(Received for publication, October 24, 1994; and in revised form, December 21, 1994)
From the
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 (), 24,113 (
), 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.
Diol dehydrase (1,2-propanediol hydro-lyase, EC 4.2.1.28) is an
enzyme that catalyzes the AdoCbl()-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
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
,
26,000), whereas component S consists of at least four polypeptides (M
, 60,000, 23,000, 15,500, and 14,000). McGee and
Richards (13) claimed that the enzyme isolated from
detergent-extract of membrane (M
, 250,000) is
composed of M
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.
The DNA
fragment encoding the N-terminal region of the M 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 pUCDD11
5 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-
-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.
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.
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.
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.
Figure 3:
Nucleotide sequences and deduced amino
acid sequences of pddA, pddB, and pddC genes
encoding the ,
, and
subunits of diol dehydrase,
respectively. Nucleotides are numbered beginning with the first
nucleotide of the translational initiation codon of the
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.
The ribosome binding sites
(Shine-Dalgarno sequences) are shown in the dotted
underlines.
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.
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 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 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
60,000, 30,000, and 19,000
subunits were designated
,
, 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 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
60,000 (
), 30,000 (
), 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).
In this study, we revealed by gene cloning and expression
that functional diol dehydrase consists of the M 60,000 (
), 30,000 (
), 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
of the
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
of the
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 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
30,000 subunit
and the M
60,000 + 19,000 subunits,
respectively (data not shown).
McGee and co-workers (13, 14) reported that the M 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
,
, and
subunits, respectively, indicating that the M
51,000 polypeptide is not a functional subunit
of diol dehydrase. The M
51,000 polypeptide was
not present in the enzyme purified from cell-free
extracts(12) . At present, the relation of the M
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D45071[GenBank].