From the Edinburgh Centre for Protein Technology, Department of Chemistry, University of Edinburgh, The King's Buildings, West Mains Road, Edinburgh EH9 3JJ, United Kingdom
Received for publication, December 21, 2000
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ABSTRACT |
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The enzyme 6-oxocamphor hydrolase,
which catalyzes the desymmetrization of 6-oxocamphor to yield
(2R,4S)- The desymmetrization of prochiral substrates in organic synthesis
remains a powerful technique for the generation of chiral intermediates
with, in principle, 100% yield with absolute optical purity. In
addition to chemical processes that have been reviewed recently (1),
enzyme-catalyzed methods have assumed an important role in
desymmetrization (2) owing to the well documented advantages of
biocatalysis in general. Such methods have usually exploited the
regioselectivity of a hydrolytic enzyme, such as a lipase or nitrilase,
to effect transformation of one of two identical functions in a
molecule (3, 34). Enzymatic desymmetrizations have for the most
part been performed using carbon-heteroatom bond hydrolases of this
type, although Taschner and Black (4) were successful in
desymmetrizing a series of prochiral cyclic ketones using an enzymatic
Baeyer-Villiger reaction.
The metabolism of (1R)-(+)-camphor by
Corynebacterium sp. T1 (now taxonomically reclassified and
deposited as Rhodococcus sp. NCIMB 9784) was described in
the 1960s by Gunsalus and co-workers (5). The pathway is distinct from
that found in Pseudomonas putida (ATCC 17453; NCIMB 10007)
in that initial hydroxylation occurs in the 6-endo position
of the camphor skeleton (Fig. 1). 6-endo-Hydroxy camphor 2 is oxidized to a
symmetrical diketone 3, which is then cleaved by a
retro-Claisen reaction to yield a keto acid 4, which was
reported by Gunsalus and co-workers (5) to have a negative
optical rotation. This last enzymatic reaction is in fact a
desymmetrization, and is interesting in that it apparently proceeds by
an unusual enzyme-catalyzed retro-Claisen reaction. Enzymes
that hydrolyze 1,3-diketo functionality, -campholinic acid, has been purified
with a factor of 35.7 from a wild type strain of
Rhodococcus sp. NCIMB 9784 grown on
(1R)-(+)-camphor as the sole carbon source. The enzyme has
a subunit molecular mass of 28,488 Da by electrospray mass
spectrometry and a native molecular mass of ~83,000 Da indicating
that the active protein is trimeric. The specific activity was
determined to be 357.5 units mg
1, and the
Km was determined to be 0.05 mM for the
natural substrate. The N-terminal amino acid sequence was obtained from the purified protein, and using this information, the gene encoding the
enzyme was cloned. The translation of the gene was found to bear
significant homology to the crotonase superfamily of enzymes. The gene
is closely associated with an open reading frame encoding a ferredoxin
reductase that may be involved in the initial step in the
biodegradation of camphor. A mechanism for 6-oxocamphor hydrolase based on sequence homology and the known mechanism of the
crotonase enzymes is proposed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-diketone hydrolases
(6) are rare, and only three reports describe their purification to
homogeneity and characterization (7, 8, 15, 35). In the first
two cases, the substrate specificity of the enzymes extends only to
3,5-diketo acids. This specificity has been recently explained by
elucidation of the x-ray crystal structure of fumarylacetoacetate
hydrolase (9), which shows a dependence on a divalent calcium ion for
substrate recognition and enolate stabilization.
View larger version (7K):
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Fig. 1.
Metabolism of
(1R)-(+)-camphor 1 by Rhodococcus sp.
NCIMB 9784. i, cytochrome P450camr; ii,
alcohol dehydrogenase; iii, 6-oxocamphor hydrolase.
In view of the ongoing interest in the biocatalytic generation of
chiral intermediates and desymmetrization in particular, we were
interested in studying the potential of the enzyme, which we have named
6-oxocamphor hydrolase, for the desymmetrization of other cyclic and
bicyclic -diketones. In this paper, we present the purification and
initial characterization of the enzyme from the wild type strain of
Rhodococcus and the cloning of the gene encoding its
activity. Our findings suggest that the enzyme does not share homology
with other
-diketone hydrolases but rather is related to a
different class of enzyme, the crotonase superfamily.
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EXPERIMENTAL PROCEDURES |
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Enzymes and Chemicals--
Restriction endonucleases were from
New England Biolabs. T4 DNA ligase, T4 polynucleotide kinase, and calf
intestinal alkaline phosphatase were from Roche Molecular
Biochemicals. RNase was purchased from Sigma-Adrich (Poole,
United Kingdom). Protein and DNA size markers were obtained from
(Amersham, United Kingdom) Pharmacia Biotech.
[-32P]ATP (3000 Ci/mmol) was purchased from Amersham
Pharmacia Biotech. Isopropyl-
-D-thiogalactopyranoside
was obtained from U. S. Biochemical Corp. All other chemicals
were purchased from Sigma-Aldrich. 6-Oxocamphor 3 was
synthesized by pyridinium chlorochromate oxidation (10) of
6-endo-hydroxy camphor derived from ethyl acetate
extractions of the mother liquor of fermentations of
Rhodococcus sp. NCIMB 9784 grown on
(1R)-(+)-camphor performed as described below.
Maintenance and Growth of Microorganisms--
Escherichia
coli XL1 Blue supercompetent cells were obtained from Stratagene
(La Jolla, CA) and grown on 1% yeast extract, 1% Tryptone, and 0.5%
NaCl at 37 °C. Rhodococcus sp. NCIMB 9784 was obtained
from the National Culture of Industrial and Marine Bacteria (Aberdeen,
United Kingdom). The bacterium was maintained on nutrient agar slopes
at room temperature. Ten 250-ml shake flasks containing 50 ml of basal
salts medium supplemented with 35 mM sodium pyruvate
were inoculated from slope and grown on an orbital shaker at 220 rpm at
30 °C for 3 days. This combined inoculum was used to seed 10 liters
of basal salts medium in a 12-liter fermentation vessel (BioFlo 1000 fermenter, New Brunswick Scientific) supplemented with 1 g
liter1 (R)-(+)-camphor (Aldrich).
Following 2 days of growth at 30 °C with an impeller speed of 250 rpm and an air flow of 4 liters min
1,
a further 1 g liter
1 was added. After an
additional 1 day of growth, the bacteria were harvested by
centrifugation to yield a typical wet weight of 4 g
liter
1.
Enzyme Assays--
Activity of 6-oxocamphor hydrolase was
monitored using a Hewlett Packard 8453 UV-visible spectrophotometer.
The assay was performed as follows: to a 3-ml stirred cuvette
containing 2990 µl of 50 mM Tris/HCl buffer, pH 7.0 (henceforth referred to as "buffer") and the appropriate
concentration of substrate (for standard assays, 100 µM)
was added 10 µl of enzyme solution, and the disappearance of
substrate was measured using a decrease in absorption at 294 nm.
Calculations were made using an value of 258 mol
dm
3 cm
3 for
6-oxocamphor. For inhibition/activation studies, 2990 µl of
buffer containing 3 units of 6-oxocamphor hydrolase and the appropriate concentration of additive were preincubated at 25 °C
prior to assay. The assay was initiated by addition of 10 µl of a 30 mM ethanolic solution of the substrate.
Enzyme Purification--
40 g of cell paste was suspended in 150 ml of buffer, and to this suspension was added 150 ml of 5-mm glass
beads. The mixture was homogenized twice for 15 min each at 4500 rpm in a Type KDL Dynomill. The resulting homogenate was centrifuged at
11,000 rpm using a GSA rotor to yield a cell extract (320 ml)
from which a 40-80% ammonium sulfate cut was derived. After
dissolution in buffer and dialysis against 6 liters of the same buffer
(with one change), the protein solution (100 ml) was taken to a
concentration of 1.7 M ammonium sulfate and loaded onto a
2.5 × 10-cm phenyl Sepharose column that was eluted with a
decreasing gradient of ammonium sulfate. Fractions exhibiting
6-oxocamphor hydrolase activity were pooled and precipitated by the
addition of 80% ammonium sulfate. After overnight dialysis against 6 liters of buffer at 4 °C, the protein solution (25 ml) was applied
to a 2.5 × 10-cm Fast Flow Q Sepharose column and eluted with an
increasing gradient of potassium chloride (0-0.5 M).
Active fractions were pooled, precipitated, and dialyzed as above. The
resulting protein solution (6 ml) was taken to a concentration of 1.7 M ammonium sulfate and divided into two aliquots, which
were separately loaded onto a prepacked H/R 5/5 phenyl Superose
column (1 ml) and eluted with a decreasing gradient of salt. Active
fractions were identified, and their purity was assessed by
SDS-polyacrylamide gel electrophoresis prior to being pooled.
The enzyme was stored at 80 °C for at least six weeks with
no discernible loss of activity.
The isoelectric point of the purified protein was determined by isoelectric focusing using an Amersham Pharmacia Biotech Phastsystem. The native molecular weight was determined by calibrated gel filtration chromatography on a Superose 12 gel filtration column against a range of commercially available standards (Amersham Pharmacia Biotech). The void volume of the column was determined using blue dextran 2000. N-terminal sequencing was performed according to the method of Hayes et al. (11), and 18 residues were unambiguously assigned as Met-Lys-Gln-Leu-Ala-Thr-Pro-Phe-Gln- Glu-Tyr-Ser-Gln-Lys-Tyr-Glu-Asn-Ile.
Liquid chromatography-mass spectrometry of pure 6-oxocamphor
hydrolase was performed on a Waters/Alliance 2690 HPLC system fitted
with a Phenomenex Jupiter C18 300-Å, 250 mm × 2 mm × 5 µm column. The flow rate was 200 µl
min1, and a gradient of 0-95%
water/acetonitrile was employed. The liquid chromatography
apparatus was fitted to a Waters 286 UV detector and a Micromass
Platform II single quadrupole mass spectrometer utilizing an
electrospray ionization source controlled via the VG Mass-Lynx software
(VG Biotechnology Ltd., Altrincham, Cheshire, United Kingdom). The
source temperature was 140 °C. Capillary voltage was 3.3 kV, and the
cone voltage was ramped from 40-90 V over a range of 500-2000
m/z. The instrument was calibrated over this
Mr range with horse heart myoglobin (Sigma).
Nucleic Acid Preparation and Cloning-- Genomic DNA was prepared by a modified version of the method described by Kulakova et al. (12). Approximately 0.5 g of wet cell paste was washed twice in 5 ml of 10 mM EDTA, pH 8.0, and then resuspended in 5 ml of 75 mM NaCl, 25 mM EDTA, pH 8.0, 20 mM Tris/HCl, pH 8.0, containing 5 mg/ml lysozyme. The mixture was incubated for 2 h at 37 °C before adding 50 µl of proteinase K solution (20 mg/ml) and 300 µl of 10% (w/v) SDS. This was incubated for a further 2 h at 55 °C with occasional inversion. The solution was extracted once with phenol (equilibrated with Tris/HCl, pH 8.0) and then twice with chloroform. DNA was precipitated with isopropanol and spooled onto a glass rod. After rinsing with 70% (v/v) ethanol, the DNA was air-dried and dissolved in 0.5 ml of TE buffer (10 mM Tris HCl, pH8, 1 mM EDTA pH8).
A partially degenerate oligonucleotide (36-mer) designed against the
N-terminal sequence (residues 7-18) of the 6-oxocamphor hydrolase
activity was synthesized as a hybridization probe:
5'-CCSTTCCAGGAGTACWSSCAGAAGTACGAGAACATC-3' (where S represents G or C,
and W represents A or T). The oligonucleotide mix was radiolabeled by a
kinase reaction using T4 polynucleotide kinase and
[-32P]ATP under standard conditions (13). Total DNA,
digested to completion with different restriction endonucleases and
blotted onto a Hybond-N membrane (Amersham Pharmacia Biotech) was
hybridized to the radiolabeled oligonucleotide mix at 55 °C for
48 h. The membrane was then washed twice at room temperature with
300 mM NaCl, 30 mM sodium citrate containing
0.1% (w/v) SDS for 15 min and twice at 55 °C with 30 mM
NaCl, 3 mM sodium citrate containing 0.1% (w/v) SDS for 15 min. Following autoradiography, a single cross-hybridizing band was
detected in each lane. EcoRI, SacI, and
SmaI digests of genomic DNA were separated on preparative gels; the regions spanning ~4.5, 3.6, and 2.0 kbp,1 respectively, were
excised; and the DNA was extracted and shotgun-cloned into pUC18
vector. Following transformation into E. coli XL1 Blue, positive clones were isolated by a colony lift procedure (13) using
hybridization and washing conditions identical to those used for the
Southern blot. Clones were verified using a dot blot procedure (13)
prior to sequencing.
DNA Sequencing and Analysis--
Double-stranded DNA
sequencing (14) of plasmid DNA prepared from positive clones was
carried out with an automated DNA sequencer (ABI PRISM 377, PerkinElmer
Life Sciences). All sequencing was carried out on both strands.
Computer-assisted sequence analysis was performed using the DNAStrider
and MacVector software packages. Data base homology searches
(SwissProt release 39 protein data base) were carried out using the
NCBI BLAST server. The nucleotide sequence data reported in
this paper has been deposited at EMBL and GenBankTM with
the accession number AF323755.
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RESULTS |
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Purification of 6-Oxocamphor Hydrolase-- Table I shows that the purification of 6-oxocamphor hydrolase from crude cell extract proceeds with a yield of 5% and a factor of 35.7. SDS-polyacrylamide gel electrophoresis of the purified protein (Fig. 2) suggested a denatured molecular mass of ~35,000 Da, but electrospray mass spectrometry confirmed a smaller subunit mass of 28,488 Da. Analysis of the native protein by gel filtration chromatography revealed an apparent native molecular mass of 83,000 Da (average of two determinations). This suggests the protein exists in solution as a trimer (assuming the molecule is not highly elongated, which would give it a much larger than expected Stokes radius). An isoelectric point of 8.5 was recorded for the enzyme.
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6-Oxocamphor hydrolase was observed to have the same pI as penta-2,4-dione hydrolase and a comparable native molecular mass, the latter being a monomer of 75,000 Da (14). The pH optimum of the enzyme was ascertained to be 7.0, and the enzyme displayed 25% higher activity in 50 mM phosphate buffer than 50 mM Tris/HCl at the same pH.
Kinetic Properties--
The specific activity of 6-oxocamphor
hydrolase was determined to be 357.5 units
mg1, the Km to be 0.05 mM, and the Kcat to be 167 s
1 for 6-oxocamphor. The high specific
activity of the enzyme is reflected in an inability to detect any
6-oxocamphor in fermentation extractions of Rhodococcus sp.
NCIMB 9784, which yield a high proportion of
6-endo-hydroxycamphor. It is evident that the transformation of the hydroxycamphor to the diketone represents a bottleneck in the
metabolism of camphor by this organism.
Effect of Metal Ions, Salt, and Inhibitors-- 1 mM Cu2+ was found to inhibit 6-oxocamphor hydrolase activity (72% relative activity), whereas 1 mM Zn2+ (104%) had no significant effect. In common with penta-2,4-dione hydrolase (15), 6-oxocamphor hydrolase was greatly inhibited by Hg2+ ions (1 mM gave only 2% relative activity). High salt concentrations were shown to strongly inhibit penta-2,4-dione hydrolase, with no observable activity at 1 M NaCl (15). No such inhibition was observed with 6-oxocamphor hydrolase. Gel filtration studies indicated no significant alteration in the apparent molecular size of 6-oxocamphor hydrolase at this concentration of NaCl.
The effect of EDTA and various inhibitors of both thiol nucleophile-dependent hydrolases and the serine hydrolase inhibitor phenylmethylsulfonyl fluoride were tested. 6-oxocamphor hydrolase was inhibited to some degree by thiol active reagents, such as 1 mM N-ethylmaleimide (68% relative activity), but most notably 1 mM hydroxymercuribenzoate (14%); 1 mM EDTA had a slight activating effect (119%). Phenylmethylsulfonyl fluoride (1 mM) had almost no effect on activity.
Gene Cloning--
The gene encoding the 6-oxocamphor hydrolase was
cloned by hybridization with a mixture of oligonucleotides designed
against the N-terminal sequence of the purified protein activity. Three overlapping fragments of DNA (2.0 kbp SmaI, 3.6 kbp
SacI, and 4.3 kbp EcoRI) were isolated by this
procedure (Fig. 3) and cloned into pUC18
(clones S2.0, Sa3.6, and E4.3, respectively). Sequence analysis of the
clones revealed several potential open reading frames (ORFs) (Fig.
4). All displayed the typical codon usage pattern found in Rhodococcus sp., with a strong bias
toward GC-rich codons.
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The deduced polypeptide translation of one such ORF (camK)
matches the N-terminal sequence obtained from the isolated 6-oxocamphor hydrolase activity. camK encodes a protein of 257 amino
acids, and the predicted ATG start codon is positioned 5 bp 3' of a
purine-rich region that may act as a ribosome binding site (Fig.
5). Furthermore, the calculated mass of
the polypeptide (28,482 Da) is very close to the experimentally
determined mass of the purified protein by electrospray mass
spectrometry (28,488 Da). Comparison of the translated sequence with
the SwissProt data base revealed significant homology to the crotonase
superfamily of enzymes from several sources. The best alignments were
obtained with crotonase (enoyl-CoA hydratase) from Clostridium
acetobutylicum (16) and E. coli (17), revealing 45 and
42% homology, respectively (data not shown). Significantly, close
homology was also observed with 2-ketocyclohexanecarboxyl coenzyme A
hydrolase from Rhodopseudomonas palustris (18) and 4-chlorobenzoyl-CoA dehalogenase from a Pseudomonas sp.
(19). A sequence comparison of the translated sequence against
representative members of the crotonase superfamily is given in Fig.
6.
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The 3' end of the gene encoding 6-oxocamphor hydrolase
(camK) has an overlap of 1 nucleotide, encompassing the TGA
stop codon and a predicted GTG start codon of another open reading
frame (ORF1). ORF1 appears to be translationally coupled to
camK and encodes a protein of 167 amino acids. A BLAST
search of the SwissProt data base indicates homology to maoC
gene from Klebsiella aerogenes (20), which belongs to the
aldehyde dehydrogenase family and to the short-chain
dehydrogenase/reductase family of enzymes (e.g. 17-estradiol dehydrogenase from rat).
Downstream of ORF1 is an ORF2 encoding a polypeptide of 408 amino acids that displays similarity to nonspecific lipid transfer proteins from various species (e.g. 49% homology to chicken protein). The function of this open reading frame in relation to camphor metabolism is not known.
Upstream of camK, an ORF encoding a polypeptide of 206 amino acids (ORF3) was identified on the opposite strand. This divergently transcribed open reading frame has a potential ribosome binding site located 5 bp 5' of the proposed ATG start codon. Sequence analysis of the translated product revealed significant homology to a number of transcriptional repressor proteins of the TetR/AcrR family from various microbial sources (e.g. 47% homology to the acrab operon repressor from E. coli (24)). A potential DNA-binding motif based on sequence homologies was identified (38SVRDLGEALGIQPGSVYAHI) that may form a helix-turn-helix motif.
Further upstream of the proposed transcriptional regulator is ORF4, transcribed in the same direction as camK. Two alternative potential ATG start codons in the same reading frame were identified at positions 492 and 498 in the nucleotide sequence (Fig. 5). Because both ATG codons are positioned just downstream (5 and 6 bp, respectively) of a potential ribosome binding site, it is not clear which one represents the start of the ORF. For clarity, we have assigned the more 5' ATG as being the start codon. The ORF encodes a polypeptide of 396 amino acids. Sequence analysis of the translated product revealed convincing homology to a number of ferredoxin reductase proteins in the data base. Conservation of sequence was particularly obvious in the regions involved in adenine nucleotide binding (data not shown). The best homology was found with the ferredoxin (rhodocoxin) reductase (ThcB) involved in the biodegradation of thiocarbamate from Rhodococcus sp. NI86/21 (21) (46% homology) and the putidaredoxin reductase involved in the hydroxylation of camphor by P. putida (22) (46% homology).
Substrate Specificity--
We have recently carried out
initial studies on the substrate specificity of 6-oxocamphor hydrolase
(23) (refer to Fig. 7). Acyclic diketones
such as pentane-2,5-dione (a substrate for the -diketone hydrolase
from Pseudomonas vesicularis var. povalolyticus (15)) and 3,3-dimethylpentane-2,5-dione were not substrates. 2,2-Disubstituted cyclohexa-1,3-diones were the only monocyclic diketones transformed, although the transformation of two of these, 2-methyl-2-propylcyclohexa-1,3-dione 5 and
2-methyl-2-butylcyclohexa-1,3-dione 6, resulted in racemic
keto acid products (which were converted to their methyl esters
7 and 8, respectively, for analysis).
Transformations of the bicyclic diketone substrates bicyclo[2.2.1]heptane 2,6-dione 9 and
bicyclo[2.2.2]octane-2,6-dione 10 were, however, shown to
yield (S)-keto acid products of 84%, (converted to methyl
ester 11) and 95% enantiomeric excess (methyl ester
12), respectively (Fig. 7). It is noteworthy that all
substrates accepted by the enzyme are nonenolizable either due
to quaternary substitution between the carbonyl groups (e.g.
3, 5, 6) or due to ring-strain-related restrictions imposed
by Bredt's rule (3, 9, 10).
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DISCUSSION |
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The recent completion of the E. coli genome (24) has
revealed seven genes encoding paralogues of enoyl-CoA hydratase, three with unknown function (17). This suggests that there remain activities
of the crotonase superfamily yet to be described. In this paper, we
present evidence that 6-oxocamphor hydrolase, which catalyzes the
asymmetric hydrolysis of -diketones, represents a new addition to
the spectrum of activities catalyzed by the crotonase superfamily.
The activity of crotonase, or enoyl-CoA hydratase (ECH), has been the
subject of intensive study over many years owing to its central role in
the -oxidation pathway. The essential activity of ECH in this regard
has been the stereospecific reversible hydration of enoyl-CoA molecules
of varying fatty acid length to yield
-hydroxy thioesters (25, 26,
30). The catalytic mechanism of ECH is dependent on stabilization of an
enolate intermediate by hydrogen bonding to an oxyanion hole created by
two peptidic NH groups in the active site of the enzyme, Ala-98
and Gly-141 (Fig. 8). In recent years,
the comparison of genetic sequence information for a wide range of
enzymatic activities has revealed that there exists a superfamily of
crotonase-like proteins, each member of which catalyzes a reaction that
is dependent on the same general stabilization of an enolate anion
(17). Activities assigned to the crotonase family include double-bond
isomerization (27), aromatic ring closure (28), 1,3-dioxo cleavage
(18), dehalogenation (19), and decarboxylation (29), in addition to
double bond hydration (Fig. 9). Although
the overall amino acid sequence is well conserved between these
enzymes, crucial active site residues have been shown to be present in
some members of the family and absent in others, suggesting a
nonconserved mechanism of enol stabilization and water transfer. In
enoyl-CoA hydratase from rat mitochondria, two glutamate residues at
the active site are responsible for acid/base catalysis of double bond
hydration: Glu-144 facilitates attack of nucleophilic water to the
carbonyl, and Glu-164 donates a proton to the
-carbon in the final
step to yield the hydrated product (Fig. 8).
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Homology between ECH and 6-oxocamphor hydrolase is conserved throughout
most of the length of the polypeptide chain. Identity is most
pronounced in the central region of the protein, which constitutes the
spiral domain of ECH, most especially in the A3 B3 -strand region
129PVIAAVNG, although the 140GGG turn
that is present in both ECH, 2-ketocyclohexanecarboxyl coenzyme A
hydrolase (18) and 4-chlorobenzoyl-CoA dehalogenase (4CBD) (19) is
notably absent in 6-oxocamphor hydrolase. Whereas homology persists
throughout the so-called first trimerization domain of ECH, T1 (Lys-185
through Thr-217), conservation between the second trimerization
domain, T2 (Lys-234 through His-290) of ECH, 4CBD, and 6-oxocamphor
hydrolase is more poorly conserved. Nevertheless, all three enzymes
exist as trimers at one level of quaternary substructure.
It is notable that of the two active site glutamate residues in ECH,
only Glu-144, which facilitates attack of water on the 3-carbon of the
enoyl-CoA substrate, is conserved in 6-oxocamphor hydrolase (Glu-124).
This residue is absent in other members of the superfamily, including
those of which the activity most closely resembles that of 6-oxocamphor
hydrolase, e.g. 2-KCH (18) and 4-chlorobenzoyl-CoA
dehalogenase, in which Asp-145 has been identified as the crucial
catalytic residue (19). Glu-144 is conserved between ECH and
methylmalonyl-CoA decarboxylase (29), but in this case, it has been
shown that this glutamate residue cannot be catalytic. Hence, it cannot
be assumed with certainty that the homologous glutamate in 6-oxocamphor
hydrolase is catalytic. The other catalytic residue of ECH, Glu-164, is
not conserved in 6-oxocamphor hydrolase. There are several candidate
residues with the required acid/base character capable of forming an
acid-base couple with Glu-124 in 6-oxocamphor hydrolase, including
Glu-136 (conserved with rat ECH and 2-KCH), Asp-142, and perhaps
Asp-154, which is one residue distant from the active Asp-145 of
4CBD. Despite the absence of structural studies or site
directed mutation experiments to determine the actual active site
residue in 6-oxocamphor hydrolase responsible for water activation, it
is nevertheless still possible to tentatively postulate a mechanism for
asymmetric -diketone hydrolysis based on the results of sequence comparison.
The activation of water in the active site of 6-oxocamphor hydrolase,
possibly by Glu-124, in concert with another residue would facilitate
nucleophilic attack of water at the pro-S carbonyl, yielding
the (S)-enantiomer of keto acid 4 (Fig.
10). It is possible that
tautomerization of the keto form is not an enzyme-catalyzed process
because the same ratio of diastereomers (predominantly cis-)
is also observed in acid-catalyzed hydrolysis of 3 to
4.
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The desymmetrization of bicyclic -diketones, and indeed the
hydrolysis of 2,2-dialkylcyclohexanones, constitutes a novel reaction
in the crotonase superfamily. The previously reported activity of a
crotonase homologue that bears the closest resemblance is that of
2-KCH, which hydrolyzes a
-dicarbonyl species. Importantly, however,
6-oxocamphor hydrolase is the first crotonase homologue not to
have an activity dependent on coenzyme A for substrate activation.
Indeed, of those residues shown to be responsible for coenzyme A
binding in ECH (e.g. Lys-92, Lys-101, and Lys-282, involved
in forming salt bridges to the phosphates of ADP) and 4CBD
(Arg-24 and Arg-67), none is conserved in 6-oxocamphor hydrolase.
Interestingly, all the compounds which act as substrates for 6-oxocamphor hydrolase have nonenolizable 1,3-diketones. This factor may explain why the retro-Claisen reaction mediated by 6-oxocamphor hydrolase is effected using a crotonase type mechanism. In contrast, similar reactions on enolizable diketones, such as fumarylacetoacetate (mediated by fumarylacetoacetate hydrolase (9)), utilize a serine hydrolytic triad type mechanism, in which the energy barrier to hydrolysis maybe greater. It remains to be seen whether other activities, such as cyclohexane-1,3-dione hydrolase (31) or polyvinylketone hydrolase (which is active against pentane-1,4-dione (15)), are of the fumarylacetoacetate hydrolase or crotonase type.
Sequence analysis of the clone encoding the 6-oxocamphor hydrolase activity revealed several proximal genes encoding proteins that may be involved in camphor metabolism. ORF1 is immediately downstream and apparently translationally coupled to camK, and it encodes a small protein that displays homology to dehydrogenase enzymes. This open reading frame encodes the sequence 40SDISMFAGLTGD, which is somewhat similar to a sequence motif typical for FAD-binding enzymes, TXXXXhhhhGD (32) (where h denotes a hydrophobic amino acid). However, it does not contain the expected GXGXXG sequence typical of both FAD-requiring and NAD-dependent dehydrogenases (33). The function of this activity therefore remains uncertain at the present time. ORF3, encoding a regulatory protein, was found upstream of camK, along with ORF4, which encodes a ferredoxin reductase. The first step in the metabolism of (1R)-(+)-camphor in Rhodococcus sp. NCIMB 9784 involves hydroxylation at the 6-endo position by a cytochrome P450 (Fig. 1). Because genes encoding enzymes involved in metabolic pathways are often linked, we speculate that the polypeptide encoded by ORF4 may be involved in furnishing the camphor hydroxylase with electrons.
In conclusion, we have demonstrated that the activity of 6-oxocamphor
hydrolase, namely the retro-Claisen reaction of (bi)cyclic -diketones, in the metabolism of camphor by Rhodococcus
sp. NCIMB 9784 is attributable to a novel activity of the crotonase
superfamily. The precise mechanism of the reaction awaits elucidation
by site directed mutagenesis and detailed kinetic analysis of mutant
activity. The wild type enzyme is already effective at performing the
desymmetrization of bicyclic
-diketones with a high degree of
enantioselectivity and hence constitutes an important addition to the
array of biocatalysts that might be employed in the synthesis of fine
chemical intermediates.
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ACKNOWLEDGEMENTS |
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Protein sequencing was performed by Andrew Cronshaw (Institute of Cell and Molecular Biology, University of Edinburgh). DNA sequencing was performed by Nicola Preston (ICMB, University of Edinburgh). We also thank Mark Scott for assistance with mass spectrometry.
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FOOTNOTES |
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* This work was supported by funds from the Biotechnology Biosciences Research Council (to G. G. and G. A. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be 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 GenBankTM/EMBL Data Bank with accession number(s) AF323755.
These authors contributed equally to this work.
§ To whom correspondence should be addressed. Tel.: 44-131-650-4737; Fax: 44-131-650-4743; E-mail: s.flitsch@ed.ac.uk; Internet: http://www.ed.ac.uk/~slf10.
Published, JBC Papers in Press, January 16, 2001, DOI 10.1074/jbc.M011538200
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ABBREVIATIONS |
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The abbreviations used are: kbp, kilobase pair; bp, base pair; ORF, open reading frame; ECH, enoyl-CoA hydratase; 4CBD, 4-chlorobenzoyl-CoA dehalogenase.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Willis, M. C. (1999) J. Chem. Soc. Perkin Trans. 1, 1765-1784 |
2. | Schoffers, E., Golebiowski, A., and Johnson, C. R. (1996) Tetrahedron 52, 3769-3826[CrossRef] |
3. | Yang, Y.-F., and Sih, C. J. (1984) Tetrahedron Lett. 25, 4999-5002[CrossRef] |
4. | Taschner, M. J., and Black, D. J. (1988) J. Am. Chem. Soc. 110, 6892-6893 |
5. | Chapman, P. J., Meerman, G., Gunsalus, I. C., Srinivasan, R., and Rinehart, K. (1966) J. Am. Chem. Soc. 88, 618-619 |
6. | Pokorny, D., Steiner, W., and Ribbons, D. W. (1997) Trends Biotechnol. 15, 291-297[CrossRef] |
7. | Hsiang, H. H., Sim, S. S., Mahuran, D. J., and Schmidt, D. E., Jr. (1972) Biochemistry 11, 2098-2102[Medline] [Order article via Infotrieve] |
8. | Davey, J. F., and Ribbons, D. W. (1975) J. Biol. Chem. 250, 3826-3830[Abstract] |
9. | Timm, D. E., Mueller, H. A., Bhanumoorthy, P., Harp, J. M., and Bunick, G. J. (1999) Structure 7, 1023-1033[CrossRef][Medline] [Order article via Infotrieve] |
10. | Corey, E. J., and Suggs, J. W. (1975) Tetrahedron Lett. 31, 2647-2650[CrossRef] |
11. | Hayes, J. D., Kerr, L. A., and Cronshaw, A. D. (1989) Biochem. J. 264, 437-445[Medline] [Order article via Infotrieve] |
12. | Kulakova, A. N., Stafford, T. M., Larkin, M. J., and Kulakov, L. A. (1995) Plasmid 33, 208-217[CrossRef][Medline] [Order article via Infotrieve] |
13. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
14. | Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467[Abstract] |
15. | Kawagoshi, Y., and Fujita, M. (1998) World J. Microbiol. Biotechnol. 14, 95-100 |
16. | Boynton, Z. L., Bennett, G. N., and Rudolph, F. B. (1996) J. Bacteriol. 178, 3015-3024[Abstract] |
17. | Haller, T., Buckel, T., Retey, J., and Gerlt, J. A. (2000) Biochemistry 39, 4622-4629[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Pelletier, D. A.,
and Harwood, C. S.
(1998)
J. Bacteriol.
180,
2330-2336 |
19. | Benning, M. M., Taylor, K. L., Liu, R.-Q., Yang, G., Xiang, H., Wesenberg, G., Dunaway-Mariano, D., and Holden, H. M. (1996) Biochemistry 35, 8103-8109[CrossRef][Medline] [Order article via Infotrieve] |
20. | Sugino, H., Sasaki, M., Azakami, H., Yamashita, M., and Murooka, Y. (1992) J. Bacteriol 174, 2485-2492[Abstract] |
21. | Nagy, I., Schoofs, G., Compernolle, F., Proost, P., Vanderleyden, J., and De Mot, R. (1995) J. Bacteriol. 177, 676-687[Abstract] |
22. | Koga, H., Rauchfuss, B., and Gunsalus, I. C. (1985) Biochem. Biophys. Res. Commun. 130, 412-417[Medline] [Order article via Infotrieve] |
23. | Grogan, G., Graf, J., Jones, A., Parsons, S., Turner, N. J., and Flitsch, S. L. (2000) Angew. Chem. Int. Ed. Engl., in press |
24. |
Blattner, F. R.,
Plunkett, G., III,
Bloch, C. A.,
Perna, N. T.,
Burland, V.,
Riley, M.,
Collado-Vides, J.,
Glasner, F. D.,
Rode, C. K.,
Mayhew, G. F.,
Gregor, J.,
Davis, N. W.,
Kirkpatrick, H. A.,
G.oeden, M. A.,
Rose, D. J.,
Mau, B.,
and Shao, Y.
(1997)
Science
277,
1453-1474 |
25. | Wakil, S. J. (1957) Biochim. Biophys. Acta 19, 497-504 |
26. | Willadesen, P., and Eggerer, H. (1975) Eur. J. Biochem. 54, 247-252[Abstract] |
27. | Müller-Newen, G., Janssen, U., and Stoffel, W. (1995) Eur. J. Biochem. 228, 68-73[Abstract] |
28. | Sharma, V., Suvarna, K., Meganathan, R., and Hudspeth, M. (1992) J. Bacteriol. 174, 5057-5062[Abstract] |
29. | Benning, M. M., Haller, T., Gerlt, J. A., and Holden, H. M. (2000) Biochemistry 39, 4630-4639[CrossRef][Medline] [Order article via Infotrieve] |
30. | Wu, W.-J., Feng, Y., He, X., Hofstein, H. A., Raleigh, D. P., and Tonge, P. J. (2000) J. Am. Chem. Soc. 122, 3987-3994[CrossRef] |
31. | Bartholomew, B. A., Smith, M. J., Trudgill, P. W., and Hopper, D. J. (1996) Appl. Environ. Microbiol. 62, 3245-3250[Abstract] |
32. | Eggink, G., Engel, H., Vriend, G., Terpstra, P., and Witholt, B. (1990) J. Mol. Biol. 212, 135-142[Medline] [Order article via Infotrieve] |
33. | Scrutton, N. S., Berry, A., and Perham, R. (1990) Nature 343, 38-43[CrossRef][Medline] [Order article via Infotrieve] |
34. | Yokoyama, M., Sugai, T., and Ohta, H. (1993) Tetrahedron: Asymmetry 6, 1081-1084[CrossRef] |
35. |
Pokorny, D.,
Brecker, L.,
Pogorevc, M.,
Steiner, W.,
Griengl, H.,
Kappe, T.,
and Ribbons, D. W.
(1999)
J. Bacteriol.
181,
5051-5059 |