Cloning of an intracellular d (−)-3-hydroxybutyrate-oligomer hydrolase gene from Ralstonia eutropha H16 and identification of the active site serine residue by site-directed mutagenesis

JOURNAL OFBIOSCIENCE ANDBIOENGINEERING Vol. 94, No. 2, 106-112.2002

Cloning of an Intracellular D(-)-3-Hydroxybutyrate-Oligomer Hydrolase Gene from Ralstonia eutropha H16 and Identification of the Active Site Serine Residue by Site-Directed Mutagenesis HARUHISA

SAEGUSA,’

MARI

SHIRAKI,2

AND TERUMI

SAIT02*

Research Institute of Innovative Technology for the Earth Branch in Kanagawa University, 2946 Tsuchiya, Hiratsuka, Kanagawa 259-1293, Japan’ and Laboratory of Molecular Microbiologv Department of Biological Sciences, Faculty of Science, Kanagawa University, 2946 Tsuchiya, Hiratsuka, Kanagawa 259-1293, Japan2 Received 21 January 2002lAccepted 10 May 2002

An intracellular D(-)-3-hydroxybutyrate (3HB)-oligomer hydrolase gene from R&toniu eutropha (formerly Alcaligenes eutrophus) H16 was cloned, sequenced, and characterized. As a hybridization probe to screen restriction digests of chromosomal DNA, an extracellular 3HB-oligomer hydrolase gene from Ralstonia pickettii strain (formerly Pseudomonas sp. strain) Al was used. A specific hybridization signal was obtained and a 6.5-kbp SmaI fragment was cloned in an Escherichiu coli phagemid vector. The crude extract from E. coli with this plasmid showed 3HB-trimer hydrolase activity. The subcloned 3.2-kbp fragment still showed 3HB-trimer hydrolase activity in E. coli and expressed an approximately 78-kDa protein in an in vitro transcription-translation system. Nucleotide sequence analysis of the 3.2-kbp fragment showed an open reading frame that encodes a polypeptide with a deduced molecular weight of 78,510. The putative amino acid sequence showed 54% identity with that of the oligomer hydrolase from R. pickettii Al. By sitedirected mutagenesis, a novel amino acid sequence (S-V-S*-N-G) containing an essential serine residue in the catalytic center of the enzyme was determined. The gene product was found in PHB-rich cells of R. eutropha by immunodetection. The expressed 3HB-oligomer hydrolase localized both in the supernatant fraction and the PHB granules of the cells. [Key words: Ralstonia eutropha, intracellular 3-hydroxybutyrate-oligomer Poly-3-hydroxybutyrate (PHB), a homopolymer of D(-)3-hydroxybutyrate (3HB), is a storage material produced by a variety of bacteria in response to nutritional stress. The genes involved in the biosynthesis of PHB have been cloned in Escherichiu coli and studied in detail (1). The degradation of PHI3 has two phases, extracellular and intracellular. The extracellular metabolism of PHB was recently studied in detail (2). However the intracellular degradation process of PHB is not fully clear. Studies of intracellular degradation system in PHB-producing bacteria may be important from the point of view of mass production of the polyester. A few intracellular poly(3-hydroxyoctanoate) depolymeraselike genes have been cloned (3, 4) but their products have not been characterized. Previously, we cloned and sequenced an intracellular PHB depolymerase (iPHB depolymerase) gene @haZ) from RaZstoniu eutropha H16, and conducted a preliminary characterization of the crude enzyme expressed in E. coli (5). Recently, a protein of Paracoccus denitrzficans homologous to the iPHB depolymerase of R. eutropha was identified to be an iPHB depolymerase (6). Interestingly iPHB depolymerase from R. eutropha digested artificial amorphous PHB granules, but did not hy-

hydrolase, serine esterase]

drolyze crystalline PHI3 or freezed-dried artifitial amorphous PHB granules (5). The main product in enzymatic degradation of amorphous PHI3 by the iPHB depolymerase was 3HB-oligomers (5, 6). Therefore, a 3HB-oligomer hydrolase may play an important role in PHI3 degradation, and it is interesting to clone such an intracellular 3HB-oligomer hydrolase (DHB-oligomer hydrolase) gene. A few i3HB-oligomer hydrolases have been characterized. Merrick and Yu demonstrated that 3HB dimeric ester was hydrolyzed to the monomer by a hydrolase partially purified from Rhodospirillum rubrum (7). Tanaka et al. purified and characterized an i3HB-dimer hydrolase from Zoogloeu ramigeru I-16-M (8). The molecular mass of the purified enzyme was 28,000 Da, as determined by Sephadex G- 100 gel filtration. In this study, we cloned an i3HB-oligomer hydrolase gene using as a probe, the extracellular 3HB-oligomer hydrolase (e3HB-oligomer hydrolase) gene of Ralstoniu pickettii Al (9). Here, a novel amino acid sequence containing an essential serine residue in the catalytic center of the enzyme was determined by site-directed mutagenesis. Furthermore, expression of the gene in R. eutropha was examined.

* Corresponding auther. e-mail: [email protected] phone: +81-(0)463-59-4111 fax: +81-(0)463-58-9684 106

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MATERIALS

AND METHODS

R. eutropha Bacterial strains, cultivation and plasmids H16 was obtained from the American Type Culture Collection (ATCC 17699). R. eutropha was grown in a nutrient-rich medium containing 1% (w/v) yeast extract, 1% (w/v) polypeptone, 0.5% (w/v) beef extract, and 0.5% (w/v) ~H~)~SO~ at 3O’C. To produce PHB, cells grown on the nutrient-rich medium were transferred to a nitrogen-free medium containing 0.27% (w/v) KH,PO,, 0.99% (w/v) K,HPO,, 0.02% (w/v) MgS0,.7H,O, 0.1% (w/v) mineral solution, and 2% (w/v) fructose and cultured at 30°C as described previously (10, 11). E. coli DWS [supE44, hsdRl7, recA1, e&Al, gvrA96, thi-I, rebAI] and charomid 9-36 (phagemide) DNA were obtained from Nippon Gene, Toyama. E. coli JM109 IrecAl, endAl, &vrA96, thi, hsdRf7, stipE44, relA1, A(~ac-proAB)~[~a~36, proAB+, lacls, lacZAM15]] and pUCl8 and pUCl9 were from Takara Shuzo, Kyoto. E. coli BLR(DE3)pLysS and pET23b were from Novagen, Madison, WI, USA. E. coli strains were grown in Luria-Bertani (LB) medium at 37°C with or without antibiotics. DNA restriction fragments were Hyb~d~ation experiments separated by horizontal electrophoresis in 1.0% (w/v) agarose gels and the denatured DNA was transferred to positively charged nylon membranes (Biodyne@B; Pall BioSupport Division, NY, USA). Prehybridization and hybridization with ““P-labeled probes were performed by standard procedures (12). Colony hybridization of genomic libraries in an E. coli charomid vector was done in accordance with the methods of Grunstein and Wallis (13). 3HB-oligomer hydrolase activity was asEnzyme assay sayed as follows. The reaction mixture (0.4 ml) contained 100 mM Tris-HCI (pH 8.0) and 3HB-dimer or trimer ester (2.5 mM). The reaction was allowed to proceed at 30°C for 15 min. Then the mixture was heated at 100°C for 5 min, and was centrifuged. The 3HB in the supernatant fraction was measured enzymatically using 3HB dehydrogenase as described previously (14). One unit of the enzyme catalyzes the formation of 1 pmol of 3HB per min under the assay conditions. Nucleotide sequences were deterDNA sequence analysis mined by the dideoxy nucleotide chain termination method (12) with single-strand templates by using an Auto Cycle Sequencing Kit (Pharmacia Biotech, Tokyo). Sequence information was analyzed with GENETYX-MAC software (Software development, Tokyo). In vitru translation system An E. coli S30-based coupled transcription-translation system was used according to the user’s manual (Promega, Tokyo). Briefly, the purified plasmid DNA was mixed with amino acids containing [U-‘4C]leucine (11 GBq/mmol, 0

t

2

4

3

I SmP

P

P

SP

18.5 kBq; Amersham, Tokyo) and S30 extract. The mixture was incubated at 37°C for 1 h. After the reaction had completed, a 5-pl aliquot was removed, dried, treated with sodium dodecyl sulfate (SDS), and applied to an 12.5% SDS-polyacrylamide gel. After electrophoresis, the gel was soaked in 10% acetic acid and 30% methanol for 1 h and then in an enhancer reagent (ENLIGHTNING; Du~n~EN Research, Boston, MS, USA) for 30 min. The treated gel was dried, and the radioactivity on the gel was detected by overnight exposure to X-ray film (Kodak X-Omat AR, New York, USA). Modification of the 3HB-oIigomer hydrolase gene for affbity purification and purification of His-tag-3HB-oligomer hydrolase The 3HB-oligomer hydrolase gene was amplified in a thermal cycling reaction with primers Nde (GAGGCATATGGCCGCG CCAGCGGI) and Xho (C~CTCGAG~CAG~ACCT~AC). The primer Nde introduces a Ndei site at the position of the translation start codon. The primer Xho introduces a XhoI site at the position of the stop codon. The amplified DNA was cloned into the protein fusion vector pET23b as a NdeI-XZzoI fragment. The fusion results in the addition of a decahistidine-confining peptide to the c~boxyl-te~inal end of the protein, which allows for the purification of the modified protein (His-tag-3HB-oligomer hydrolase) from inclusion bodies on a metal chelation column under denaturing conditions (15). The cells harboring His-tag-3HB-oligomer hydrolase gene in pET23b were cultivated in 2 I of LB broth. After a 9-h induction period with 2 mM isopropyl-1-thio-P-D-galactopyranoside (IPTG), they were harvested and disrupted. After centrifugation (12,000xg; IO min), the precipitates were dissolved in 6 M guanidine_HCl and separated on a nickel chelation column as recommended by the supplier (Novagen). The protein was finally purified by a continuous elution SDS-polyacrylamide gel electrophoresis (Model 491 Prep Cell; Bio-Rad, Tokyo) to electrophoreticat homogeneity. The purified protein was used to prepare antisera against the His-tag-3HB-oligomer hydrolase as described below. Preparation of cell extract Cells harvested from an overnight culture in LB were suspended in 50 mM Tris-HCl (pH 7.5) (5 ml/g [wet weight] of cells). The cell suspension was disrupted by sonication (20-kHz tip, 30 W for 5 min). The sonicated cells were centrifuged at 10,000 xg for 10 min, and the supernatant fraction was used as the crude extract. Pu~Bcatio~ of PHB granules R. e~~oph~ cells confining PHB were suspended in 20 mM Tris-HCl (PH 7.5) (5 ml/g [wet weight] of cells) and disrupted by sonication (20~kHz tip, 40 W for I5 min). The resulting suspension (5 ml) was loaded onto a discontinuous glycerol gradient, which was prepared from 3 ml each of 88% and 44% glycerol. After centrifugation for 30 min at 210,000 5

6

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f

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f

1 PSm

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107

W’p) BHB-oligomer hydrolase Activity

+ + +

(SHEoligomer hydroiase) FIG. 1. Restriction endonuclease sites of the cloned DNA (6.5-kbp) in pUC 18 and the ability of recombinant plasmids to express 3HB-oligomer hydrolase activity in E. coli JM109. Sm, SmaI; P, PvuII; Sp, SphI; E, &&I.

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xg and 4°C the granules were collected between 44% and 88% glycerol. They were washed with 20 mM Tris-HCl (pH 7.5) and loaded onto another discontinuous sucrose gradient, which was prepared from 4 ml each of 1.66 and 1.50 M sucrose. After centrifugation for 2 h at 210,000 xg and 4”C, the granules were collected between 1.66and 1.50M sucrose.The purified granuleswere with-

drawn,washed,and suspendedin 20 mM Tris-HCI @H 7.5). Electrophoresis and immunoblotting SDS-polyacrylamide gel electrophoresis was performed by the procedure of Laemmli (16). Electroblotting of proteins was performed according to the method of Towbin et al. (17). The antisera to the purified His-tag3I-Boligomer hydrolase was prepared as follows. The rabbits used were 3 months old, male, and about 1.8 kg. After 2 weeks of growing in good condition, 1 ml of the enzyme (1 mg) and 1 ml of Freund’s complete adjuvant (Wako Pure Chemical Industries, Tokyo) were mixed and injected in the center of the back subcutaneously. Finally, some blood was obtained by puncturing the aorta. The blood was kept at room temperature for 1 h and centrifuged. The supematant was preserved at -20°C. Site-directed mutagenesis was Site-directed mutagenesis performed by the gapped duplex method described by Kramer et al. (18). Fractionation of the protein by an anion exchange chromaRecombinant cells were suspended in 5 volumes of tography 50 mM Tris-HCl (pH 7.5) and disrupted in a sonic disintegrator for 10 min. After centrifugation at 12,000 xg for 10 min, the supematant was precipitated with solid ammonium sulfate (45%). After centrifugation (10 min at 12,000 xg), the precipitate was dissolved in 20 mM Tris-HCl @H 7.5) (buffer A) and dialyzed overnight against the same buffer. The dialyzed enzyme solution was applied to an anion-exchange column (RESOURCE Q 1 ml; Pharmacia, Tokyo) equipped with a Pharmacia FPLC System. The enzyme was eluted with a linear gradient of 0 to 0.5 M NaCl-buffer A at a flow rate of 1 ml/min. The nucleotide seNucleotide sequence accession number quence data reported in this paper have been deposited in the

DDBJ, EMBL, and GenBank nucleotide sequence databases under

accession no. ABOO3701.

RESULTS Identification and cloning of a 3HB-oligomer hydroA 2. l-kbp SphIlKpnI-restriction fragment which lase gene

harbored the structural gene for extracellular 3HB-oligomer hydrolase from R. pickettii Al was used as a heterologous hybridization probe to detect the homologous gene of R. eutropha. When SmaI-digested genomic DNA of R. eutropha separated by agarose gel electrophoresis was hybridized with the probe, a single signal representing a 6.5-kbp restriction fragment appeared. The DNA corresponding to the positive signal was extracted from the agarose gel, ligated to

97 kDa --+ 69 kDa + 46 kDa 30 kDa

14 kDa

FIG 2. In vitro transcription-translation of DNA fragments from in pUCl8. The products of DNA cloned in pUC18 were analyzed by E. coli S30 coupled translation system. Translation reactions were performed with pUC18 containing DNA fragments according to the manufacture’s instruction as described in Materials and Methods. “‘C-labeled translation products were analyzed on SDSpolyacrylamide gel (12.5%) and then detected using an overnight exposure to X-ray film. Lanes: 1 and 4, molecular size markers; 2, pUC 18 (2 pg); 3, pUCl8 containing 3.1-kbp fragment (SmaI-SphI, 2 pg). The band of around at 30 kDa in lanes 2 and 3 was E-lactamase expressed by the pUC vector.

R. eurropha

FIG. 3. Nucleotide sequence of a 3173-bp region carrying the oligomer hydrolase gene. The underlined sequence is the postulated Shin+Dargarno sequence. The facing arrows indicate inverted repeat sequences.

VOL. 94,2002 R. e Al

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FIG. 5. SDS-polyacrylamide gel electrophoresis of the purified His-tag-3HB-oligomer hydrolase. Proteins in the gel was stained with Coomassie Brilliant Blue R-250. Lanes: 1, molecular size markers; 2, the total protein of E. coli BLR(DE3)pLysS containing pET23b+Histag-3HB-oligomer hydrolase gene (20 pg); 3, precipitate of the disrupted cells of E. coli BLR(DE3)pLysS containing pET23b+His-~g3HB-oligomer hydrotase gene (20 pg); 4, the eluate from the nickel chelation column (10 pg); 5, the purified His-tag-3HB-oligomer hydrolase from continuous elution SDS-polyacrylamide gel electrophoresis (5 W).

757 722

FIG. 4. Alignment of the deduced amino acids sequences from two 3HB-oligomer hydrolases. Asterisks show the identical amino acid residues between two enzymes. Arrows show the positions of the candidate of active-site serine residues. Underlined sequences show the lipase box (AXSXG or GXSXG). Identical sequence between two enzymes which contain a serine residue (s) were boxed. R. e, R. eutrupha 3HB-oligomer hydrolase; Al, R. pickettii sp. Al 3HB-oligomer hydrolase. Nucleotide sequence of 3HB-oligomer hydrolase gene from Al was revised resulting in 5 nucleotide decrease and 18 amino acid increase (total 722 amino acids) from the original sequence (16). The revised sequence have been deposited in the DDBJ, EMBL, and Gene Bank nucleotide sequence databases.

0

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S&-digested charomid vector 9-36, and introduced into E. coli DH5 by transfection. After colony hybridization with the probe, a few transformants were purified to homogeneity. All harbored the same genomic DNA fragment of 6.5kbp. The crude extract from an E. coii colony containing this plasmid showed high 3HB-trimer hydrofase activity. The restriction site of this fragment is shown in Fig. 1. Subcloning and determination of the nucleotide sequence The 6.5-kbp fragment was subcloned in pUC 18. The 3.2-kbp SmdSphI fragment obtained still showed 3HB-trimer hydrolase activity (Fig. 1) and expressed about a 78 kDa protein ipl vitro (Fig. 2). We determined the nucleotide sequence of the 32-kbp ~rna~~p~~ fragment (Fig. 3). DNA sequence analysis of the 3.2-kbp fragment From a computer analysis of the determined nucleotide sequence, one open reading frame was found. It started with the ATG start codon at nucleotides 166 to 168 and ended with the TGA stop codon at nucleotides 2437 to 2439. The CIRF was preceded by a tentative ribosomal binding site. However, there was no region that revealed significant ho-

FIG. 6. Inhibition by DFP. The crude extract (0.3 mg) of E. coli with pUC18 carrying the 3.2-kbp fragment was treated with DFP for 30 min at room temperature and assayed as described in Materials and Methods. Control value (100%) was 3.5 units/mg protein.

mology to the E. coli consensus sequence for a promoter 160 bp upstream of this gene (Fig. 3). The putative gene product translated from the ORF was composed of 757 amino acids and had a molecular weight of 78,5 10. Analysis of the amino acid sequence deduced from the ORF revealed significant homology with the primary structure of the extracellular oligomer hydrolase of Al (54.3% amino acid identity) (Fig. 4). Properties of the expressed ORF product in E. c&i The 3HB-dimer and -trimer hydrolase activity of the crude extract from E. coli DH5 transformed with pUC 18 carrying the 3.2-kbp fragment were 0.20 and 0.15 unit/mg protein, respectively. On the other hand, those of E. coli DH5 transformed with pUCl8 were 0.0005 and 0.0003 unit’mg, respectively.

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After treatment of the crude extract of E. coli carrying the 3HB-oligomer hydrolase gene with various concentrations of diisopropyl fluorophosphate (DFP) at room temperature, enzymatic activity was measured (Fig. 6). DFP strongly inhibited the activity at low concentrations. Purification of the His-tag-3HB-oligomer hydrolase The cells harboring the pET23b+His-tag-3HB-oligmer hydrolase gene were cultured in LB broth with IPTG, and the expressed His-tag-3HB-oligomer hydrolase was purified from inclusion bodies as described in Material and Methods. The final preparation was apparently homogeneous on SDS-polyacrylamide gel electrophoresis (Fig. 5). From the result of DFP inSite-directed mutagenesis hibition, the active center of the cloned 3HB-oligomer hydrolase seems to have an active serine residue (19, 20). So we searched for the motifs GXSXG and AXSXG usually conserved at the active-site of hydrolytic enzymes containing an active serine, and found that Ser-218 (A-T-S-S-G)

and Ser-630 (G-R-S-D-G) fitted. We constructed two mutated DNA sequences in which a serine residue was changed to an alanine residue. After verification of the DNA sequences, the mutated genes were cloned into pUC 18 or 19. To confirm the expression of these mutants in E. coli, we performed SDS-polyacrylamide gel electrophoresis/immunostaining (Fig. 7A). Two mutants expressed the enzymes to a similar extent judging from stained protein bands. But these two mutants revealed the same activity as the wild-type (Fig. 7B). Therefore we selected another four candidates according to an alignment of the two enzyme sequences from R. eutropha and Al (Fig. 4). In the four candidate sequences which were relatively long and contained at least one serine residue, Ser-191, Ser-306, Ser-356 and Ser-359, four mutated DNA sequences (S191A, S306A, S356A, S359A) were constructed in a similar manner, and the DNA sequences verified and the expressions of a protein confirmed (Fig. 7A). Among the four mutants, only S359A C wild type

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FIG 7. Analyses of wild-type and mutants of 3HB-oligomer hydrolases expressed in E. coli. (A) Western blotting and immunostaining of the expressed 3HB-oligomer hydrolase in E. coli. Each lanes are containing about 10 pg protein of crude extract from E. coli containing various pUC19 derivatives. Lanes: 1, pUC19; 2,3.2-kbp fragment (wild type); 3, S191A mutant; 4, S218A mutant; 5, S306A mutant; 6, S356A mutant; 7, S359A mutant; 8, S630A mutant. (B) 3HB-oligomer hydrolase activities of the samples used in (A). 3HB-trimer was used for substrate as described in Materials and Methods. (C) Elution patterns of the wild-type and S359A mutant. Western blotting and immunostaining of the collected fraction (hatched fractions) were shown in the lower. Lanes: 1, molecular size markers; 2, fraction from the wild-type (15 pg); 3, fraction from S359A mutant (15 ug).

INTRACELLULAR 3HB-OLIGOMER HYDROLASE

VOL. 94,2002

any immunoreactive

A

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band at all (data not shown).

Expression of 3HB-oligomer hydrolase in R. eutropha kDa

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Using antiserum against the gene product, the expression of 3HB-oligomer hydrolase in R. eutropha was examined (Fig. 8). The 3HB-oligomer hydrolase was detected in R. eutropha cells accumulating PHB but not in PHB-poor cells. In PHB-rich cells, the hydrolase protein was expressed both in the supematant and PHB granules (Fig. 8A). The immunoreactive band was detected after one day of incubation in PHB-accumulation medium and its intensity increased with time (Fig. 8B). The expression of 3HB-oligomer hydrolase in PHB granules was relatively constant with time, indicating that hydrolase is an essential component of PHB granules formed in R. atrophy cells. The gene product was not detected in the culture supematant by inspection of 3HBoligomer hydrolase activity and immunoreactivity (data not shown).

kDa

DISCUSSION 94 67 43 30

20

FIG. 8. Western blotting and ~mm~os~ining of the expressed 3HB-oligomer hydrolase in R. eutropha. (A) Localization of expressed 3HB-oligomer hydrolase in R. eu&pha cdls. Lanes: M, molecular size markers; 1, purified His-tag-3HB oligomer hydrolase (5 up); 2, disrupted PHB-poor cells (90 ug); 3, disrupted PHB-rich cells (80 pg); 4, supematant fraction of the disrupted PHB-rich cells (70 ug); 5, PHB granules from PHB-rich cells (200 ug of PHB). (B) Time course of expression of 3HB-oligomer hydrolase in R. eutropha. Lanes: M, molecular size markers; 1 and 2, disrupted PHB-poor cells; 3 and 7, cells grown for 1 d in PHB accumulation medium; 4 and 8, cells grown for 2 d in PHB accumulation medium; 5 and 9, cells grown for 3 d in PHB accumulation medium; 6 and 10, cells grown for 4 d in PHB accumulation; lane I, total protein (90 ug of protein); lanes 2-6, supematant fraction (70 ug of protein each); 7-10, PHB granules (20 ug of protein, 180 ug of PHB each).

showed no activity (Fig. 7B).

Fractionation by an anion exchange chromatography The crude extracts from the wild-type and S359A mutant cells were fractionated with RESOURCE Q, an anion exchange column. The activity of the wild-type enzyme was found in fractions eluted with 80-100 mM NaCl (data not shown). The Western blot of this fraction is shown in Fig. 7C, lane 1. The crude extract from the S359A mutant was fractionated under the same conditions. The Western blot of the fraction eluted with 80-100 mM NaCl showed that the expressed mutant protein migrated in a similar manner to the wild-type (Fig. 7C, lane 2). Other fractions did not show

Zhang ef al. (9) have cloned an extracellular 3HB-oligomer hydrolase gene from R. picke~~i~Al (IFrom the putative amino acid sequence, it was found to be composed of 722 amino acids (73,824Da). An alignment of deduced amino acid sequences of the oligomer hydrolases from Al and R. eu~ro~~ais shown in Fig. 4. Although two enzymes from R. eutropha and Al showed significant overall similarity, the N-terminal region of the intracellular 3HB-oligomer hydrolase from R. eutropha has relatively low homology to the Al enzyme. This may reflect the difference between intracellular and extracellular enzymes. Since the central region has high homology, this region probably contains the catalytic part, which was confirmed by site-direct mutagenesis. Another intracellul~ 3HB-oligomer hydrolase has been purified from Z. ramigeru 1-16-M by Tanaka et al. (8). The enzyme had a molecular mass of 28 kDa as determined by gel filtration and 30 kDa as estimated by SDS-polyacrylamide gel elec~ophoresis, both values being much smaller than that of the hydrolase in this study (78 kDa). There may be two types of intracellular oligomer hydrolases with low and high molecular masses. Poly-(R)-3-hy~oxyalk~oates (PHA) depolymerases, lipases and other esterases have the conserved serine-containing pentapeptide, Gly-X-Ser-X-Gly, which is called the “lipase box” and plays an essential role in catalysis by these enzymes (2, 21). Oligomer hydrolases and PHA depolymerases are similar in catalytic properties in terms of the hydrolysis of ester bonds of 3HB. Inspection of the sequence of the structural gene of the oligomer hydrolase from R. eutropha revealed the presence of the pentapeptide, Gly-X-Ser-X-Gly, residues 628 to 632 (Gly-Arg-Ser-AspGly) near the C-terminal and another pentapeptide, Ala-XSer-X-Gly, the sequence reported in a lipase of Bacillus subtiZi.s(22), at residues 216 to 220 (Ala-Thr-Ser-Ser-Gly). But the site-directed mutagenesis expe~ment showed that Ser-2 18 and Ser-630 were not the active-site serine. Among the other candidates, only the S359A mutant showed no activity. Substitution of a single residue may cause a decrease in enzymatic activity not directly related to the desertion of the catalytic site. Such critical residues may influence

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protein folding. However, in the anion exchange chromatography, the protein from S359A behaved in a similar manner to the wild-type enzyme. Therefore, we assume that the introduced substitutions did not change the overall protein conformation, and conclude that Ser-359 in the sequence, Ser-Val-Ser-Asn-Gly, was indeed the active-site serine. These results show the possibility that an active-site serine is located in not only the reported motif, Gly-X-SerX-Gly (21) or Ala-X-Ser-X-Gly (22), but also in another amino acid sequence, Ser-X-Ser-X-Gly, as indicated in this study. The remaining problem is to determine which histidine and aspartate make a classical charge-relayed triad together with Ser-359 in this hydrolase as in serine proteases (23) and extracellular PHB depolymerases (24). The biological role of the 3HB-oligomer hydrolase studied here is not clear, This enzyme seems to be induced when the cells accumulate PHB and is expressed both in the supernatant and in PHB granules. Since PHB depolymerase localized to PHB granules produces mainly 3HB-oligomers (5,6), the hydrolase bound to PHB granules may play a role in hydrolyzing 3HB-oligomers produced during the degradation of PHB. Certainly, further investigation is required to clarify this point. ACKNOWLEDGMENTS This study was performed as part of the Development of Biodegradable Plastics supported by the New Energy and Indus~~ Technology Development Organization (NEDO), and a Grant-inAid for Scientific Research on Priority Areas, “Sustainable Biodegradation Plastics,” no. 1127214 (1999), t?om the Ministry of Education, Science, Sports and Culture, Japan.

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