Vol. 60, No. 3APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1994, p. 888-8950099-2240/94/$04.00+0Copyright © 1994, American Society for Microbiology

Cloning, Sequencing, and Expression in Escherichia coli of theD-Hydantoinase Gene from Pseudomonas putida and

Distribution of Homologous Genes inOther Microorganisms

GISELE LAPOINTE, STEPHANE VIAU, DANIELLE LEBLANC, NORMAND ROBERT,AND ANDRE MORIN*

Bio-Ingredients Section, Food Research and Development Centre, Agriculture Canada,St. Hyacinthe, Quebec, Canada J2S 8E3

Received 17 September 1993/Accepted 31 December 1993

Pseudomonas putida DSM 84 produces N-carbamyl-D-amino acids from the corresponding D-5-monosubsti-tuted hydantoins. The gene encoding this D-hydantoinase enzyme was cloned and expressed in Escherichia coli.The nucleotide sequence of the 1.8-kb insert of subclone pGES19 was determined. One open reading frame of1,104 bp was found and was predicted to encode a polypeptide with a molecular size of 40.5 kDa. Local regionsof identity between the predicted amino acid sequence and that of other known amidohydrolases (two otherD-hydantoinases, allantoinase and dihydroorotase) were found. The D-hydantoinase gene was used as a probeto screen DNA isolated from diverse organisms. Within Pseudomonas strains of rRNA group I, the probe wasspecific. The probe did not detect D-hydantoinase genes in pseudomonads not in rRNA group I, other bacteria,or plants known to express D-hydantoinase activity.

Pure enantiomers of amino acids can be produced byring-opening hydrolysis of D- or L-5-monosubstituted hydanto-ins (Fig. 1). This reaction is catalyzed by the enzyme hydan-toinase and yields N-carbamyl-amino acids. N-carbamyl-aminoacids are converted chemically or enzymatically (N-carbamyl-amidohydrolase; EC 3.5.1.6) to the corresponding amino acids.For example, D-p-hydroxyphenylglycine is used in the produc-tion of semisynthetic penicillin. The 1988 market estimate ofthe biocatalyst D-p-hydroxyphenyl-hydantoinase was U.S.$2,000,000 (27). Where optically pure products are required,the stereospecific enzyme provides a clear advantage overchemical hydrolysis.

Hydantoinase hydrolyzes cyclic amide bonds (36). The threetypes of hydantoinases are D-enantiomer specific (45), L-enantiomer specific (44), and nonspecific (17, 38) for hydro-lysis of 5-substituted hydantoins. The D-hydantoinase enzymewas considered a dihydropyrimidinase (EC 3.5.2.2), since ithydrolyzes 5,6-dihydropyrimidine, 5,6-dihydrouracil, and 5,6-dihydrothymine (37). Recently, however, a D-hydantoinaseprotein that does not exhibit dihydropyrimidine amidohydro-lase activity has been purified from an Agrobacterium sp. (30).Dihydropyrimidinase functions in the reductive pyrimidinesalvage pathway in bacteria (6, 37) and in pyrimidine biosyn-thesis in plants (16).

D-Hydantoinase activity has been detected in plants, ani-mals, and bacteria. However, hydantoinases for industrialapplications are obtained mainly from microorganisms.Screening of hydantoinase-producing microorganisms with hy-dantoin derivatives requires quantifying one of two end prod-ucts, the N-carbamyl-amino acid (20, 21) or the correspondingamino acid. If the microorganism produces N-carbamyl-amidohydrolase, then the N-carbamyl-amino acid might not bedetected, even by high-performance liquid chromatography(HPLC) (35). Polyclonal antibodies have been used to detect

* Corresponding author. Phone: (514) 773-1105. Fax: (514) 773-8461.

the L-hydantoinase enzyme (33, 34). The antibodies did notrecognize the D-hydantoinase of the Agrobacterium sp.The purpose of our work was to detect potential new sources

of D-hydantoinases. The D-hydantoinase gene from Pseudomo-nasputida DSM 84 was cloned and sequenced. Its homology toother known hydantoinases and cyclic amidases is described.The gene was used as a DNA probe to detect other potentialsources of D-hydantoinase.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions. Hydan-toinase-producing P. putida DSM 84 was obtained from theGerman Collection of Microorganisms (Deutsche Sammlungvon Mikroorganismen und Zellkulturen, Braunschweig, Ger-many). Apart from Escherichia coli DH5a and vector pT7/T3a18 (GIBCO-BRL, Burlington, Ontario, Canada) and E.coli JM109 and vector pGEM-7Zf(+) (Promega Corp., Mad-ison, Wis.), other bacterial strains are listed in Table 1. Allbacteria were grown in brain heart infusion broth (DifcoLaboratories, Detroit, Mich.) or in 2 x TY medium containing(per liter) 16 g of tryptone, 10 g of yeast extract, and 5 g ofNaCl. Media were supplemented with 0.2% hydantoin (SigmaChemical Co., St. Louis, Mo.), 0.2% thymine, or 0.2% 5,6-dihydrouracil and solidified when appropriate with 1.5% Bactoagar (Difco Laboratories). After sterilization, ampicillin (100,ug/ml) (Sigma Chemical Co.) and X-Gal (5-bromo-4-chloro-3-indolyl-fi-D-galactopyranoside; 50 jig/ml) (GIBCO-BRL)were added when necessary. The minimal medium was high-cell-density cultivation medium (28). Incubation was at 27 or37°C for 18 to 72 h with agitation at 150 rpm.Assays for D-hydantoinase activity. Bacteria were tested for

conversion of hydantoins or 5,6-dihydrouracil to N-carbamyl-amino acids as follows. Single colonies were subcultured onbrain heart infusion solid medium supplemented with 0.2%hydantoin and incubated at 37°C for 18 to 24 h and thensuspended in 100 Rl of 100 mM carbonate-bicarbonate buffer,pH 9.5, supplemented with 50 mM hydantoin or dihydrouracil

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D-HYDANTOINASE GENE SCREENING 889

A

R- O

HNyNH0

D-hydantoinase

'H0

R,iCOOH

HN

I-12N

N-carbamyl-amidohydrolase

H20

ORR,rCOOH

H2N

5'-monosubstitutedhydantoin

N-carbamyl-D-amino acid

B

>-NH 0

H2N )-(

aNt NH

allantoin

$Y0

FOOCrOFiN N

0

5,6-dihydro-orotate 5,6-dihydrouracil

FIG. 1. Scheme of hydrolysis of 5-monosubstituted hydantoins to D-amino acids (A) and the chemical structures of the substrates of some otherenzymes of EC group 3.5.2 (B).

(Sigma Chemical Co.) and incubated at 37°C for 1 h. Cellswere removed by centrifugation at 14,000 x g for 10 min. Tenmicroliters of filtered (0.22-jim syringe filter units; Chromato-graphic Specialities, Inc., Brockville, Ontario, Canada) super-natant was injected on an HPLC NH2 column (Degussa A.G.,Hanau, Germany) and eluted with acetonitrile-KH2PO4 (0.05M):H3PO4 (0.1 M), 650:250:100 (vol/vol/vol), at a flow rate of1 ml min- . The HPLC system included an automatic sampler(model AS-48; Bio-Rad, Mississauga, Ontario, Canada), apump (Bio-Rad model 1350T) and a UV monitor (Bio-Radmodel 1305A) set at 205 nm. The N-carbamyl-amino acidproduct formed was also quantified colorimetrically. Afterincubation, 175 ,lI of 12% (wt/wt) trichloroacetic acid and 12.5,ul of 10% (wt/wt) para-dimethylaminobenzaldehyde in 6 NHCl were added to 100 ,lI of cell suspension. A yellow productindicated a positive reaction. The reaction mixture was thencentrifuged at 14,000 x g for 10 min and the optical density at450 nm of the supernatant was measured by spectrophotome-ter (Lambda Reader automated microplate reader; Perkin-Elmer, Montreal, Quebec, Canada), and the resulting valueswere compared to a standard curve of the expected N-carbamyl-amino acid. One unit of hydantoinase produces 1,Imol of N-carbamyl-amino acid in 1 min. Hydrolysis ofhydantoin produces N-carbamyl-glycine while the hydrolysis of5,6-dihydrouracil produces N-carbamyl-,B-alanine.DNA extraction. Bacterial total DNA was extracted as

described by Lewington et al. (15) or by the guanidiumthiocyanate method of Pitcher et al. (26). Plant tissues wereground as previously described (19). Total DNA from groundtissue of the green field pea, Pisum sativum (whole peas or peahulls) and feed culls from Phaseolus vulgaris was isolatedaccording to the CTAB method (23). Plasmid extraction wasdone as described by Sambrook et al. (31). The rapid proce-dure of Kado and Liu (10) was used to isolate native bacterialplasmids.DNA manipulations. Restriction endonuclease digestions

were done according to the manufacturer's recommendations.Ligations and agarose gel electrophoresis techniques wereconducted by the methods of Sambrook et al. (31). E. coli cells

were transformed with plasmid DNA by electroporation usinga Gene Pulser system (Bio-Rad). Native bacterial plasmidswere detected by the protocol described by Eckhardt (7), asmodified by Wheatcroft et al. (42).DNA hybridization. Probe DNA was prepared with a

1,521-bp MluI-PvuII fragment obtained from recombinantplasmid pGEC51 (see Results for details). The DNA waslabeled with digoxigenin according to the procedure for thenonradioactive DNA labeling and detection kit (BoehringerMannheim, Laval, Quebec, Canada). The labeled probe washybridized to DNA blots produced either with a slot blotapparatus (Millipore MilliBlot-S system; Montreal, Quebec,Canada) or by Southern transfer using the protocols describedby Sambrook et al. (31). Hybridization conditions tolerated a39% mismatch. Nonstringent wash conditions were at 37°C in1.0 x SSPE (1 x SSPE is 0.18 M NaCl, 10 mM NaPO4, and 1mM EDTA [pH 7.7]). Stringent wash conditions were at 55°Cin 0.1 x SSPE, tolerating a 21% mismatch.DNA sequencing. Unidirectional deletions of the cloned

DNA were generated by using exonuclease III (Pharmacia,Laval, Quebec, Canada). E. coli was transformed with theresulting recombinants. Template DNA for sequencing waspurified by using the Magic Miniprep kit (Promega Corp.) anddenatured by alkali when necessary. Nucleotide sequences ofprogressive deletions in both directions were determined bythe dideoxy chain-termination method of Sanger et al. (32) byusing the T7 sequencing kit (Pharmacia). Both strands weresequenced. Computer analysis was conducted, using the DNA-SIS and PROSIS programs (Hitachi Software Engineering Co.Ltd., San Bruno, Calif.). Comparisons with protein and nucle-otide sequence data bases were performed with the basic localalignment search algorithms (1) of the Blast Network service atthe National Center for Biotechnology Information, Bethesda,Md. (BLASTN, BLASTP, and BLASTX programs). Align-ment of protein sequences was performed with the CLUSTALV program (8), using the percentage scoring method and thedefault parameters (k-tuple = 1; gap penalty = 3; and windowsize = 5).

Nucleotide sequence accession number. The nucleotide se-

HNO2, HCI D-amino acid

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890 LAPOINTE ET AL.

TABLE 1. Bacterial strains used, D-hydantoinase activity, and hybridization with the molecular probea

Family or genus and species rRNA Strain (reference or Activity Hybridizationgroup source)"Pseudomonadaceae

P. aeruginosa I PA01162 (M. Sylvestre) + +P. alcaligenes I ATCC 14909 + +P. aureofaciens I 211 (2) + +P. cepacia II ATCC 25416 +P. chlororaphis I ATCC 17414 + +P. diminuta IV ATCC 11568P. fluorescens I ATCC 948 + +P. fragi I CRDA 037 + +P. mendocina I ATCC 25411P. paucimobilis V ATCC 29837P. pickettii II ATCC 27511P. pseudoalcaligenes I ATCC 17440 + +P. putida I DSM 84 + +P. putida I ATCC 795 + +P. putida I CRDA 324 + +P. putida I CRDA 325 + +P. stutzeri I ATCC 17588P. vesicularis IV ATCC 11426 - -Comamonas acidovorans III ATCC 15668 +Comamonas testosteroni III ATCC 11996Xanthomonas maltophilia V ATCC 13637

AgrobacteriumA. radiobacter K84 (11)A. rhizogenes A4 (18)A. tumefaciens LM136 (14) +A. tumefaciens LM58 (14) +A. tumefaciens ATCC 15955

Other speciesCorynebacterium pseudodiphteriticum CRDA 317 +Serratia liquefaciens CRDA 327 +E. coli DHSaE. coli JM109a The D-hydantoinase gene from P. putida DSM 84 was used as the probe. +, characteristic present; -, characteristic absent.bATCC, American Type Culture Collection, Rockville, Md.; CRDA, Food Research and Development Centre, Agriculture Canada, St. Hyacinthe, Quebec, Canada;

DSM, Deutsche Sammlung von Mikroorganismen und Zellkulturen; M. Sylvestre, Institut National de la Recherche Scientifique, Pointe Claire, Quebec, Canada.

quence data reported in this paper have been submitted toGenBank and assigned the accession number L24157.

RESULTS

Chromosomal location of the D-hydantoinase gene of DSM84. No plasmids were revealed after electrophoresis andstaining of the Eckhardt type agarose gels. In addition, plasmidextractions by the method of Kado and Liu (10) performed onDSM 84 cultures never revealed any plasmid DNA. Therefore,the D-hydantoinase gene was assumed to be located on thechromosome of DSM 84.

Cloning of the gene coding for D-hydantoinase. High-molec-ular-weight total DNA extracted from P. putida DSM 84 waspartially digested with either EcoRI, HindIII, PstI, or BamHIand ligated to pT7/T3ao18 previously digested with the corre-sponding enzyme. Three thousand E. coli clones were assayedfor hydantoinase activity by using dihydrouracil and hydantoinas test substrates. One clone carrying 27.5 kb of EcoRI-digested DNA inserted into pT7/T3cxl8 showed hydantoinaseactivity with the chromogenic assay. This clone was designatedpT22. Complete removal of the substrates was confirmed byHPLC. By subcloning of the pT22 insert, hydantoinase activitywas found associated with a 3.8-kb EcoRI-HindIlI fragment,and this subclone inserted in pT7/T3al8 was designated

pT22H2. Detailed restriction analysis was performed onpT22H2 (Fig. 2).

Effect of potential inducer and substrate on the hydantoin-ase activity coded by pT22H2. D-Hydantoinase activity of clonepT22H2 in E. coli was assayed after the cultivation of bacteriain minimal media with and without a potential-inducing sub-stance, either hydantoin or thymine. The same experimentswere carried out on the parent strain, P. putida DSM 84.Hydantoin and thymine both induced enzyme activity in P.putida DSM 84, although thymine induced higher enzymeactivity (Table 2). In contrast, expression of the hydantoinasein E. coli carrying pT22H2 was constitutive. Potential inducershad no effect on the activity of the clone. Average (± standarddeviation) induced activity of DSM 84 on hydantoin (1.69 +0.81 U/ml) was not significantly different from that of the clone(1.74 ± 0.53 U/ml; t = 0.15, P > 0.05, where t is Student's tstatistic). With dihydrouracil as the substrate, the averageinduced activity of DSM 84 (4.95 ± 0.66) did not differ fromthat of the clone (5.17 ± 0.23 U/ml; t = 0.99, P > 0.05). E. coliDH5cx harboring either of the two cloning vectors did notexpress any dihydropyrimidinase or hydantoinase activity.

Subcloning and construction of deletion derivatives ofpT22H2. The D-hydantoinase activity of pT22H2 was associ-ated with the 1.8-kb EcoRI-SmaI fragment subcloned inpT7/T3a18 or in pGEM-7Zf(+) to generate pGES19 (Fig. 2).

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D-HYDANTOINASE GENE SCREENING 891

I Ii ~~~~~~ HydantoinasO- _ z~~~ C-I CL E0co (A C0O 0(c v Clone activity-" L pT22H2 +

ORFI~ pTSPHS +

pTSMA4 +

pTPSr3- pGES19 +

_ pGSB24 +

- pGEC51 +

* pT73calf8 -

m pGEL-7Zf(+) -

I kb

FIG. 2. Restriction map of clone pT22H2 and selected deletionderivatives. Vector DNA (black boxes), insert DNA (open boxes), anddeleted segments (lines) are indicated. Restriction endonucleases usedin mapping and to construct the deletion derivatives are shown at thetop. The presence (+) or absence (-) of hydantoinase activity isindicated.

The inserted 1.8-kb fragment of pGES19 was digested withexonuclease III from either the SmaI or the EcoRI end andthen religated. The shortest construct that retained hydanto-inase activity after digestion from the SmaI extremity wasdesignated pGSB24. The shortest subclone of pGSB24 thatretained hydantoinase activity after digestion from the EcoRIextremity was designated pGEC51 (Fig. 2). Hydantoinaseactivity was abolished by removing either 268 bp from theEcoRI extremity of pGES19 or 156 bp from the SmaI extrem-ity.

Sequence analysis of the D-hydantoinase gene from P. putidaDSM 84. The nucleotide sequence of the 1.8-kb insert of theclone pGES19 was determined (Fig. 3). The overall G+Ccontent of the insert was 61.4%, which is about average for P.putida (5) and not significantly different from the value of63.47% ± 0.70% measured for the entire native DNA of DSM84 by Morin et al. (22). Analysis of the sequence revealed oneopen reading frame (ORF) of 1,104 bp. The G+C content ofthe ORF was 63.2%, while that of the upstream region wasonly 44.7% and that of the 3' region was 66.2%. The ORF waspreceded by a possible ribosome-binding site (GAGG) andwas predicted to encode a peptide of 368 amino acids with acalculated molecular mass of 40.5 kDa. Upstream of the ORFat position 112, a possible promoter sequence was found with-10 and - 35 motifs for some Pseudomonas promoters. The

TABLE 2. Effects of inducer and substrate on the D-hydantoinaseactivity of P. putida DSM 84 and E. coli harboring clone pT22H2

Potential Enzyme activity' (U/ml)Bacterial straininueBelrInducer Hydantoin Dihydrouracil

P. putida DSM 84 None 0.13 ± 0.01 0.02 ± 0.02Hydantoin 0.89 ± 0.06 4.29 ± 0.01Thymine 2.50 ± 0.02 5.61 ± 0.04

E. coli(pT22H2) None 1.91 ± 0.32 4.72 ± 0.24Hydantoin 2.01 ± 0.65 5.31 ± 0.23Thymine 1.46 ± 0.03 5.02 ± 0.12

aThe values of enzyme activity are presented in units per milliliter of cellsuspension and represent the averages of two experiments, with the standarderrors.

- 35 region has the consensus sequence for promoters recog-nized by rpoD-type sigma factors (YSTTGR) (29). The -10region is identical to that of the algQ promoter recognized bythe same sigma factor (TCTAG) (29). Downstream of theORF (352 bp), a palindromic sequence (positions 1701 to1717) was found which predicted a GC-rich stem-loop mRNAstructure with a 10-bp stem, not followed by any U residues,and believed to function in transcription termination. Analysisof the secondary structure of the putative D-hydantoinaseprotein revealed that the average hydrophobicity was -0.41,generated by using the algorithm of Kyte and Doolittle (13).The isoelectric point of the putative protein was predicted tobe 7.04.DNA hybridization with the cloned D-hydantoinase gene.

The 1.5-kb insert of recombinant pGEC51 (Fig. 2) was chosenas the DNA probe. This fragment hybridized with total DNAfrom Pseudomonas strains of rRNA group I that demonstratedD-hydantoinase activity (Table 1; Fig. 4). The same results werefound when we used either stringent or nonstringent washconditions, tolerating, respectively, 21 or 39% mismatch inbase pairing. The probe did not hybridize to DNA from rRNAgroup I pseudomonads that did not demonstrate hydantoinaseactivity, nor did it hybridize to DNA from strains belonging toother rRNA groups, whether or not those strains demon-strated hydantoinase activity. There was no hybridization ofthe cloned hydantoinase gene with DNA from any bacterialstrain outside members of the family Pseudomonadaceae orfrom plant material with D-hydantoinase activity (from theseeds of three different legumes [19]).

DISCUSSION

This report describes the development of a DNA probe ableto detect some D-hydantoinase or dihydropyrimidinase genes.Our study is the first wherein the putative amino acid se-quences of D-hydantoinases are compared with those of anonspecific D,L-5-substituted hydantoinase (38), other D-hy-dantoinases, and other amidohydrolases of EC group 3.5.2.

P. putida DSM 84 belongs to the rRNA homology group I ofpseudomonads (24, 25). Within this group, not all strainspossess D-hydantoinase activity. The strains that were notinduced by hydantoin were also not induced by dihydrothy-mine, which has been shown to induce dihydropyrimidinaseactivity in Pseudomonas stutzeri (43), Pseudomonas pseudoal-caligenes (40), and Pseudomonas diminuta (41). Dihydrothy-mine did not induce higher levels of dihydropyrimidinaseactivity in Pseudomonas aeruginosa (12). In each of the casescited, a low constitutive level of dihydropyrimidinase activitywas detected in the absence of inducing substances. D-Hydan-toinase activity induced by hydantoin was also detected outsidethe pseudomonad group, in bacteria belonging to the generaAgrobacterium, Corynebacterium, and Serratia.

The genes coding for production of enzymes responsible forthe transformation of D- and L-5-substituted hydantoins intothe corresponding N-carbamyl-D- and L-amino acids have beencloned from a plasmid of 172 kb harbored by a Pseudomonasstrain (38). The plasmid also harbors the gene involved in theconversion of N-carbamyl-L-amino acid to its correspondingL-amino acid, as well as the gene coding for the racemization ofhydantoins. Sequence analysis of the 7.5-kb insert of therecombinant plasmid revealed five ORFs. Two ORFs, desig-nated hyuA and hyuB, were essential for the conversion of D-and L-5-substituted hydantoins to N-carbamyl-amino acids.When the entire sequence obtained by Watabe et al. (38, 39)

was compared with that obtained in our study, no significantnucleotide or amino acid homology was revealed. Therefore,

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892 LAPOINTE ET AL.

EcoRI1 CATCTTGTCAATAGGAAAATTTTCTTGAAGATTCCCAAATCAGCATCTAGCCTCGAATCTTGTCAGGTCTGACAGGATTAACCGCCCTGCCTGCCCTACACCCGAGACAAT

ORF1N S L L I R G A T V V T H E E S Y P A

TCCAAGAACCGGCCCAGACCGGTCAGCCTGCGAGGAAAAACGGCATGTCCCTGTTGATCCGTGGCGCCACCGTGGTCACCCACGAAGAGAGTTACCCCGC

D V L C V D G L I R A I G P N L E P P T D C E I L D G S G Q Y L N301 CGATGTCCTGTGTGTCGATGGCCTGATCCGTGCCATCGGGCCAAACCTCGAACCGCCCACCGACTGTGAAATCCTCGACGGCAGCGGCCAGTACCTGATG

P G G I D P H T H M Q L P F M G T V A S E D F F S G T A A G L A G G401 CCCGGCGGCATCGACCCGCATACCCACATGCAGTTGCCATTCATGGGCACCGTGGCCAGCGAGGATTTCTTCAGCGGCACCGCAGCGGGCCTTGCCGGCG

T T S I I D F V I P N P Q Q S L L E A F H T W R G W A Q K S A S D501 GCACCACGTCGATCATCGACTTCGTCATCCCCAACCCGCAGCAGTCATTGCTGGAGGCCTTCCACACCTGGCGCGGCTGGGCGCAGAAGAGCGCCAGCGA

Y G F H V A I T W W S E Q V A E E M G E L V A K H W G E Q L Q A L601 CTACGGCTTCCACGTTGCCATCACCTGGTGGAGCGAACAGGTGGCTGAAGAAATGGGCGAACTGGTAGCCAAGCATTGGGGTGAACAGCTTCAAGCACTT

H G L Q E C N H G R R R H P G G Q L R A L P A T G C R A H R A C P R701 CATGGCTTACAAGAATGCAATCATGGCCGCCGACGACACCCTGGTGGCCAGCTTCGAGCGCTGCCTGCAACTGGGTGCCGTGCCCACCGTGCATGCCCGA

T A N W C T T C R K N C L P R G M T G P E A H P L S R P S Q V E G801 GAACGGCGAACTGGTGTACCACCTGCAGAAAAAACTGCTTGCCCAGGGGCATGACCGGACCAGAGGCTCACCCCCTTTCGCGCCCTTCACAAGTGGAAGG

E A A S R A I R I A E T I G T P L Y V V H I S S R E A L D E I T Y901 TGAAGCGGCCAGCCGCGCCATCCGTATTGCCGAAACCATTGGTACGCCGCTGTATGTGGTGCCATTTCCAGCCGTGAAGCACTGGATGAAATCACCTAT

A R A K G Q P V Y G E V L P G H L L L D D S V Y R D P D W A T A A G1001 GCACGCGCCAAGGGCCAGCCGGTTTACGGCGAAGTCTTGCCCGGCCACCTGCTGCTGGACGACAGCGTCTACCGTGACCCGGACTGGGCCACTGCCGCTG

Y V M S P P F R P R E H Q E A L W R R L Q S G Q P A H H G H R P L1101 GCTACGTGATGAGCCCGCCGTTCCGCCCGCGCGAGCACCAGGAGGCGCTGTGGCGCCGCTTGCAGTCGGGCCAACCTGCACACCACGGCCACCGACCACT

L F L R R T E S H G P R R L Q S H P Q Q A P P A S K T A W R C C G1201 GCTGTTTCTGCGCCGAACAGAAAGCCATGGGCCGCGACGACTTCAGTCGCATCCCCAACAGGCACCGCCGGCATCGAAGACCGCATGGCGGTGCTGTGGG

M P V S T A G D C R C M S S L R *1301 ATGCCGGTGTCAACAGCGGGTGACTGTCGATGCATGAGTTCGTTGCGCTGACCTCCACCAACACGGCAAAAATCTTCAACCTTTTCCCACGCAAGGGCGC1401 CATCCGCGTGGGTTGCCGACGCCGACCTGGTGCTGTGGGACCCGCAGGGCACTCGCACTCTATCGGCCCAGACCCACCACCAGCGGGTGGACTTCAATAT1501 CTTTGAAGGCCGCACTGTGCGCGGGGTCACCCCAGCCACACCATCAGCCAGGGCAAGGTGCTCTGGGCCGATGGCGACCTGCGCGCCGAGGCCGGGGCGG1601 GGCGGTATGTGGAACGGCCGGCGTATCCGTCGGTGTACGAGGTGCTGGGGCGACGCGCCGAACAGCAGCGCCCGACGCCCGTTCAGCGCTGAGGCCATTG1701 GGGCTGCTGCGCAGCCCATCGCCGGCAAGCCAAATATAATAAAGAGAGAGGCTACAACCGTGATCGACGCCCTGAACCACTTGCCGCGCCCC

SmaI

FIG. 3. Nucleotide sequence of the insert DNA of pGES19 and deduced amino acid sequence of the ORF. The putative sequence of thetranscription terminator is underlined. Sequences similar to the - 10 and -35 sequences for Pseudomonas promoters are double underlined.

the probe consisting of the D-hydantoinase gene would not

detect sequences coding for nonspecific D,L-5-substituted hy-dantoinases. According to Watabe et al. (38), the amino acidsequences of hyuA and hyuB did not show homology to any

1213 1415 16

23.1

9.4

6.6

4.4

2.32.0

FIG. 4. Southern blot analysis of EcoRI-digested chromosomalDNA, using a probe consisting of the 1.5-kb insert of recombinantplasmid pGEC51, which contains the ORF of the D-hydantoinase fromP. putida DSM 84. Lanes: 1, Pseudomonasfragi; 2, P. putida 325; 3, P.putida 324; 4, Pseudomonas aureofaciens 211; 5, P. putida ATCC 795;6 and 16, Pseudomonas fluorescens; 7 and 15, Pseudomonas chlorora-phis; 8 and 14, P. pseudoalcaligenes; 9 and 13, P. putida DSM 84; 10, E.coli DH5ao; 11 and 12, HindIII-digested lambda DNA. Numbers in thecenter indicate the size in kilobases of lambda DNA fragments. Theorganisms in lanes 1 through 9 and 13 through 16 showed hybridiza-tion. Nonstringent wash conditions were used.

known sequences in the National Biomedical Research Foun-dation and SWISS protein data bases. However, the proteinsequence of hyuB did show local similarity (33 identical aminoacids of 105) to an unidentified ORF located 3' to the uralgene of Saccharomyces cerevisiae. Dihydroorotate dehydroge-nase is the product of ural (GenBank accession numberX59371).

Runser and Meyer (30) purified a D-hydantoinase from aAgrobacterium sp. and have sequenced the first 22 amino acids.Only 3 of the 22 amino acids sequenced from the D-hydantoi-nase of the Agrobacterium sp. (30) were identical to theN-terminal region of the protein sequence from our study (P.putida DSM 84). Further elucidation of the evolutionaryrelationship with the D-hydantoinase from the Agrobacteriumsp. will be possible once the gene is cloned and sequenced.

Jacob et al. (9) first reported the isolation of D-hydantoinasegenes from two thermophiles, one strain gram negative(CBS30380) and the other gram positive (Lu1220). Neitherstrain was identified further to genus or species level. Compar-ison of the nucleotide sequences did not reveal significantidentity. However, comparison of the first 19 amino acids ofthe putative proteins revealed a 53% identity.When the two D-hydantoinase nucleotide sequences of

Jacob et al. (9) were compared with that from P. putida DSM84, overall identity was less than 30%. There are several stopcodons within the putative amino acid sequence of the clonefrom one strain (CBS30380 [9]), which indicates a possiblesequencing error. Therefore, for comparisons with CBS30380,we utilized only the uninterrupted N-terminal half of thesequence from this strain.

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D-HYDANTOINASE GENE SCREENING 893

SEGMENT 1

DSM 84LU1220CBS30380DAL1DHO-BAC

1 MSL-LIRGATVVTHNZSYPA--DVLCVDGLIRAIGPNLEPPTDCEI--------LDGSGQY 501 MTK-IIQNGTIVTATDTYKA--DLLIXDGKIAMIGQHLEZXA-EV--------VIDAKGCY 491 MPL-LIXNGEIITADSRYKA--DIXAZGETITRIGQNLEAPPGTZV------ I-ATGKY 491 NPINAITSDHVIINQANKPATIVYSTESGTILDVLEGSVVIKTEITRYEIHTLENVSPCT 611 MSY-LIKNGWILNENQZKTQ-ADIRVTGETITAIGQLDAT---------DNETVIDAKGLL 50

SEGMENT 2

DSM 84 51 LMPGGIDPHTHXQLPFMGTVASEDFFSGTAAGLAGGTTSIIDFVIPNPQQSLLEAFHT ----WRGWAQ--- KSASDYGFHVAI 126LU1220 50 VFPGGIDSETHLDKPFQGTVTKDDFESGTIAAAFOGTTTIIDFCLTNKGZPLKXAIET----WmNKAK--GKAVIDYGFHLXI 126CBS30380 50 VFPGFIDPHVHIYLPFMATFAKDT!ETGSKAALMGGTTTYIE-VLPQPQRRALEGYQL ---- WKSKPR--ATATADYTFIDAV 125DAL1 62 ILPGLVDSHVHLNEP--GRTSWErFETGTQAAISGGVTTVVDKPLNAIPPTTNVKNF-----RIKLEAAEZQQWCDVGFWQGL 137DHO-BAC 51 VSPGFVDLWHVFREP--GGEKRETINTGAKAAGRGGYTTVAAXP-NTRPVPDTREQMEWVQNRIK-ETSCVRVLPYASITIRQ 129DHO-CAD 61 RLPGLIDVHVHLREP--GGT!gEDFASGTAAALAGGVTNVCAKP-NTRPPIIDAPAL-----ALAQKLAEAARCDFALFLGA 135DRO-PYR 260 RLPGLVDVHVHLRZP--GATHKEDWDSGTATALAGGFTMVGAMP-NTNPAINDDASF-----ELCKSLAASKARCDYGIFIGA 334

SEGMENT 3

DSM 84 199 LPRGQTGP3AHPLSRPSQVEGZAASRAIRIAETIGTPL 236.LU1220 198 LAEGNTEPIYHALTRPPZVEGEATGRACQLTELAGSQL 235

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

DSM 84 237 YVVHISSREALDEITYARAIKGQPVYGEVLPGHLLLDDSVYRDPDWATAAGYVMSPPFRPREHQEALWRRLQSGQ 310LU1220 236 YVVEVTCAQAVEKIAQARNKGLDVWGETCPQYLLLDQSYLZRPDFE-GAKYVWSPPLRRKWHQEVLWNALRNGQ 308DAL1 247 HIVHILASIKAIPLIRKARASGLPVTTETCFHYLCIAAEQIPD----GATYFKCCPPIRSZSNRQGLWDALRRGV 316DHO-BAC 227 HVCHISTKESVRVVRDAIQAGIRVTAEVSPRRLLLCDEDIPG----LDTNY7O4PPLRSPEDRAALI1ELLDGT 296DHO-CAD 209 HICHVARKEEILLIXTAKAQGLPVTCNVAPIHLFLNREDLERLGPGRGEVR--- PELGSREDNEALWENMAVID 279DHO-PYR 409 HVCHVSHKEEIDIIRDAKRGI.:LSCNVSPJHLTLCDRDIPRIGAGQSEVR--- PKWGTEEDLNALWDNIDYID 479

* * * * * * *

FIG. 5. Optimal alignment by the CLUSTAL V program of high-scoring segments from the predicted amino acid sequences of variousamidohydrolases. DALI, allantoinase from S. cerevisiae (GenBank accession number M69294); DHO, DHO protein coded by the gene pyrC fromB. subtilis (GenBank accession number M59757); DHO-CAD, DHO domain from the multifunctional CAD protein of the hamster, Cricetulus sp.(GenBank accession number M33702); DHO-PYR, DHO domain of the multifunctional protein Pyrl-3b involved in pyrimidine biosynthesis in theslime mold Dictyosteliunm discoideuli (GenBank accession number X14634). Identical amino acids are indicated with an asterisk.

Alignment of the putative amino acid sequences of the threeD-hydantoinase proteins revealed three individual high-scoringsegments (Fig. 5). These three regions are the first 50 aminoacids (segment 1), a consecutive region of 76 amino acids in theN-terminal half (segment 2), and, finally, a 74-amino-acidregion in the carboxy half of the proteins (segment 3). Al-though segments 1 and 2 are consecutive, they have been keptseparate because segment 1 was generally not a high-scoringsegment in comparisons with other enzymes. The region ofhighest identity between the two sequences of Jacob et al. (9)is indeed the N-terminal segment 1 (Table 3). However, theputative amino acid sequence of the D-hydantoinase fromDSM 84 shows greatest identity to the sequences of Jacob et al.(9) in segment 2 (Table 3). Identity between the sequences ofDSM 84 and the gram-positive strain Lu1220 in the thirdsegment extended a further 38 amino acids (47% identicalresidues out of 38 [Fig. 5]).

Both our nucleotide sequence and our putative amino acidsequence were compared with the data available throughGenBank, EMBL, Swiss-Prot, and the National BiomedicalResearch Foundation-Pir. Two high-scoring segments repre-senting regions of identity were revealed with the amino acidsequences of two other amidohydrolases (allantoinase anddihydroorotase [Fig. 5; Table 3]). The three D-hydantoinasesequences (for P. putida DSM 84, Lu1220, and CBS30380)showed identity with the sequence of the allantoin amidohy-drolase (Dall) from S. cerevisiae (GenBank accession numberM69294) in segments 2 and 3. All three D-hydantoinasesequences showed identity with the dihydroorotate amidohy-drolase, or dihydroorotase (DHO), family of proteins in seg-ment 2 (Table 3; Fig. 5). The D-hydantoinase sequences fromLu1220 and CBS30380 showed additional identity with thebacterial DHO (DHO-Bacillus subtilis) in segment 1.

The evolutionary relationships among the enzymes of pyri-midine metabolism have been described previously (4). Inparticular, the DHO proteins have all diverged from a com-mon ancestor. The DHO of mammals and of the slime moldDictyostelium discoideum is part of a multifunctional proteininvolved in de novo pyrimidine biosynthesis. In bacteria andyeast cells, the DHO is coded by a separate gene. Theeukaryotic sequences (DHO-CAD and DHO-PYR) retain ahigh degree of identity but have diverged significantly from thebacterial DHO. For example, the E. coli DHO is only 19%identical with the hamster DHO (4). The sequence of theallantoinase enzyme (Dall) shows slightly more identity withthe various DHO sequences than the bacterial DHO (DHO-B.subtilis) shows to the eukaryotic counterpart (DHO-CAD[Table 3]). The highest identity of the allantoinase to thesequence of DHO enzymes is found in segment 2, the sameregion described by Buckholz and Cooper (3). These authorsproposed that region 2 could represent the substrate recogni-tion site, given the similarity in the substrates of these enzymes(Fig. 1).Among the D-hydantoinases for which the full sequence is

known, segment 2 is also the highest-scoring identity segmentbetween DSM 84 and each of the thermophile strains. How-ever, the sequences from Jacob et al. (9) are most similar insegment 1. Although the D-hydantoinases generally show themost identity with the DHOs in segment 2, they are still morehomologous among themselves than with this group of en-zymes or with the allantoinase. Hydantoin is also a substratesimilar to allantoin, differing only in the 5' substitution (Fig. 1).The dihydropyrimidines also resemble DHO. These compari-son data thus support the hypothesis of Buckholz and Cooper(3) that the sequence within region 2 could represent thesubstrate recognition site.

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894 LAPOINTE ET AL.

TABLE 3. Comparison of high-scoring segments between selected pairs of predicted protein sequences

% of identical amino acidsbPairwise comparison"

Segment 1 Segment 2 Segment 3

DSM 84 vs Lu1220 37 53 48DSM 84 vs CBS30380 37 43 NACLu1220 vs CBS30380 49 41 NA

DSM 84 vs DAL1 NA 26 31Lu1220 vs DALi NA 34 32CBS30380 vs DALi NA 26 NA

DSM 84 vs DHO-B. subtilis NA 21 36DSM 84 vs DHO-CAD NA 33 30DSM 84 vs DHO-PYR NA 33 28

Lu1220 vs DHO-B. subtilis 34 25 26Lu1220 vs DHO-CAD NA 36 27Lu1220 vs DHO-PYR NA 33 20

CBS30380 vs DHO-B. subtilis 44 29 NACBS30380 vs DHO-CAD NA 26 NACBS30380 vs DHO-PYR NA 25 NA

DALI vs DHO-B. subtilis NA 41 33DALi vs DHO-CAD NA 45 33DALI vs DHO-PYR NA 43 25

DHO-B. subtilis vs DHO-CAD NA 37 40DHO-B. subtilis vs DHO-PYR NA 35 48DHO-CAD vs DHO-PYR NA 67 54

" DAL1, allantoinase from S. cerevisiae. DHO-CAD, DHO domain of the CAD protein from the hamster (Cricetulus sp.). DHO-PYR, DHO domain ofmultifunctional enzyme Pyrl-3b involved in pyrimidine biosynthesis from the slime mold Dictyostelium discoideum.

b Values reported are the percentages of identical amino acids within each segment (see Fig. 4 for residue numbers).'NA, not applicable.

The probe constructed in this study could be used to rapidlyscreen bacteria from environmental samples for those possess-ing a functional or potential D-hydantoinase gene related tothe pseudomonads of rRNA homology group I. The lack ofhybridization of our probe with DNA from bacteria that areoutside the pseudomonad group but that also demonstratehydantoinase activity is an indication of the divergence ofgenes coding for proteins with this type of activity.

ACKNOWLEDGMENTSWe thank Khalil Mehindate and Tran Trung Nguyet Hong for their

technical assistance. We are grateful to Michel Sylvestre (InstitutNational de la Recherche Scientifique-Santd, Pointe Claire, Quebec,Canada) for advice during the course of this work and during thepreparation of the manuscript. We thank Carmelle Perron for herparticipation in text editing.

G. LaPointe gratefully acknowledges the receipt of a visiting fellow-ship from NSERC.

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