©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning, Sequencing, and Expression of the Genes Encoding Adenosylcobalamin-dependent Diol Dehydrase of Klebsiella oxytoca(*)

(Received for publication, October 24, 1994; and in revised form, December 21, 1994)

Takamasa Tobimatsu Tetsuya Hara Munetoh Sakaguchi Yasuhiro Kishimoto Yukihisa Wada Masaki Isoda Takahiro Sakai Tetsuo Toraya (§)

From the Department of Biotechnology, Faculty of Engineering, Okayama University, Tsushima-Naka, Okayama 700, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The pdd genes encoding adenosylcobalamin-dependent diol dehydrase of Klebsiella oxytoca were cloned by using a synthetic oligodeoxyribonucleotide as a hybridization probe followed by measuring the enzyme activity of each clone. Five clones of Escherichia coli exhibited diol dehydrase activity. At least one of them was shown to express diol dehydrase genes under control of their own promoter. Sequence analysis of the DNA fragments found in common in the inserts of these five clones and the flanking regions revealed four open reading frames separated by 10-18 base pairs. The sequential three open reading frames from the second to the fourth (pddA, pddB, and pddC genes) encoded polypeptides of 554, 224, and 173 amino acid residues with predicted molecular weights of 60,348 (alpha), 24,113 (beta), and 19,173 (), respectively. Overexpression of these three genes in E. coli produced more than 50-fold higher level of functional apodiol dehydrase than that in K. oxytoca. The recombinant enzyme was indistinguishable from the wild-type one of K. oxytoca by the criteria of polyacrylamide gel electrophoretic and immunochemical properties. It was thus concluded that these three gene products are the subunits of functional diol dehydrase. Comparisons of the deduced amino acid sequences of the three subunits with other proteins failed to reveal any apparent homology.


INTRODUCTION

Diol dehydrase (1,2-propanediol hydro-lyase, EC 4.2.1.28) is an enzyme that catalyzes the AdoCbl(^1)-dependent conversion of 1,2-diols to the corresponding deoxy aldehydes(1, 2) . It has been established that AdoCbl participates as an intermediate hydrogen carrier in the reactions catalyzed by diol dehydrase and other AdoCbl-requiring enzymes(3, 4, 5, 6, 7, 8, 9) . Although the mechanism of action as well as the structure-function relationship of the coenzyme has been extensively studied with diol dehydrase(7, 8) , only a little information is available about the structure and function of the apoenzyme in the catalysis.

In the previous papers(10, 11, 12) , we have reported that diol dehydrase purified from cell-free extracts (M(r) 230,000) of Klebsiella oxytoca (formerly Klebsiellapneumoniae and Aerobacter aerogenes) ATCC 8724 is composed of two dissimilar protein components, F and S. Poznanskaja et al.(12) reported that component F is a single polypeptide (M(r), 26,000), whereas component S consists of at least four polypeptides (M(r), 60,000, 23,000, 15,500, and 14,000). McGee and Richards (13) claimed that the enzyme isolated from detergent-extract of membrane (M(r), 250,000) is composed of M(r) 60,000, 51,000, 29,000, and 15,000 polypeptides(13, 14) . In contrast, a paper appeared that reported that component F is an essential constituent of the enzyme from detergent-extract of membrane as well(15) . We attempted to solve this apparent discrepancy by cloning the genes encoding this enzyme.

In this paper, we report cloning and sequence analysis of the pdd genes encoding diol dehydrase. Overexpression systems for the diol dehydrase genes in Escherichia coli are also described here, which promise us to get the enzyme in a large quantity for structural analysis.


EXPERIMENTAL PROCEDURES

Materials

[-P]ATP (specific activity, 5,000 Ci/mmol) and [alpha-S]dATPalphaS (specific activity, 1,500 Ci/mmol) were purchased from DuPont NEN. Crystalline AdoCbl was a gift from Eizai, Co., Ltd., Tokyo, Japan. Restriction endonucleases and the enzymes used for the construction of DNA library and expression plasmids and DNA sequencing were obtained from Toyobo Co., Osaka, Japan.

DNA Manipulations

Standard recombinant DNA techniques were performed as described by Sambrook et al.(16) . Genomic DNA from K. oxytoca was isolated by the procedure of Marmur (17) .

Construction and Screening of the K. oxytoca Genomic DNA Library

Random 6-20-kb fragments of genomic DNA from K. oxytoca ATCC 8724 were obtained by partial digestion with Sau3AI and cloned into the BamHI site of plasmid pUC119(18) . The transformation of E. coli HB101 and screening procedures were carried out as described previously (19) . A synthetic 50-mer oligodeoxyribonucleotide (5`-TCTTTCACGAAGCCGTCCTGGTTCACCGGGCGTTTCGCCAGCGCTTCGAA-3`), which is complementary to the DNA sequence deduced from the reported N-terminal amino acid sequence of the M(r) 60,000 subunit (FEALAKRPVNQDGFVKE) of diol dehydrase (13, 14) was used as a probe since this subunit was found in common by all the investigators (12, 13, 14, 15) . This probe labeled with P at the 5`-end was used for hybridization at 60 °C. Positive clones were isolated and characterized by restriction endonuclease mapping.

Enzyme and Protein Assays

Hybridization-positive E. coli clones as well as E. coli carrying plasmid pUC119 were cultivated aerobically at 37 °C in a modified glycerol/1,2-propanediol medium (20) supplemented with 40 µg/ml ampicillin and harvested in the late logarithmic phase. Cell homogenates prepared by sonication were assayed for diol dehydrase activity by the 3-methyl-2-benzothiazolinone hydrazone method(21) . One unit of diol dehydrase is defined as the amount of enzyme activity that catalyzes the formation of 1 µmol of propionaldehyde/min. Protein was assayed by the method of Lowry et al.(22) with crystalline bovine serum albumin as a standard. Specific activity is expressed as units/mg of protein.

Polyacrylamide Gel Electrophoresis and Activity Staining of Diol Dehydrase

Polyacrylamide gel electrophoresis of cell-free extracts was performed under nondenaturing conditions as described by Davis (23) in the presence of 0.1 M 1,2-propanediol or under denaturing conditions as described by Laemmli(24) . Protein staining was carried out with Coomassie Brilliant Blue R250. Activity staining for diol dehydrase was performed as follows. The gel was immersed in a solution consisting of 0.035 M potassium phosphate buffer (pH 8.0), 0.1 M 1,2-propanediol, 0.05 M KCl, and 15 µM AdoCbl and incubated at 37 °C for 10-15 min in the dark. The gel was immediately transferred to a solution of 0.4% (w/v) 2,4-dinitrophenylhydrazine in 2 N HCl and allowed to stand for 15 min. Excess 2,4-dinitrophenylhydrazine was removed by immersing in 2 N HCl solution with several changes. The band of diol dehydrase can be located by the precipitate of 2,4-dinitrophenylhydrazone of propionaldehyde.

Nucleotide Sequencing

The plasmid recovered from the E. coli strain that showed the highest activity of diol dehydrase was designated pUCDD11 and used for analysis of the nucleotide sequence. pUCDD11 was digested with SacI, treated by T4 DNA polymerase with dNTPs, ligated to HindIII linker CCAAGCTTGG, and cleaved with HindIII. The resulting 7.0-kb fragment was subcloned into pUC119(18) . pUCDD11 was digested with KpnI, treated by T4 DNA polymerase with dNTPs, and digested with EcoRI. The resulting 9.3-kb fragment was ligated with the 31-base pair HincII-EcoRI fragment of pUC119 to produce plasmid pUCDD11-1. The 6.2-kb XbaI fragment from pUCDD11-1 was inserted into XbaI site of pUC119 and selected for the plasmid having an insert DNA in the opposite direction (pUCDD11-2). Other restriction fragments obtained from pUCDD11 were also subcloned into pUC119. Deletion mutants were produced by exonuclease III and mung bean nuclease(25, 26) . DNA sequencing was performed by the dideoxynucleotide chain termination method of Sanger et al.(27) , as modified by Johnston-Dow et al.(28) , using Klenow fragment of E. coli DNA polymerase I or Sequenase from U. S. Biochemical Corp.

Construction of Expression Plasmid for Diol Dehydrase Genes

pUSI2(29) , an expression vector containing the tac promotor, was digested partially with EcoRI, treated by T4 DNA polymerase with a mixture of four dNTPs, and ligated. The remaining EcoRI site of the resulting DNA was eliminated by repeating the same treatments, and the plasmid possessing no EcoRI sites was selected. The plasmid was then digested with SmaI and ligated with EcoRI linker GGAATTCC. Digestion with EcoRI followed by self-ligation yielded expression vector pUSI2E. The 9.4-kb EcoRI-KpnI fragment from pUCDD11 and a 0.3-kb EcoRI-KpnI fragment from a deletion mutant (pUCDD11d505) containing nucleotide residues 2708-2960 (taking the first nucleotide of the translational initiation codon of ORF2 as 1) were ligated to produce plasmid pUCDD11Delta5. A DNA segment encoding the N-terminal region of the M(r) 28,000 polypeptide was amplified by PCR using a 5` oligonucleotide primer TCGGATCCTAGGAGGTTTAAACATATGAGCAGCAATGAGCTGGTGG (the Shine-Dalgarno sequence and the initiation codon of this ORF are underlined), and a 3` oligonucleotide primer GGGCGCCGAGAATGCCGATGGAGCG (complimentary to the nucleotide sequence 18-42 bases downstream of the unique HindIII site). The PCR product digested with BamHI and HindIII was cloned into HindIII-BamHI-digested pUC119 (pUC28N). The 0.2-kb BamHIHindIII fragment from pUC28N and the 3.6-kb HindIII-EcoRI fragment from pUCDD11Delta5 were ligated to pUSI2E previously linearized with BamHI and EcoRI to construct expression plasmid pUSI2E(1DD).

The DNA fragment encoding the N-terminal region of the M(r) 60,000 polypeptide was amplified by PCR using a 5` oligonucleotide primer GACATATGAGATCGAAAAGATTTGA (the initiation codon is underlined) and a 3` oligonucleotide primer GCATTTTCTGCATCGCCATC (complimentary to the nucleotides 378-397 of ORF2 encoding the 60,000 polypeptide). The PCR product was digested with NdeI and inserted into the NdeI site of pUC28N. A plasmid containing Shine-Dalgarno sequence and the insert DNA in the same direction was selected. The plasmid obtained was digested partially with NdeI and completely with BamHI. The resulting 0.4-kb BamHI-NdeI fragment and the 2.6-kb NdeI-EcoRI fragment from pUCDD11Delta5 were ligated to the 5.0-kb BamHI-EcoRI fragment from pUSI2E to construct pUSI2E(DD), another expression plasmid for diol dehydrase. It was confirmed by sequencing that no undesired mutations occurred during PCR reactions. Expression plasmids constructed were transformed to E. coli JM109 by the electroporation method as described by Dower et al.(30) . Recombinant E. coli strains harboring expression plasmids were aerobically grown in LB medium (16) containing 1,2-propanediol (0.1%) and ampicillin (50 µg/ml). When the culture reached an A of approximately 0.7, isopropyl-1-thio-beta-D-galactopyranoside was added for induction to a concentration of 1 mM. Cells were harvested in the late logarithmic phase.

The T7 expression system constructed by Studier et al.(31) was also examined for overproduction of diol dehydrase. pUSI2E(DD) was digested partially with NdeI and completely with EcoRI. The resulting 3.0-kb fragment was ligated to the 2.4-kb NdeI-EcoRI fragment from pRK172 to construct pRK172(DD). This expression plasmid was transformed to E. coli BL21(DE3)/pLysS by the electroporation method, and the transformant was cultured as described above, except that chloramphenicol (30 µg/ml) was also added together with ampicillin.

Western Blot Analysis

Cell-free extracts of transformed E. coli cells were subjected to 11% SDS-polyacrylamide gel electrophoresis (24) and transferred onto nitrocellulose membrane BA 85 (Schleicher & Schuell) by electroblotting. The diol dehydrase-derived proteins were probed with rabbit antiserum raised against K. oxytoca diol dehydrase (32) followed by treatment with alkaline phosphatase-conjugated goat anti-rabbit IgG antibody and located by reaction with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium.


RESULTS

Cloning of the Diol Dehydrase Genes

Genomic DNA from K. oxytoca ATCC 8724 was digested partially with restriction enzyme Sau3AI, and resulting 6-20-kb fragments were inserted into the BamHI site of plasmid pUC119(18) . E. coli HB101 was transformed with the plasmids. Ampicillin-resistant transformants were screened using the synthetic 50-mer oligodeoxyribonucleotide probe, which was complementary to the deduced DNA sequence from the N-terminal amino acid sequence of the M(r) 60,000 subunit of diol dehydrase(13, 14) . Twelve hybridization-positive clones were isolated from 4 times 10^3 transformants.

Twelve hybridization-positive E. coli clones cultivated aerobically in a glycerol/1,2-propanediol medium were examined for diol dehydrase activity. As shown in Table 1, specific activity of homogenates of the clones carrying plasmid pUCDD11 or pUCDD3 was rather higher than that of K. oxytoca ATCC 8724 grown in the same medium under aerobic conditions (0.55 unit/mg of protein). Clones carrying pUCDD4, pUCDD7, or pUCDD12 exhibited low but definite activity. Enzyme activity of the other seven clones as well as E. coli HB101 transformed with parent vector pUC119 was less than 0.001 unit/mg of protein. Plasmid pUCDD11, expressing the highest diol dehydrase activity in E. coli, was used for further analysis.



Identification of the Gene Products Expressed in E. coli Strain Carrying Plasmid pUCDD11

Cell-free extract of the recombinant E. coli strain carrying plasmid pUCDD11 was analyzed by polyacrylamide gel electrophoresis under nondenaturing conditions according to the method of Davis (23) in the presence of 1,2-propanediol(12) . The extract of E. coli carrying plasmid pUCDD11 contained a new protein band (marked with arrowhead in Fig. 1A, lane3). When the gel was subjected to activity staining by visualizing propionaldehyde through reaction with 2,4-dinitrophenylhydrazine, this band was the only one that converted 1,2-propanediol to propionaldehyde in the presence of AdoCbl (Fig. 1C, lane3). The propionaldehyde formation was not observed in the absence of AdoCbl (Fig. 1B, lane3). The cell-free extract of K. oxytoca showed the band of diol dehydrase in the same position (Fig. 1, A and C, lane1), whereas the extract of E. coli HB101 transformed with pUC119 did not (Fig. 1, A and C, lane2). The recombinant diol dehydrase formed in E. coli co-migrated with diol dehydrase of K. oxytoca (Fig. 1, A and C, lane4). Furthermore, diol dehydrase activity in the extract of the recombinant E. coli strain was immunoprecipitated by rabbit antiserum against diol dehydrase of K. oxytoca(32) (data not shown). Therefore, it was suggested that the diol dehydrase formed in E. coli carrying plasmid pUCDD11 is identical to the wild-type enzyme of K. oxytoca.


Figure 1: Expression of diol dehydrase in E. coli HB101 carrying plasmid pUCDD11. Cell-free extracts of K. oxytoca (lane1), E. coli HB101 carrying vector pUC119 (lane2), or plasmid pUCDD11 (lane3), and a mixture of extracts of K. oxytoca and E. coli HB101 carrying pUCDD11 (lane4) were electrophoresed on 5% polyacrylamide gel under nondenaturing conditions. Resulting gel was subjected to protein staining (A) or activity staining without (B) or with (C) AdoCbl. Experimental details are described in the text.



Characterization of the Diol Dehydrase Genes

Restriction map of pUCDD11 is shown in Fig. 2. pUCDD11 carried a 10.5-kb DNA insert which gave 1.3-, 0.4-, 1.6-, 0.3-, 0.9-, 1.8-, 2.0-, and 0.5-kb fragments upon digestion with PstI. Among them, the 0.4-, 1.6-, 0.3-, and 0.9-kb PstI fragments were found in common in the inserts of all the five clones expressing diol dehydrase activity in E. coli, whereas the 1.8-, 2.0-, and 0.5-kb fragments were not found in pUCDD3 and pUCDD7, and the 1.3-kb PstI fragment not found in pUCDD12 (data not shown). Therefore, it was suggested that diol dehydrase is encoded within the region containing the 1.3-, 0.4-, 1.6-, 0.3-, 0.9-, and 1.8-kb PstI fragments. The E. coli strain carrying pUCDD11 expressed diol dehydrase activity when grown in the glycerol/1,2-propanediol medium, but not in a glucose medium. The addition of isopropyl-1-thio-beta-D-galactopyranoside to the medium did not increase but rather decreased the level of enzyme activity. These results suggest that the diol dehydrase genes in pUCDD11 are under control not of the lac promoter of pUC119 but of their own promoter.


Figure 2: Restriction map of the insert DNA of pUCDD11 and sequencing strategy. The restriction map is drawn to scale. ORFs are indicated by the open boxes. The direction and extent of sequence determinations are shown by the horizontal arrows.



Nucleotide Sequencing of Plasmid pUCDD11 and Assignment to Protein Sequence

The DNA region containing the 1.3-, 0.4-, 1.6-, 0.3-, 0.9-, and 1.8-kb PstI fragments was subjected to nucleotide sequence analysis according to the strategy shown in Fig. 2. As summarized in Fig. 2and Fig. 3, there existed four successive open reading frames (ORF1-ORF4) encoding polypeptides of 270, 554, 224, and 173 amino acid residues with predicted molecular weights of 28,071, 60,348, 24,113, and 19,173, respectively. The fourth ORF was followed by another ORF (ORF5), whose putative initiation codon (GTG) was found 41-bases downstream of the termination codon of ORF4. However, the termination codon of ORF5 did not appear within the 1.8-kb PstI fragment. ORF2-ORF4 were separated from their upstream ORFs by 18, 10, and 14 bases, respectively. Shine-Dalgarno sequences were found 8-12 bases upstream of the putative initiation codon (ATG) for each polypeptide. The deduced N-terminal amino acid sequences of 60,000, 24,000, and 19,000 polypeptides shown in Fig. 3coincided with the N-terminal 36 residues of the M(r) 60,000 subunit, 39 residues of the M(r) 29,000 subunit, and 40 residues of the M(r) 15,000 subunit of diol dehydrase reported by McGee et al.(14) , respectively. The deduced sequence of the 60,000 polypeptide contained the same sequences as two proteolytic peptides. (^2)Amino acid compositions of those polypeptides are in reasonable agreement with those reported by McGee et al.(14) . The N-terminal amino acid sequence of the M(r) 51,000 subunit reported by McGee et al.(14) was not encoded in the DNA region shown in Fig. 2.


Figure 3: Nucleotide sequences and deduced amino acid sequences of pddA, pddB, and pddC genes encoding the alpha, beta, and subunits of diol dehydrase, respectively. Nucleotides are numbered beginning with the first nucleotide of the translational initiation codon of the alpha subunit. Amino acids are numbered beginning with the N-terminal residue of each subunit. The solid underline indicates the region corresponding to the probe (50-mer), which was used for screening of the genomic DNA library. The broken underlines show the sequences that match the N-terminal amino acid sequences reported by McGee et al.(14) and amino acid sequences of two proteolytic peptide fragments.^2 The ribosome binding sites (Shine-Dalgarno sequences) are shown in the dotted underlines.



Construction of Expression Plasmid for Diol Dehydrase Genes

As the diol dehydrase genes were suggested to exist within the region containing the six PstI fragments listed above, we constructed a deletion mutant plasmid that was defective in most of ORF5 and the downstream region. The mutant plasmid, pUCDD11Delta5, which carried ORF1-ORF4 and lacked ORF5 except for the first 34 nucleotides was transformed into E. coli HB101. The homogenate of the transformant exhibited diol dehydrase activity of 0.74 unit/mg of protein, which was comparable with that of E. coli HB101 carrying pUCDD11 (1.2 units/mg of protein). It is therefore evident that the polypeptide encoded by ORF5 is not a functional subunit of diol dehydrase. In order to confirm this conclusion and to determine whether the M(r) 28,000 polypeptide is essential for diol dehydrase activity, we constructed two expression plasmids. As shown in Fig. 4, pUSI2E(1DD) contained the first four ORFs encoding the M(r) 28,000, 60,000, 24,000, and 19,000 polypeptides downstream of the tac promoter. pUSI2E(DD) containing ORF2-ORF4 encoding the M(r) 60,000, 24,000, and 19,000 polypeptides in the same position was also constructed. For overexpression of the diol dehydrase genes, T7 expression system of Studier et al.(31) was also examined. pRK172(DD) containing the same ORFs as pUSI2E(DD) downstream of T7 promoter was constructed. All of the three expression plasmids retained the first 34 nucleotides of ORF5.


Figure 4: The plasmids constructed for high level expression of the diol dehydrase genes. The construction of pUSI2E is described in the text. Open boxes, ORFs; small open boxes, Shine-Dalgarno sequences; lpp3`, E. coli lipoprotein gene 3`-region including transcriptional terminator.



High-Level Expression of Diol Dehydrase Genes and Characterization of the Gene Products

E. coli JM109 cells transformed with an expression vector were cultured as described above in LB + 1,2-propanediol medium in the presence of isopropyl-1-thio-beta-D-galactopyranoside, and diol dehydrase activity of cell homogenates was determined. As shown in Table 2, when cultivated at 30 °C, specific activity of homogenates of both E. coli stains carrying pUSI2E(1DD) and pUSI2E(DD) were 38 and 53 times higher, respectively, than that of K. oxytoca ATCC 8724 cultivated aerobically in the glycerol/1,2-propanediol medium. E. coli carrying pUSI2E(DD) grown at 37 °C showed almost the same specific activity as that grown at 30 °C.



The E. coli BL21(DE3)/pLysS cells transformed with pRK172(DD) were cultivated in a similar manner at 30 or 37 °C, and diol dehydrase activity of homogenates was determined. As shown in Table 2, specific activity of the cells grown at 30 °C was higher than that of the cells grown at 37 °C. The former was 35 times higher than that of K. oxytoca ATCC 8724 but a little lower than that of the cells transformed with pUSI2E(1DD) or pUSI2E(DD). In the following experiments, characterization of recombinant diol dehydrase was performed using E. coli JM109 carrying pUSI2E(DD) or pUSI2E(1DD).

Diol dehydrase in cell-free extracts was subjected to polyacrylamide gel electrophoresis under nondenaturing conditions and located by protein staining and activity staining. As shown in Fig. 5A and B, electrophoretic mobility of recombinant diol dehydrase in cell-free extracts of E. coli carrying pUSI2E(1DD) (Fig. 5, A and B, lane3) or pUSI2E(DD) (Fig. 5, A and B, lane4) was in good agreement with that of diol dehydrase in the extract of K. oxytoca (Fig. 5, A and B, lane 1) (marked with arrowhead in the left) upon both protein staining and activity staining. These results suggest that inclusion of the three ORFs (ORF2-ORF4) encoding M(r) 60,000, 24,000, and 19,000 polypeptides in an expression plasmid was sufficient to form high levels of functional diol dehydrase. It is therefore strongly suggested that ORF2, ORF3, and ORF4 are the genes encoding the subunits of diol dehydrase. These were designated pddA, pddB, and pddC genes, respectively.


Figure 5: Polyacrylamide gel electrophoresis of cell-free extracts of E. coli carrying expression plasmids. Cell-free extracts of K. oxytoca (lane1) and E. coli JM109 carrying pUSI2E (lane2), pUSI2E(1DD) (lane3), or pUSI2E(DD) (lane4) were electrophoresed on 7% polyacrylamide gel under nondenaturing conditions. Resulting gel was subjected to protein staining (A) or activity staining (B). Experimental details are described in the text.



For further characterization of the gene products, Western blot analysis of cell-free extracts was performed using anti-K. oxytoca diol dehydrase antiserum (32) as probe. When polyacrylamide gel electrophoresis was performed under denaturing conditions, the extract of E. coli carrying pUSI2E(DD) contained the three thick protein bands with M(r) of 60,000, 30,000, and 19,000 (marked with arrowhead in Fig. 6A, lane3), which reacted with anti-diol dehydrase antiserum (Fig. 6B, lane3). These three bands were the same in size as the three subunits detected in the cell-free extract of K. oxytoca with the antiserum (Fig. 6B, lane1). No bands reactive with the antiserum were observed in the cell-free extract of E. coli carrying pUSI2E (control) (Fig. 6B, lane2). The M(r) 60,000, 30,000, and 19,000 subunits were designated alpha, beta, and subunits, respectively.


Figure 6: Western blot analysis of homogenates of E. coli carrying expression plasmids. Cell-free extracts of K. oxytoca (lane1) and E. coli JM109 carrying pUSI2E (lane2) or pUSI2E(DD) (lane3) were electrophoresed on 11% SDS-polyacrylamide gel. Resulting gel was subjected to protein staining (A) or Western blotting with anti-diol dehydrase antiserum (B). Experimental details are described in the text. LaneM, molecular weight markers (Sigam Dalton Mark VII-L).



Recombinant diol dehydrase formed in E. coli carrying pUSI2E(DD) was further analyzed by two-dimensional gel electrophoresis, i.e. polyacrylamide gel electrophoresis in the presence of 1,2-propanediol (nondenaturing conditions) followed by SDS-polyacrylamide gel electrophoresis (denaturing conditions). Functional diol dehydrase migrated as a single band under nondenaturing conditions in the presence of substrate (marked with arrowhead on the top of Fig. 7A). As shown in Fig. 7A, the band of diol dehydrase then dissociated into the three polypeptides upon SDS-polyacrylamide gel electrophoresis. Their M(r) of 60,000, 30,000, and 19,000 coincided with those of the subunits recognized with anti-diol dehydrase antiserum in the Western blot analysis (Fig. 6). When the extract of E. coli carrying pUSI2E(1DD) was analyzed in a similar manner, essentially the same results were obtained (data not shown). None of these bands were detected in the gel with the extract of E. coli carrying pUSI2E (control). From all of these results, it can be concluded that diol dehydrase apoenzyme is composed of the M(r) 60,000 (alpha), 30,000 (beta), and 19,000 () subunits that are encoded by the pddA, pddB, and pddC genes, respectively. It is also evident that the gene product of ORF1 is not a functional subunit of the enzyme.


Figure 7: Two-dimensional gel electrophoresis of cell-free extract of E. coli carrying expression plasmids. Cell-free extracts of E. coli JM109 carrying pUSI2E(DD) (A) or pUSI2E (B) were electrophoresed on 7% polyacrylamide gel under nondenaturing conditions (first dimension, from left to right) and then on 11% SDS-polyacrylamide gel under denaturing conditions (second dimension, from top to bottom).




DISCUSSION

In this study, we revealed by gene cloning and expression that functional diol dehydrase consists of the M(r) 60,000 (alpha), 30,000 (beta), and 19,000 () subunits. No additional subunits were required for activity, since specific activity of the recombinant diol dehydrase purified from E. coli carrying pUSI2E(DD) (86 units/mg of protein) was essentially the same as that of the enzyme purified from cell-free extracts of K. oxytoca (88 units/mg of protein). Apparent M(r) of the beta subunit obtained from the calibration curve (30,000) was a little larger than calculated (24,000) from the deduced amino acid sequence from the pddB gene, although M(r) of the alpha and subunits (60,000 and 19,000) were just as calculated from the pddA and pddC genes, respectively.

We have previously reported that diol dehydrase is separated into components F and S upon chromatography on DEAE-cellulose (11) or DEAE-Sephadex A-50 in the absence of substrate(12, 15) . Neither component alone is inactive, but enzymic activity is restored when both are combined(11) . M(r) of F and S determined by gel filtration are 26,000 and 200,000, respectively(12) . We confirmed that recombinant diol dehydrase was also separated into F and S and that F and S included the M(r) 30,000 subunit and the M(r) 60,000 + 19,000 subunits, respectively (data not shown).

McGee and co-workers (13, 14) reported that the M(r) 51,000 polypeptide is also a subunit of diol dehydrase. However, the nucleotide sequence corresponding to the N-terminal 40 amino acid residues described by them was not found in the insert DNA of pUCDD11. Functional diol dehydrase was formed by expression of the pddA, pddB, and pddC genes encoding the alpha, beta, and subunits, respectively, indicating that the M(r) 51,000 polypeptide is not a functional subunit of diol dehydrase. The M(r) 51,000 polypeptide was not present in the enzyme purified from cell-free extracts(12) . At present, the relation of the M(r) 51,000 polypeptide to diol dehydrase remains obscure.

Four ORFs lie adjacent to each other on the genomic DNA and seem to constitute an operon. Expression of ORF1 using plasmid pUSI2E(1DD) resulted in overproduction of another 30,000 polypeptide (data not shown). High-level expression of ORF5 in E. coli has not yet been attempted. Functions of the products of these two genes have not yet been characterized. They may play certain roles in the fermentation of 1,2-propanediol(33) .

The deduced amino acid sequences of the diol dehydrase subunits did not show significant homology to those of proteins listed in the PIR and SWISSPROT data bases when analyzed using FASTA program(34) . No significant similarity was found between the subunits of diol dehydrase and other cobalamin-dependent enzymes or cobalamin-binding proteins. Motifs of a binding site for a large molecule, such as AdoCbl, may not be apparent.


FOOTNOTES

*
This work was supported in part by Grants-in-aid for Scientific Research on Priority Areas (04220227 and 06240237) and for Encouragement of Young Scientists(04750798) from the Ministry of Education, Science, and Culture, Japan, and research grants from Nagase Science and Technology Foundation and from Okayama Foundation for Science and Technology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D45071[GenBank].

§
To whom correspondence should be addressed: Fax: 81-86-253-7399.

(^1)
The abbreviations used are: AdoCbl, adenosylcobalamin; kb, kilobase(s); ORF, open reading frame; pdd, genes encoding diol dehydrase or propanediol dehydratase; dATPalphaS, 2`-deoxyadenosine 5`-(alpha-thio)triphosphate; PCR, polymerase chain reaction.

(^2)
K. Tanizawa and H. Kimimoto, personal communication.


ACKNOWLEDGEMENTS

We thank Drs. Y. Morimoto and O. Shibui, Mitsubishi Chemical Industries for supplying expression vector pUSI2 and Associate Professor K. Tanizawa and H. Kumimoto, Osaka University, for providing information concerning amino acid sequences of proteolytic peptide fragments from the 60,000 subunit (Fig. 3). Computer analysis of the amino acid sequences was carried out using programs and data bases in DNA Data Bank of Japan (DDBJ), Mishima, Japan. We also thank Y. Kurimoto for assistance in manuscript preparation.


REFERENCES

  1. Lee, H. A., Jr., and Abeles, R. H. (1963) J. Biol. Chem. 238, 2367-2373 [Free Full Text]
  2. Toraya, T., Shirakashi, T., Kosuga, T., and Fukui, S. (1976) Biochem. Biophys. Res. Commun. 69, 475-480 [Medline] [Order article via Infotrieve]
  3. Frey, P. A., and Abeles, R. H. (1966) J. Biol. Chem. 241, 2732-2733 [Abstract/Free Full Text]
  4. Frey, P. A., Essenberg, M. K., and Abeles, R. H. (1967) J. Biol. Chem. 242, 5369-5377 [Abstract/Free Full Text]
  5. Frey, P. A., Essenberg, M. K., Abeles R. H., and Kerwar, S. S. (1970) J. Am. Chem. Soc. 92, 4488-4489 [Medline] [Order article via Infotrieve]
  6. Essenberg, M. K., Frey, P. A., and Abeles, R. H. (1971) J. Am. Chem. Soc. 93, 1242-1251 [Medline] [Order article via Infotrieve]
  7. Abeles, R. H. (1979) in Vitamin B (Zagalak, B., and Friedrich, W., eds) pp. 373-388, Walter de Gruyter, Berlin
  8. Toraya, T., and Fukui, S. (1982) in B (Dolphin, D., ed) Vol. 2, pp. 233-262, John Wiley & Sons, Inc., New York
  9. Babior, B. M. (1988) Biofactors. 1, 21-26 [Medline] [Order article via Infotrieve]
  10. Toraya, T., Uesaka, M., Kondo, M., and Fukui, S. (1973) Biochem. Biophys. Res. Commun. 52, 350-355 [Medline] [Order article via Infotrieve]
  11. Toraya, T., Uesaka, M., and Fukui, S. (1974) Biochemistry 13, 3895-3899 [Medline] [Order article via Infotrieve]
  12. Poznanskaja, A. A., Tanizawa, K., Soda, K., Toraya, T., and Fukui, S. (1979) Arch. Biochem. Biophys. 194, 379-386 [Medline] [Order article via Infotrieve]
  13. McGee, D. E., and Richards, J. H. (1981) Biochemistry 20, 4293-4298 [Medline] [Order article via Infotrieve]
  14. McGee, D. E., Carroll, S. S., Bond, M. W., and Richards, J. H. (1982) Biochem. Biophys. Res. Commun. 108, 547-551 [Medline] [Order article via Infotrieve]
  15. Tanizawa, K., Nakajima, N., Toraya, T., Tanaka, H., and Soda, K. (1987) Z. Naturforsch. 42, 353-359
  16. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  17. Marmur, J. (1961) J. Mol. Biol. 3, 208-218
  18. Vieira, J., and Messing, J. (1987) Methods Enzymol. 153, 3-11 [Medline] [Order article via Infotrieve]
  19. Tobimatsu, T., Kameshita, I., and Fujisawa, H. (1988) J. Biol. Chem. 263, 16082-16086 [Abstract/Free Full Text]
  20. Toraya, T., and Ishida, A. (1988) Biochemistry 27, 7677-7681 [Medline] [Order article via Infotrieve]
  21. Toraya, T., Ushio, K., Fukui, S., and Hogenkamp, H. P. C. (1977) J. Biol. Chem. 252, 963-970 [Abstract]
  22. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 [Free Full Text]
  23. Davis, B. J. (1964) Ann. N. Y. Acad. Sci. 121, 404-427 [Medline] [Order article via Infotrieve]
  24. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  25. Henikoff, S. (1984) Gene (Amst.) 28, 351-359 [CrossRef][Medline] [Order article via Infotrieve]
  26. Barnes, W. M. (1987) Methods Enzymol. 152, 538-556 [Medline] [Order article via Infotrieve]
  27. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 [Abstract]
  28. Johnston-Dow, L., Mardis, E., Heiner, C., and Roe, B. A. (1987) BioTechniques 5, 754-765
  29. Shibui, T., Uchida, M., and Teranishi, Y. (1988) Agric. Biol. Chem. 52, 983-988
  30. Dower, W. J., Miller, J. F., and Ragsdale, C. W. (1988) Nucleic Acids Res. 16, 6127-6145 [Abstract]
  31. Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990) Methods Enzymol. 185, 60-89 [Medline] [Order article via Infotrieve]
  32. Toraya, T., and Fukui, S. (1977) Eur. J. Biochem. 76, 285-289 [Abstract]
  33. Toraya, T., Honda, S., and Fukui, S. (1979) J. Bacteriol. 139, 39-47 [Medline] [Order article via Infotrieve]
  34. Pearson, W. R., and Lipman, D. J. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 2444-2448 [Abstract]

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