Diversity of 2,3-dihydroxybiphenyl dioxygenase genes in a strong PCB degrader, Rhodococcus sp. strain RHA1

JOURNAL OF BIOSCIENCE AND BIOENGINEERING

Vol. 93, No. 4, 421~427.2002

Diversity of 2,3-Dihydroxybiphenyl Dioxygenase Genes in a Strong PCB Degrader, Rhodococcus sp. Strain RHA 1 M A S A Y U K I SAKAI, 1 EIJI MASAI, 1 H I R O K I ASAMI, 1 K A T S U M I S U G I Y A M A , 1 K A Z U H I D E K I M B A R A , 2 AND M A S A O F U K U D A 1.

Department of Bioengineering, Nagaoka Universityof Technologv, Kamitomioka, Nagaoka, Niigata 940-2188, JapanI and Environmental Biotechnology Laboratory, Rail-way TechnicalReseareh Institute, Kokubunji, Tokyo 185-8540, Japan2 Received 18 October2001/Accepted8 February2002 Two 2,3-dihydroxybiphenyl (23DHBP) dioxygenase genes, bphC1 and etbC involved in the degradation of polychlorinated biphenyi(s) (PCBs) have been isolated and characterized from a strong PCB degrader, Rhodococcus sp. RHA1. In this study, fonr new 23DHBP dioxygenase genes, designated as bphC2, bphC3, bphC4, and bphC5 were isolated from RHA1, and their nucleotide sequences were determined. Based on amino acid seqnence similarities, all of the newly isolated bphC genes could be categorized into type I along with BphC1 and EtbC [Eltis, L. D. and Bolin, J. T., J. Bacteriol., 178, 5930-5937 (1996)]. Six bphC genes, incinding bphC1, etbC, and four new genes, were expressed in Escherichia coli to determine their substrate specificity. The activifies of BphC2, BphC3, BphC4, and BphC5 were found to be specific to 23DHBP, while BphC1 and EtbC exhibited activities towards compounds other than 23DHBP, including catechol (CAT) and 3-methylcatechol (3MC). RNA siot blot hybridization analysis indicated that only bphC5 was transcribed among the newly isolated bphC in RHA1 cells grown on biphenyl and ethylbenzene. The nncleotide sequence of the flanking region of each bphC revealed a homolog of the 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPD) hydrolase gene, bphD, just npstream of bphC5. The bphC5 and putative bphD genes may constitute an operon and p|ay a role in the degradation of biphenyl and PCBs together with bphC1 and etbC. In contrast, the bphC2, bphC3, and bphC4 genes may not be involved in biphenyl and PCB degradation. [Key words: Gram-positive bacterium, biodegradation, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase] PCBs have been widely used as industrial materials because of their chemical stability, strong insulating properties, and high levels of heat conductivity. The use and synthesis of PCBs has been discontinued, however, in view of their toxicity and recalcitrance in the environment. The degradation of PCBs by microorganisms is regarded as one of the most effective procedure in removing them from the environment. Many PCB-degrading bacteria have been isolated, and they commonly cometabolize PCBs through the biphenyl catabolic pathway (Fig. 1). In this pathway, biphenyl is transformed to 2,3-dihydroxy-l-phenylcyclohexa-4,6diene (dihydrodiol compound) by a multicomponent biphenyl dioxygenase (BphA). Dihydrodiol compound is then converted to 23DHBP by dihydrodiol dehydrogenase (BphB), and the resulting 23DHBP is cleaved at the 1,2 position by 23DHBP dioxygenase (BphC). The ring-cleavage product, HOPD, is hydrolyzed to benzoate and 2-hydroxypenta-2,4dienoate (HPD) by HOPD hydrolase (BphD), and the resulting HPD is further metabolized to pyruvate and acetylCoA by successive reactions catalyzed by BphE, BphF, and BphG. Rhodococcus sp. RHAI was isolated from y-hexachlorocyclohexane-contaminated soff, and this strain can efficient-

ly transform PCB48, which consists primarily of tetrachlorobiphenyl (1). In previous studies, we have characterized the bphAC1B genes responsible for the degradation of biphenyl to the meta-cleavage compound (2) and etbCbphD1EF encoding the downstream pathway of biphenyl degradation in RHAI (3). Each of these two gene clusters contains a 23DHBP dioxygenase gene, bphC1 and etbC, respectively, and both appear to be involved in biphenyl and PCB catabolism (4). Recently, a multiplicity of 23DHBP dioxygenase genes has been reported in some rhodococcal strains, Rhodococcus erythropolis TA421 (5) and R. globerulus P6 (6) have seven and three bphC genes, respectively. Kulakov et al. (7) have also reported multiple 23DHBP dioxygenase genes in rhodococcal aromatic degraders. Thus, a multiplicity of23DHBP dioxygenase genes may be common in rhodococcal strains, and the multiple catabolic enzymes may contribute to an effective degradation of aromatic compounds. In this study, we screened 23DHBP dioxygenase genes and isolated four new bphC genes in addition to bphC1 and etbC from Rhodococcus sp. RHA1. To address the significance of multiple bphCs in RHA1, the substrate specificities of their products and the induction profiles of their expression in RHA1 were examined.

* Corresponding author, e-mail: [email protected] phone: +81-(0)258-47-9405 fax: +81-(0)258- 47-9450 421

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MATERIALS AND M E T H O D S Bacterial strains, plasmids, and eulture conditions The bphC1 insertion mutant of RHAI, RDC1 was grown in LB or W medium (2) supplied with vapor of ethylbenzene. Cloning and nucleotide sequencing Genomic DNA from RDC1 was prepared as described previously (2). RDC1 ehromosomal DNA was partially digested with Sau3AI, and the entire mixture was ligated into the BamHI site of pUC 119. The resulting plasmids were used to transform Escherichia coli JMI09 eells, which were spread onto LB agar plates containing 100 mg/l ampieillin and 1 mM isopropyl-13-D-thiogalaetopyranoside(IPTG). After the development of colonies, a diethyl ether solution of 23DHBP was sprayed onto the plates. Transformants expressing 23DHBP dioxygenase activity were identified by the formation of the yellow meta-eleavage metabolite, HOPD, from 23DHBP. To determine the nucleotide sequences of the positive elones, the positive clone DNAs were reeovered from the cells grown from the positive ¢olonies, and the deletion derivatives of eaeh clone were generated by using a kilo-sequence deletion kit (Takara shuzo, Kyoto). Nucleotide sequences of deletion derivatives were determined by the dideoxy termination method using an ALFred DNA sequeneer (Pharmacia Biotech, Milwaukee, WI, USA). Sequence analyses were earried out using the GeneWorks programs (IntelliGenetics Inc., Mountain View, CA, USA). The sequences were aligned by using CLUSTALW with all parameters set at their default values. Phylogenetic analyses were performed with the neighbor-joining method (8). Graphics for phylogenetic trees were produeed by using the TreeView program (9). Deteetion of the gene produets Expressions of bphC genes and bphD2 gene in E. coli JM109 were examined by SDS-PAGE (10). E. coli JM109 harboring pC4A, pC4B, pC4C, pC4F, and pUDS18 were incubated in 3tal of LB containing 100mg/l of ampicillin at 37°C. When the optieal density of the cultures at 600 nm reached 0.6, IPTG was added to a final concentration of 1 mM. After 4 h, the cells from l-tal eultures were harvested and suspended in 100 p.l of lysis buffer (100 mM Tris, 10% glycerol, 5% SDS and 1 mM mercaptoethanol [pH 6.8]). After boiling, 4 ~tl

of the samples was applied onto the gel, and the separated proteins were visualized by staining with Coomassie brilliant blue R250. Enzyme assays E. coli JMI09 harboring the recombinant plasmids were grown as described above. The teils were harvested by centrifugation, washed with 50 mM Tris-HC1 buffer (pH 7.5), and resuspended in the same buffer. Cells were disrupted by sonication, and cell debris was removed by centrifugation at 15,000 rpm for 10 min at 4°C. The supernatant was then used as a crude extract. The meta-ring cleavage activity of crude extracts for various diol-aromatic compounds, including 23DHBP, were spectrophotometrically determined by using DU-640 speetrophotometer (Beckman, Fullerton, CA, USA). Reactions were performed in 3 ml of 50 mM Tris-HCl buffer (pH 7.5) containing a 500 ~tM substrate at 25°C. One unit of enzyme activity was defined as the amount that catalyzed the formation of 1 ~tmol of product per min at 25°C. The molecular extinction coefficients of the products under the assay conditions (pH 7.5) were as follows: 23DHBP, e43«=13,200M-lcm 1; CAT, ~375=36,000M-~cm~; 3MC, ~3ss= 32,000 M-~cm-t; 4-methylcatechol (4MC), e3s2= 17,000 M lcm~; 3-chlorocatechol (3CC), g37s=33,000M-Icm-1; and 4-chloroeatechol (4CC), e379=40,000 M-~cm-t (6). The HOPD hydrolase aetivity o f a erude extract was determined as described by Yamada et al. (11). RNA slot blot, Southern blot, and colony hybridizations RHA1 was grown in LB until the optieal density of the eulture at 600 nm reaehed 0.5. The cells from 10-tal cultures were harvested, washed twice with 10 ml of W minimal medium, and resuspended with 10 ml of the same medium containing one of the following carbon sources; 0.2% of biphenyl, sodium succinate, phenylalanine, tyrosine, naphthalene, 2,3-dihydroxynaphthalene, naphthalenesulfonate, and phenanthrene, and the vapor of ethylbenzene, toluene, propylbenzene, butylbenzene, cumene, o-xylene, aniline, phenol, carvone, and p-cymene. After a 15-h incubation with these substrates, the eells were harvested, and RHA1 total RNA was prepared from the cells as described by Ausubel et al. (12). RNAs (2 ~tg eaeh) were blotted onto a nylon membrane by using a slot blot apparatus (Bio-Rad, Riehmond, CA, USA). Hybridization was carried out using the digoxigenin-11-dUTP labeled bphC probes

VOL. 93, 2002

MULTIPLE BPHC GENES IN A RHODOCOCCUS PCB DEGRADER

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shown in Fig. 2 by using the DIG system (Boehringer Mannheim Biochemicals, Indianapolis, IN, USA) according to the procedure recommended by the manufacturer. Southern and colony hybridizations were also performed using the DIG system. All the hybridizations were carried out at 65°C, and each blot was washed with 0.2 x SSC containing 0.1% SDS at 65°C. Nucleotide sequence aecession numbers The nucleotide sequences determined in this study have been deposited in the DDBJ, EMBL and GenBank databases under ac¢ession nos. AB030669 (bphC2), AB030670 (bphC3), AB030671 (bphC4) and AB030672 (bÆhD2and bphCS). R E S U L T S AND DISCUSSION Cloning and sequencing o f the bphC genes To isolate new species of bphC genes other than bphC1 and etbC from Rhodococcus sp. RHA1, the gene library of the bphC1 insertion mutant, RDC 1 (2), was constructed using pUC 119 in E. coli JM109. Approx. 30,000 E. coli transformants containing the RDC 1 library were screened to isolate the genes conferring the meta-ring cleavage activity toward 23DHBP to E. coli. Ten colonies showed bright yellow derived from the meta-cleavage compound of 23DHBP. Plasmid DNAs were recovered flora these colonies, and categorized into

five species according to the results of restriction digestion and Southern hybridization analysis. The nucleotide sequence of the insert DNA in the plasmid representing each group was determined. Each of the clones contained an open reading ffame (ORF) showing amino acid sequence similarity to bphC genes. The cloned ORFs were separated into five species again, which contained four new gene species and etbC reported previously. These four new genes were designated as bphC2, bphC3, bphC4, and bphC5. The partial restriction maps of the insert DNA of the plasmids, pC4A, pC4B, pC4C, and pC4F carrying bphC2, bphC3, bphC4, and bphC5, respectively, are illustrated in Fig. 2, and the features of each ORF are shown in Table 1. The deduced amino acid sequence identity between the bphC genes in RHA1 ranged from 16% to 47% (Table 2). All of them conserved two histidine and one glutamate residues of iron ligands and a histidine residue of a catalytic base, which were indicated to be essential for the enzyme activity. Based on amino acid sequence similarities, a phylogenetic tree of the 23DHBP dioxygenases was constructed and is depicted in Fig. 3. The extradiol dioxygenases have been classified into two types (13). The overall structures of type I and type II enzymes are totally different, and they were

424

SAKAIET AL.

J. BIosct. BIOENG., TABLE 1. Characteristicsof the RHA1 bphCgenes and gene products

Gene

Nt sequence (bp) bphC1 948 etbC 912 bphC2 900 bphC3 927 bphC4 927 bphC5 900 ATG indicates initiation codon.

%G+C in ORF 60.6 61.6 67.6 69.4 66.1 66.8

No. of aa residues 316 304 300 309 309 300

thought to have originated from separate ancestors (14). Most of meta-ring cleavage dioxygenases are included in type I. The family 1 enzymes of type I have nearly half the size of other family enzymes of type I. The families 2 and 3 enzymes of type I preferencially contain monocyclic and polycyclic aromatic ring-cleavage dioxygenases, respectively. All the RHAI BphCs belong to the type I enzymes, and BphC2 and BphC5 belong to the family 3 in this type together with BphC1 and EtbC. In contrast, BphC4 belongs to the family 4, which includes BphC2 of Sphingomonas sp. BN6 (15) and MpcII ofAlcaligenes eutrophus JMP222 (16). BphC3 seems to belong to another family that we designated family 6. The distribution of RHA1 BphCs in the phylogenetic tree presented in Fig. 3 is fundamentally different from that of TA421 BphCs, suggesting that RHA1 bphC genes have evolved separately from TA421. These results indicate that RHA1 possesses at least six bphC genes for which the primary structures differ from each other. RHA1 bphC gene produets expressed in E. coli The four newly isolated bphC genes were expressed in E. coli JM109 transformants, each harboring pC4A, pC4B, pC4C, and pC4F. The putative products of bphC2, bphC4, and bphC5 were observed on SDS-PAGE (Fig. 4), and their molecular masses were estimated to be 29, 32, and 33 kDa, respectively. These values are close to those predicted from the deduced amino acid sequences of bphC2, bphC4, and bphC5 (Table 1). The putative product ofbphC3 was not detected on SDS-PAGE. The cell extract prepared from each transforrnant was subjected to the enzyme assay using 23DHBP, CAT, 3MC, 4MC, 3CC, and 4CC as substrates. We have previously purified and characterized BphC 1 and EtbC from RHA1 cells. Both enzymes showed meta-ring cleavage activity toward 23DHBP, CAT, and 3MC. In addition, EtbC also showed activity toward 4MC and 4CC (4). All the products of the newly isolated bphC genes showed meta-ring cleavage activity only toward 23DHBP. The bphC2 gene product showed distinct activity (2 U/mg protein in crude extract). Although the bphC3 gene product was TABLE 2. Identitybetween the deduced aa sequences ofRHA1 bphCgenes Gene

bphC1 etbC bphC2 bphC3 bphC4 bphC5

%Identity with

bphC1 100

etbC bphC2 bphC3 bphC4 bphC5 31 100

33 30 100

21 23 24 100

19 16 23 24 100

35 32 47 25 24 100

Molecular mass (kDa) Predicted Estimated 34.9 34.0 34.0 34.0 32.8 29.0 33.8 34.5 32.0 33.5 33.0

Putative ribosome binding sites AAGAAAGGTAAGAAAATGa GGAAAGGAGTAACCAATG GTAGAAGGACAACACATG AACTGGAGGCATGCGATG TGGACGGTTGAGCCGATG TGGACGGGGTAAGTGATG

not observed on SDS-PAGE, its product, BphC3, exhibited activity (0.01 U/mg protein in crude extract). BphC4 exhibited trace activity, although the putative product of bphC4 gene was detected on SDS-PAGE. Similarly, the BphC5 activity was not so high (0.13 U/mg protein in crude extract) in comparison to the amount of putative bphC5 product detected on SDS-PAGE.

Transeriptional induetion of bphC genes in RHA1 To obtain insight into the roles played by the newly isolated bphCs in the degradation of arõmatic compounds, we examined ea¢h bphC transcript in RHA1 grown on various carbon sources. Total RNAs were extracted from RHA1 cells grown on biphenyl, ethylbenzene, LB, and succinate, and were subjected to a RNA slot blot hybridization (Table 3) using bphC gene fragments as probes (Fig. 2). bphC1 and etbC were transcribed in the cells grown on both biphenyl and ethylbenzene. Interestingly, bphC1 expression was induced much more by biphenyl than by ethylbenzene, and etbC expression was induced much more by ethylbenzene than by biphenyl. Among the newly isolated bphC genes, only the bphC5 probe hybridized to the total RNAs derived from biphenyl- and ethylbenzene-grown cells (Table 3). Taking the 23DHBP dioxygenase aetivity of the bphC5 product into account, these results suggest that bphC5 is involved in biphenyl and PCB degradation together with bphC1 and etbC. In addition to biphenyl and ethylbenzene, bphC1 and etbC expression was induced by toluene, propylbenzene, butylbenzene, cumene, o-xylene, and phenanthrene (Table 3). The inducing-substrate specificities of bphC1 and etbC were almost the same, suggesting that the same regulatory system may be involved in bphC1 and etbC induction when these substrates are used as inducers. Transcription of the bphC5 gene was induced by toluene and o-xylene in addition to biphenyl and ethylbenzene. But it was not induced by propylbenzene, butylbenzene, cumene, and phenanthrene, which are inducers of bphC1and etbC-coding enzymes. These results suggest that the regulatory system responsible for bphC5 induction is different from that responsible for the induction of bphC1 and etbC. The transcription of bphC2 and bphC3 genes was induced only by o-xylene, while that of the bphC4 gene was induced by toluene and butylbenzene in addition to o-xylene. In conclusion, the inducing-substrate specificities of the six bphC genes, including etbC, can be categorized into four types. The first type includes bphC1 and etbC, the second type bphC5, the third bphC4, and the fourth bphC2 and bphC3. Transcription of all six bphC genes was induced by

VOL.93, 2002

MULTIPLEBPHC GENES IN A RHODOCOCCUSPCB DEGRADER

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FIG. 3. Phylogenetic tree for RHA1 BphCs and other extradiol dioxygenases. RHAI BphCs are presented with dark backgrounds. Gene product and strain name abbreviations in alphabetical order: MndD AGLOB, mndD product ofArthrobacter globiformis CM-2 (U19817); BphC2 BN6, bphC2 product ofSphingomonas sp. strain BN6 (U38978); DmpB CF600, dmpB product ofPseudomonas sp. CF600 (M33263); PcbC DJI2, pcbC product of Pseudomonas sp. strain DJI2 (D44550); EdoC I 1, edoC product of Rhodococcus sp. strain I 1 (AJ006126); MpcII JMP222, mpcH product of Alcaligenes eutrophus JMP222 (X52415); BphC LB400, bphC produet of Burkholderia sp. LB400 (X66122); XylE MT2, xylE product of P. putida MT-2 (V01161); EdoA NCIMB 13064, edoA product of R. rhodochrous NCIMB 13064 (L77225); EdoB NC1MB13064, edoß product of R. rhodochrous NCIMBI3064 (AJ003244); BphC 1 P6, bphCl product of R. globerulus P6 (X75633); BphC2 P6, bphC2 product of R. globerulus P6 (X75634); BphC3 P6, bphC3 product of R. globerulus P6 (X75635); BztE PS104, bztE product of P. aeruginosa JI104 (X60740); BphE PSIC, bphE product of Pseudomonas sp. strain IC (U01825); NahH P SG7, nahH product of P putida PpG7 (X06412); BphC Q 1, bphC product of Sphingomonaspaucimobilis QI (M20640); DbfB RWI, dbfB product of S. paucimobilis RWI (X72850); BphCl TA421, bphCl product ofR. erythropolis TA421 (D88013); BphC2 TA421, bphC2 product of R, erythropolis TA421 (D88014); BphC3 TA421, bphC3 product of R. erythropolis TA421 (D88015); BphC4 TA421, bphC4 product of R. erythropolis TA421 (D88016); BphC 5 TA42I, bphC5 product of R. erythropolis TA421 (D88017); BphC6 TA421, bphC6 product ofR. erythropolis TA421 (D88018); BphC7 TA421, bphC7 product ofR. erythropolis TA421 (D88019), o-xylene (Table 3). The induction by o-xylene may be governed by a more common regulatory system. In the case of Arthrobacter sp. B 1B, an effective induction of PCB degradation activity was observed in the presence of plant-derived terpenoids such as carvone and p-cymene (17). However, these terpenoids did not induce any RHA1 bphC gene expression, We also examined the aromatic amino acids phenylalanine and tyrosine, but neither of them induced bphC gene expression (Table 3). A eatabolie gene linking to bphC5 gene To examine whether four newly isolated bphC genes are accompanied by other catabolic genes for aromatic compounds, the nucleotide sequences o f the adjacent regions of each bphC were determined. We found that the DNA region upstream of bphC5 in pC4F is similar to that o f HOPD hydrolases of Burkholderia sp. LB400 (18) and RHA1 (11). No other adjacent regions of newly isolated bphC genes showed similarity to any other aromatic catabolic genes. Because, the 5'terminus of the newly found putative bphD gene in pC4F was truncated, a fragment containing its 5'-terminus was screened and identified by Southern hybridization analysis using the DIG-labeled BstXI-SmaI fragment (probe 7 in Fig. 2). pDE17 containing a 1.7-kb EcoRI fragment was obtained from the RHA1 gene library by colony hybridization using the same probe. The nucleotide sequence of the 1.7-

kb EcoRI fragment revealed an 876-bp ORF whose initiation codon is GTG (Fig. 2). A possible ribosome-binding sequence, AGGAG, was found to be located 10-bp upstream 1

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426

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SAKAIET AL. TABLE 3. RNA slot blot hybridization analysis of bphCtranscripts in Rhodococcus sp. strain RHA 1

bphCgenes

Substrate Biphenyl Ethylbenzene Succinate LB Toluene Propylbenzene Butylbenzene Cumene o-Xylene Aniline Phenol Naphthalene 2,3-Dihydroxynaphthalene Naphthalene sulfonate Phenanthrene Carvone p-Cymene Phenylalanine Tyrosine

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.

.

FIG. 5. Alignment of the deduced amino terminal sequences between RHA1 bphD2 and its homologs. The amino terminal sequence from the 4th to 184th amino acid residues is omitted. The residues conserved among three sequences are boxe& The possible initiation codons for the first and 187th amino acid residues of bphD2 gene are indicated by vertical arrows above the deduced amino acid sequence of bphD2. Gene product and strain name abbreviations: BphD2 and BphD RHA 1, bphD2 and bphD (D78322) products of Rhodococcus sp. strain RHA1; BphD LB400, bphD product ofBurkholderia sp. LB400 (X66123). from the initiation codon.The deduced amino acid sequence of this ORF showed 34% and 29% identity with those of LB400 bphD and RHA1 bphD, respectively, and included a nucleophile motif conserved in t~/13 hydrolase fold enzyme, Gly-X-Ser-X-GIy. The molecular mass of a putative product was estimated to be 32 kDa. The TGA stop codon for this ORF overlaps the ATG start codon for bphC5, which suggests that these two genes are translationally coupled and constitute an operon. However, pDE17 did not confer HOPD hydrolase activity to E. coli. Then we cloned the 1.8kb SalI fragment in pUC119 to generate pUDS18 (Fig. 2). This fragment contains an 1434-bp ORF with an 558-bp in-frame amino terminal extension to the 876-bp ORF in pDE17. This 1434-bp ORF was preceded by a putative ribosome-binding sequence, A A G G at 6-bp upstream from the initiation codon, and pUDS 18 conferred HOPD hydrolase activity to E. coli. Together with bphC5, the transcriptional induction by biphenyl of this ORF was observed in RNA slot blot hybridization, and seems to be partly involved in biphenyl and PCB degradation. These results suggest that this 1434-bp ORF encodes an alternative BphD enzyme, which we have designated bphD2. When the bphD2 gene in pUDS 18 was expressed in E. coli, a product of 48 kDa was detected on SDS-PAGE. This molecular mass of the bphD2 product is in agreement with 52 kDa that is estimated from the deduced amino acid sequence of bphD2. The deduced amino acid sequence of 1434-bp bphD2 showed 26% and

18% identity with those of LB400 bphD and RHA 1 bphD, respectively. The percentages of identity were lower than those for the 876-bp ORF, which are 42% and 31%, respectively, suggesting that the 558-bp amino terminal extension in bphD2 product may be an extra peptide that is functionally insignificant for the activity itself (Fig. 5). The HOPD hydrolase activity exhibited by the E. coli cells containing pUDS 18 was weak, suggesting the low specific activity of bphD2 product or the small proportion of active form in entire bphD2 product produced in E. coli. ACKNOWLEDGMENTS This study was partly supported by the Promotion of Basic Research Activities for Innovative Bioscience (PROBRAIN) in Japan.

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