expression vector pHMGR-1. LambdaHMGR-5, which contained mvaA, was cleaved with KpnI,digested with the Klenow fragment of DNA polymerase I togive a blunt end, and then cleaved with PstI. The 1.35-kbpKpnI-PstI fragment that contained the coding sequence ofmvaA was isolated from a low-melting-point agarose gel asspecified by the manufacturer. The pKK177-3 expressionvector was cleaved with EcoRI, digested with mung beannuclease to give a blunt end by removing the 5' overhang,and digested with PstI. The vector and the KpnI-PstI mvaAfragment were then ligated. This derivative of pKK177-3 wasdesignated pHMGR-1.Computer programs. DNA sequences were entered on a

program written by William Schwindinger (Albert EinsteinCollege of Medicine, New York, N.Y.). Protein compari-sons were performed by using the FASTP program (27).

RESULTS

Preparation and purification of cyanogen bromide peptides.HMG-CoA reductase was cleaved with cyanogen bromide,and the resulting peptides were size fractionated on Sepha-dex G-SOSF. On the basis of the A260 and A280 profiles of theeluate, fractions were combined in seven pools, designatedcyanogen bromide pools CB-I through CB-VII (Fig. 1). Thepeptides in these pools were further fractionated by reverse-phase HPLC. CB-I and CB-II contained partial digestionproducts and were not used further. Eight peptides werepurified from CB-III, CB-IV, and CB-V (Fig. 2).Primary structures of peptides. Automated Edman degra-

dation of eight peptides (double underlined in Fig. 3) estab-lished the sequence of 177 (45%) of the 391 predicted (17)residues. CB-III(3) and CB-IV(5) had identical sequences at

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2996 BEACH AND RODWELL

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their amino termini, and CB-V(7) contained the previouslyidentified amino-terminal sequence (17).

Design and synthesis of oligonucleotide probes. On the basisof two sequences suitable for probe design (Fig. 3), a 16-fold

degenerate 14-mer, 5'-AC(AG)TC(AG)TG(AG)TA(TC)TC-3', and a 32-fold degenerate 17-mer, 5'-ACCCA(AG)TCNAC(TC)TC(AG)TC-3', were synthesized.

Cloning and structural analysis of the HMG-CoA reductase

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GGC ATGGly Met

CAG ATCGln Ile

GCT GCTAla Ala

GCC CCGAla Pro

CTG AGCLeu Ser

CTCLeu

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GAC GATAsp Asp

ATC GAAIie GluAAT GGCAsn Gly

TCG TACSer Tyr

CTG ATGLeu Met

CTG CTGLeu Leu

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AAT ACCAsn Thr

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CAG GTG CGG ATT ACT CCG CAG CAA CTG GAAGln Val Arg ole Thr Pro Gln Gln Leu Glu

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CTC GGG GCC ATGLeu Gly Ala Met

GCG CGC AAT ATTAla Ara Asn Ile

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*** *** *** *** **GAA TAC CAC GAC GTG CGC GCCGlu TYr His As) Val Ara Ala

GAC CGC GCC GTA GCAAsp Ara Ala Val Ala

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PstIGGC CTG CAGIGly Leu Gln

FIG. 3. Nucleotide sequence of the 1.35-kbp Kpnl-to-Pstl fragment containing mnv'aA and portions of its flanking regions. Asterisks areabove the two peptide sequences used to design synthetic probes. Locations of the KpnI, Sall, and PstI sites are indicated. The deducedprimary structure of HMG-CoA reductase is given below the nucleotide sequence. Double-underlined sequences were corroborated bysequencing the indicated peptides: residues 2 to 29, CB-V(7); 113 to 150, CB-I1I(2); 202 to 227, CB-IV(5) and CB-111(3); 271 to 284, CB-V(4);324 to 348, CB-IV(7); 370 to 377, CB-II(1); and 383 to 425, CB-IV(1). Single-underlined residues within the indicated peptides were notidentified by peptide sequence data. Numbers 1 to 428 enumerate amino acid residues of HMG-CoA reductase; numbers o-1 through o-16designate residues of ORF1. #, Proposed ORF1 ribosome-binding site.

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2998 BEACH AND RODWELL

TABLE 1. Codon usage for inv'aA

Amino No. of . Amino No. of . Amino No. of Amino No. ofCodon acid times used Codon acid times used Codon acid times used Codon acid times used

TTT Phe 0 TCT Ser 0 TAT Tyr 2 TGT Cys 0TTC Phe 7 TCC Ser 4 TAC Tyr 5 TGC Cys 2TTA Leu 0 TCA Ser 0 TAA End 0 TGA End 1TTG Leu 4 TCG Ser 4 TAG End 0 TGG Trp 3CUT Leu 0 CCT Pro 2 CAT His 5 CGT Arg 5CTC Leu 9 CCC Pro 4 CAC His 8 CGC Arg 20CTA Leu 0 CCA Pro 1 CAA Gln 5 CGA Arg 1CTG Leu 37 CCG Pro 10 CAG Gln 12 CGG Arg 2ATT Ile 5 ACT Thr 2 AAT Asn 5 AGT Ser 2ATC Ile 18 ACC Thr 15 AAC Asn 15 AGC Ser 8ATA Ile 1 ACA Thr 1 AAA Lys 4 AGA Arg 0ATG Met 14 ACG Thr 1 AAG Lys 5 AGG Arg 1GTT Val 2 GCT Ala 5 GAT Asp 7 GGT Gly 2GTC Val 10 GCC Ala 44 GAC Asp 16 GGC Gly 33GTA Val 6 GCA Ala 4 GAA Glu 14 GGA Gly 0GTG Val 17 GCG Ala 11 GAG Glu 6 GGG Gly 2

gene. Since both oligonucleotide probes hybridized to an840-bp Sall-HindIll fragment of genomic DNA, a P.mevalonii(pUC19) library enriched in fragments of this sizewas screened with a mixture of these oligonucleotides.Positive clones contained an identical 840-bp insert, desig-nated HMGR, which was found to overlap 500 bp at the 3'end of the HMG-CoA reductase gene. Nick-translatedHMGR was next used to screen a partial Sau3AI lambdalibrary. One clone (lambda HMGR-5) contained a 14-kbinsert that included a 1.35-kbp KpnI-PstI fragment contain-ing the entire HMG-CoA reductase gene. The nucleotidesequences of both strands of this fragment were then deter-mined (Fig. 3).Codon usage. The codon usage in mvaA (Table 1) was

similar to that for other Pseudomonas genes (10, 37, 38). Thehigh G+C content (65%) reflects a strong preference (81%)for G or C in the wobble position. There is extreme codonbias; 25% of the codons are used once or not at all. Anexception is the use of GAA for glutamate 70% of the time.Usage of GAA by other Pseudomonas genes, although high,has not previously been observed to exceed 50% (10, 37, 38).

Structure of HMG-CoA reductase. HMG-CoA reductase isa protein of Mr 45,538 (428 amino acid residues), consistentwith the previously estimated subunit molecular weight of43,000 (17). The amino acid composition of the protein (17)fully supports the DNA-derived and experimentally deter-mined protein sequences (Fig. 3).Comparison with mammalian HMG-CoA reductase. The

nucleotide-derived sequence of P. mevalonii HMG-CoAreductase revealed two regions of similarity to that of theChinese hamster enzyme (11). The first, situated in theamino-terminal portion (residues 52 to 88) of P. mevalonjiHMG-CoA reductase, exhibited 38% identity over a 37-residue segment. The second region, present in the carboxyterminus (residues 304 to 389), exhibited 35% identity over86 residues (Fig. 4). The two blocks of similarity made up29% of the P. mevalonii sequence.

Overexpression of mvaA in E. coli. E. coli JM103 cellstransformed with pHMGR-1 were induced with 1 mM iso-propyl-p-D-thiogalactopyranoside (IPTG). Log-phase cellswere harvested and lysed as previously described (17) andcentrifuged for 1 min in an Eppendorf microfuge. Thesupernatant liquid was then examined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (Fig. 5) and as-sayed for HMG-CoA reductase activity. Extracts of IPTG-

induced cells contained a major polypeptide whose mobilitycoincided with that of HMG-CoA reductase (Fig. 5). Thespecific activities (nanomoles per minute per milligram ofprotein) of crude extracts of IPTG-induced E. coli recombi-nants and of P. mevalonii were as follows: P. mevaloniigrown on 25 mM mevalonate, 1,530; P. mevalonii grown on12.5 mM glucose, 6.4; E. coli JM103(pHMGR-1), 10,300; E.coli JM103, 7.0; and E. coli JM103(pKK177-3), 7.4.

DISCUSSION

The Pseudomonas gene mvaA encodes HMG-CoA reduc-tase, a slightly acidic protein (53% of the charged residuesare negative) of Mr 45,538 (428 amino acids). The hydropa-thy profile (data not shown) is representative of a soluble,hydrophilic protein. Unlike mammalian HMG-CoA reduc-tase (11, 30, 46), the Pseudomonas enzyme lacks the multi-ple hydrophobic segments thought to function as membraneanchor domains. The more than 1,500-fold induction ofHMG-CoA reductase activity when pHMGR-1 was inducedin E. coli by IPTG provides perhaps the most convincingevidence that we hatVe indeed cloned and expressed the genethat encodes Pseudomonas HMG-CoA reductase. In addi-tion, the requirement for growth of cells on mevalonate, anexpensive and occasionally scarce carbon source, has nowbeen eliminated, reducing the expense of producing homo-geneous enzyme by at least an order of magnitude.The limited sequence similarity between mammalian and

Pseudomonas HMG-CoA reductases, while not by itselfparticularly striking, should be considered in conjunctionwith certain additional facts. First, both enzymes catalyzethe same reaction. Second, when the sequences of themammalian and Pseudomonas enzymes are aligned, the twoblocks of similarity are out of phase by only a few aminoacids. Third, it is precisely the C-terminal domain of themammalian enzyme that is most likely to exhibit homologywith P. mevalonii HMG-CoA reductase, since this solubledomain is known to contain the entire active site (15, 28, 29).We would anticipate that any primary structural similaritiesmight be confined to regions crucial either to the active siteor to appropriate folding of the molecule. Utimately, crys-tallographic analysis of P. mevalonii and of mammalianHMG-CoA reductase should reveal, at a three-dimensionallevel, the related structural and functional domains of theseenzymes.

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PSEUDOMONAS HMG-CoA REDUCTASE GENE STRUCTURE

PseudomonasMammalian

***** * ** * ** * **

IENVIGTFELPYAVASNFQINGRDVLVPLVVEEPSIVAAASYMAKLARCENVIGYMPIPVGVAGPLCLDGKEYOVPMATTEGCLVASTNRGCRAIGLG

** * * *

Pseudomonas GGETTSSSAPMHAQVQIVGIQDPLNARLSLLRRKDEIIELANRKDQUSNMammalian GGASSRVLADGMTRGPVVRLPRACDSAEVKAWLETPEGFAVIKDAFDSTS

Pseudomonas SLGGGCRDIEVHTFADTPRGPMLVAHLIVDVRDAMGANTVNmTAEAVAPLMammalian RFARLQKLHVTMAGRNLYIRFQSKTGDAMGMNMISKGTECALUKLQEFFP

* * *Pseudomonas MEAITGGQVRLRILSNLADLRLARAQVRITPQQLETAEFSGEAVIEGILDMammalian EMQILAVSGNYCTDKKPAAINWIEGRGKTVVCEAVIPAKVVREVLKTTTE

PseudomonasMammalian

PseudomonasMammalian

PseudomonasMammalian

* * * * *

AYAFAAVDPYRAATHNKGIMNGIDPLIVATGNDWRAVEAGAHAYACRSGHAMIDVNINKNLVGSAMAGSIGGYNAHAANIVTAIYIACGQDAAQNVGSSN

* * * ** * *** * * * * ***

YGSLTTWEKDNNGHLVGTLEMP MPVGLVGGATKTHPLAQLSLRILGVKTCITLEASGPTNEDLYISCTMPSIEIGTVGGGTNLLP OOACLOMLGVOG

* ** * * * *** * ** *

A QAIAEIAVAVGLAQNLGAMALATEGIQRGHMALHARNIAACKDNPGENAROLARIVCGTVMAGELSMALAAGHLVRSHMVlBRSKIN

Pseudomonas VVAGARGDEVDWVARQLVEYHDVRADRAVALLKQKRGQMammalian LQDLQGTCTKKSA

100575

150625

200675

250725

300775

349824

390874

428887

FIG. 4. Apparent similarity between P. mevalonii and mammalian HMG-CoA reductases. The degree of similarity was determined byusing the FASTP program (27). Either the entire Pseudomonas sequence or 75-residue overlapping segments were compared with the Chinesehamster HMG-CoA reductase sequence (11). Asterisks indicate regions of identity between the two enzymes. For the double-underlinedregions, the homologies are 38 and 35%, respectively.

1 2 3 4 5

FIG. 5. Overexpression of HMG-CoA reductase protein in trans-formed E. coli. E. coli transformed with pHMGR-1 was grown in thepresence (lane 1) or absence (lane 2) of IPTG. P. mei'alonii wasgrown on mevalonate (lane 3) or glucose (lane 4). Lanes 1 through 4contained 15 ,ug of protein. After sodium dodecyl sulfate-polyacryl-amide gel electrophoresis, the gel was stained with Coomassie blue.-*, Band corresponding to HMG-CoA reductase. Lane 5 containedprotein standards with the indicated Mr values: phosphorylase b(94,000), bovine serum albumin (67,000), ovalbumin (43,000), car-bonic anhydrase (30,000), soybean trypsin inhibitor (20,100), anda-lactalbumin (14,400).

Reagents that modify sulfhydryl groups inactivate P.mevalonii HMG-CoA reductase (17, 26; T. C. Jordan-Starckand J. Gill, unpublished observations) and other four-elec-tron oxidoreductases (8, 12; 13. Franzen, C. Carrubba, J.Ashcom, J. S. Franzen, and D. S. Feingold, Fed. Proc.39:2000, 1980). However, no similarity was apparent be-tween the sequence flanking either cysteine residue of P.mevalonii HMG-CoA reductase and those of active-sitepeptides from the four-electron oxidoreductases UDP-glu-cose dehydrogenase (Franzen et al., Fed. Proc. 39:2000,1980) and histidinol dehydrogenase (8).An open reading frame (ORF1) begins 12 bp downstream

from mvaA and extends for at least 350 bp beyond the end ofmvaA (Fig. 3; M. Beach, unpublished observations). mvaAmay therefore be linked to other genes involved in mevalo-nate catabolism. HMG-CoA reductase and mevalonatetransport are coregulated (16, 17) and may be geneticallylinked, as are enzymes of other catabolic pathways inpseudomonads (19). In addition, a putative ribosome-bindingsite of ORF1 that has 75% identity with the complement tothe 3' end of the 16S rRNA of P. aeruginosa (AAGGAGAG)(48) overlaps the end of mvaA by six nucleotides. In E. coli,close proximity or overlap of a ribosome-binding site withthe preceding gene in a polycistronic mRNA can reflecttranslational coupling of the two genes (41, 47). Experimentsare therefore in progress to determine whether ORF1 en-codes another protein involved in mevalonate catabolism.

ACKNOWLEDGMENTS

Technical assistance was provided by Norma Salinas. We ac-knowledge the notable assistance of Mark A. Hermodson, whodetermined and interpreted peptide sequences and detected thepotential 280,000-fold degenerate probe sequence RLSLLRR. The

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3000 BEACH AND RODWELL

data in Fig. 5 were provided by our colleague Jack Green. Finally,we acknowledge the assistance of David Ann, Scott Buckel, JackDixon, John Gill, Tuajuanda Jordan-Starck, Pete Kennelly, RonaldSomerville, and Yuli Wang, who provided both material assistanceand much thoughtful advice.

This work was funded by Public Health Service research grantGM 33457 from the National Institutes of Health. Michael J. Beachwas supported by Public Health Service training grant GM 07211from the National Institutes of Health.

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