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Applied and Environmental Microbiology, April 1999, p. 1589-1595, Vol. 65, No. 4
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
The Alkene Monooxygenase from Xanthobacter Strain Py2
Is Closely Related to Aromatic Monooxygenases and Catalyzes Aromatic
Monohydroxylation of Benzene, Toluene, and Phenol
Ning-Yi
Zhou,
Alister
Jenkins,
Chan K. N.
Chan Kwo Chion, and
David J.
Leak*
Department of Biochemistry, Imperial College
of Science, Technology and Medicine, London SW7 2AZ, United Kingdom
Received 22 September 1998/Accepted 5 January 1999
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ABSTRACT |
The genes encoding the six polypeptide components of the alkene
monooxygenase from Xanthobacter strain Py2 (Xamo) have been located on a 4.9-kb fragment of chromosomal DNA previously cloned in
cosmid pNY2. Sequencing and analysis of the predicted amino acid
sequences indicate that the components of Xamo are homologous to those
of the aromatic monooxygenases, toluene 2-, 3-, and 4-monooxygenase and
benzene monooxygenase, and that the gene order is identical. The genes
and predicted polypeptides are aamA, encoding the
497-residue oxygenase 
-subunit (XamoA); aamB, encoding
the 88-residue oxygenase 
-subunit (XamoB); aamC,
encoding the 122-residue ferredoxin (XamoC); aamD, encoding
the 101-residue coupling or effector protein (XamoD); aamE,
encoding the 341-residue oxygenase 
-subunit (XamoE); and aamF, encoding the 327-residue reductase (XamoF). A
sequence with >60% concurrence with the consensus sequence of
54 (RpoN)-dependent promoters was identified upstream of
the aamA gene. Detailed comparison of XamoA with the
oxygenase -subunits from aromatic monooxygenases, phenol
hydroxylases, methane monooxygenase, and the alkene monooxygenase from
Rhodococcus rhodochrous B276 showed that, despite the
overall similarity to the aromatic monooxygenases, XamoA has some
distinctive characteristics of the oxygenases which oxidize aliphatic,
and particularly alkene, substrates. On the basis of the similarity
between Xamo and the aromatic monooxygenases, Xanthobacter
strain Py2 was tested and shown to oxidize benzene, toluene, and
phenol, while the alkene monooxygenase-negative mutants NZ1 and NZ2 did
not. Benzene was oxidized to phenol, which accumulated transiently
before being further oxidized. Toluene was oxidized to a mixture of
o-, m-, and p-cresols (39.8, 18, and 41.7%, respectively) and a small amount (0.5%) of benzyl alcohol,
none of which were further oxidized. In growth studies
Xanthobacter strain Py2 was found to grow on phenol and
catechol but not on benzene or toluene; growth on phenol required a
functional alkene monooxygenase. However, there is no evidence of genes
encoding steps in the metabolism of catechol in the vicinity of the
aam gene cluster. This suggests that the inducer
specificity of the alkene monooxygenase may have evolved to benefit
from the naturally broad substrate specificity of this class of
monooxygenase and the ability of the host strain to grow on catechol.
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INTRODUCTION |
Xanthobacter strain Py2
is a gram-negative bacterial strain which was isolated on propene as a
sole carbon and energy source (34). The metabolism of
propene involves an alkene-specific monooxygenase which converts
propene to epoxypropane. Further metabolism involves isomerization and
carboxylation of the epoxide, ultimately yielding acetoacetate (Fig.
1), which feeds into the central
metabolism (1, 6, 36).
The Xanthobacter strain Py2 alkene monooxygenase (Xamo) will
catalyze the epoxidation of a range of alkenes, some with a high degree
of stereospecificity, but will not hydroxylate the homologous alkanes
(35). This contrasts with alkane monooxygenases such as
those based on cytochrome P-450 (28) or nonheme iron (e.g., 
-hydroxylase [27] and methane monooxygenase [MMO]
[11]), which appear to generate a highly reactive
iron-oxygen intermediate which can attack both unactivated C-H bonds
and carbon-carbon double bonds. This reaction specificity and
stereospecificity make Xamo an enzyme of considerable interest as a
biocatalyst for the production of chiral 1,2-epoxides. Additionally,
Xamo has been shown to be responsible for catalyzing the initial step in the cometabolic degradation of a number of chlorinated alkenes of
environmental concern, including vinyl chloride, trichloroethene, and
1,3-dichloropropene (9), and it can even be induced by the
presence of these chlorinated alkenes, although there is no evidence
for growth on these substrates (10). Xamo has been resolved
into four components: an NADH-dependent reductase, a Rieske-type
ferredoxin, an oxygenase, and a small protein which may be a coupling
or effector protein (29). The oxygenase is an
222 hexamer which was
reported to contain approximately four atoms of nonheme iron per
hexamer on the basis of colorimetric iron analysis and the lack of a
significant UV- or visible-light chromophore. Sequence (25)
and electron paramagnetic resonance evidence (12) has
demonstrated that an alkene-specific monooxygenase derived from the
gram-positive bacterium Rhodococcus rhodochrous (formerly
Nocardia corallina) B276 has a binuclear nonheme iron center
of the type found in soluble MMO. However, the Xanthobacter enzyme is more complex than that from Rhodococcus, having a
two-component (reductase and ferredoxin) redox system typical of
aromatic dioxygenases (3) and a more complex oxygenase structure.
We have recently reported the sequencing of the first open reading
frame (ORF) in the Xamo gene cluster (42). The predicted polypeptide sequence showed strong homology to the nonheme iron binding
subunit in aromatic monooxygenases and MMO, particularly around the
iron binding domain. Modelling of the predicted Xanthobacter polypeptide on the coordinates of the -subunit in the MMO crystal structure (24) allowed us to conclude that Xamo is also a
nonheme iron monooxygenase. In this paper, we report the entire
sequence of the six ORFs which encode Xamo. The predicted amino acid
sequences of all six polypeptides encoded by these ORFs show strong
homology with those of aromatic monooxygenases, including benzene
monooxygenase and toluene 4-monooxygenase. This led us to investigate
whether Xamo could catalyze the monohydroxylation of aromatic
hydrocarbons or grow on them as sole sources of carbon and energy.
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MATERIALS AND METHODS |
Bacterial strains.
The following strains were used:
Escherichia coli DH 10B F
mcrA
(mrr hsdRMS mcrBC) 
80d lacZM15
lacX74 deoR reclA1 endA1 araD139 (ara
leu)7697 galU galK 
rpsL
nupG (GIBCO BRL) and Xanthobacter strains Py2
(34), NZ1, and NZ2. The latter two are independently
isolated alkene monooxygenase-negative (Amo) mutants of
Xanthobacter strain Py2 (41).
Materials.
Isopropyl--D-thiogalactopyranoside
(IPTG) and
5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal)
were obtained from Promega. Restriction enzymes were supplied by
Boehringer and used as recommended by the manufacturer. DNA ligase was
purchased from New England Biolabs.
Bacterial growth.
E. coli cells were routinely grown
in liquid Luria-Bertani medium (10 g of casein peptone/liter, 5 g
of yeast extract/liter, 5 g of NaCl/liter [pH 7.2]) or on
Luria-Bertani plates at 37°C. For biotransformation assays,
Xanthobacter strain Py2 was grown on an ammonia mineral
salts medium (AMS) supplemented with 10% (vol/vol) propene or 0.5%
fructose, and the mutants NZ1 and NZ2 were grown on AMS supplemented
with 10% (vol/vol) propene and 0.01% (vol/vol) propene oxide, as
previously described (41).
For testing growth on aromatic substrates,
Xanthobacter
strain Py2 and its mutants were grown on AMS supplemented with 2 and
4 mM benzene, toluene, or phenol. Where growth was not seen, the
toxicity
of aromatic substrates was tested by comparing the growth
of
Xanthobacter strain Py2 on 0.5% fructose in AMS with and
without
aromatic substrate
supplementation.
Sequencing strategy and sequence analysis.
An 11.2-kb
EcoRI fragment from the cosmid clone pNY2, previously
subcloned as pNY2C and shown to complement Amo mutants of
Xanthobacter strain Py2 (41), was used to prepare nested deletions by using the Deletion Factory system, version 2 (GIBCO
BRL), as described previously (42). These were sequenced in
both directions by using BigDye Terminator Cycle Sequencing (Perkin-Elmer) with AmpliTaq FS DNA polymerase and were
analyzed on an ABI 377 automated sequencer (Applied Biosystems). DNA
and deduced amino acid sequences were analyzed by using the MacVector (version 4.5.3) and AssemblyLIGN (version 1.07) software packages (Eastman Kodak Co.). Homologous protein searches were carried out with
FASTA 3 (22) and PSI-BLAST (2). Pairwise
alignments were made with the GAP program in the Wisconsin package
(7). Multiple alignments were run under Clustal W
(32). The search for 54-dependent promoter
sequences was done remotely with the SEQSCAN program (26a).
Biotransformation assays with alkene monooxygenase (AMO).
Assays were carried out on resting cell suspensions of
Xanthobacter strain Py2 and the mutants NZ1 and NZ2. Cells
were harvested in late-exponential phase, washed once, and resuspended
in 25 mM phosphate buffer, pH 7.5, to an optical density at 600 nm of 10. Cell suspensions (1 ml) were preincubated for 2 min at 30°C in
7-ml Suba-sealed (W. H. Freeman, Barnsley, United Kingdom) flasks
before addition of either benzene, toluene, or phenol to a final
concentration of 2 mM. The rate of product formation was assayed by gas
chromatography (Philips PU4500) by taking 5-µl liquid samples at
suitable time intervals, separating them on a Tenax TA (60/80 mesh)
column (Phase Separations, Deeside, United Kingdom) at 210°C with
nitrogen as the carrier gas at 40 ml · min1, and
carrying out flame ionization detection. Product concentrations were
determined by reference to external standards, by using a Shimadzu CR3A
recording integrator. The identities of the reaction products were
confirmed by coretention with authentic standards on both the Tenax
column and (for phenol) 5% (wt/vol) OV-17 on a Chromosorb W.HP (80/100
mesh) column at 130°C with nitrogen as the carrier gas at 40 ml
· min1. To separate and identify o-,
m-, and p-cresol and benzyl alcohol, the samples
were analyzed by capillary gas chromatography (model 436; United
Technologies Packard) on a 50-m by 0.25-mm Lipodex C column
(Macharey-Nagel) at 120°C with helium as the carrier gas at 0.77 ml · min1. The split ratio was 1:100. For analysis
on the latter two columns, the reaction mixtures were extracted with
0.5 ml of ethyl acetate and the extract was dried over sodium sulfate.
Propene oxide formation and degradation were measured as previously
described (
41).
Nucleotide sequence accession number.
The sequences
described in this report have been deposited with the EMBL data bank
and are available under accession no. AJ012090.
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RESULTS AND DISCUSSION |
Preliminary sequence analysis of Xanthobacter aam
genes.
The genes encoding Xamo have previously been cloned as a
25.7-kb insert in the broad-host-range cosmid vector pLAFR5 and shown to express propene-inducible AMO when transferred to Xanthobacter autotrophicus JW33, although it was not expressed in E. coli (41). Mapping of the cosmid showed that it
overlapped with a region sequenced by Swaving et al. (31)
which contained the genes encoding components of the epoxide
isomerase-carboxylase complex. Additionally, deletions from one end of
the cosmid resulted in loss of expression of the AMO and
complementation of Amo mutants, suggesting that the AMO
was encoded in a fragment of 11.2 kb flanked on one side by the cosmid
junction and on the other by the isomerase-carboxylase genes. By using
a nested deletion strategy, a large part of this fragment has now been
sequenced, revealing the presence of six closely spaced ORFs in a
4.9-kb DNA fragment, consistent in size and predicted amino acid
sequence with the reported components of Xamo (29). These
ORFs have been designated aamA through aamF
(Table 1); the designations refer to
"alkene and aromatic monooxygenase," for reasons that will become
clear in this manuscript. We have already reported (42) that
the predicted amino acid sequence of the aamA gene product (497 residues; predicted mass, 58,037 Da; gene previously referred to
as xamoA [42]) has a high degree of
sequence similarity to the -subunit of nonheme iron monooxygenases,
particularly the aromatic monooxygenases, allowing us to model part of
the sequence of this polypeptide on the coordinates of the MMO
-subunit, obtained from the crystal structure.
aamB encodes a polypeptide of 88 amino acids (9,740 Da) with
a high degree of overall similarity to the
-subunit of the
222 hexameric nonheme iron
monooxygenases;
aamC encodes a polypeptide
of 122 amino
acids (13,359 Da) with a high degree of similarity
to the ferredoxins
of four-component nonheme iron monooxygenases
and aromatic
ring-hydroxylating bacterial dioxygenases;
aamD encodes
a
polypeptide of 101 amino acids (11,193 Da) which is most similar
to the
small "coupling" proteins of four-component nonheme iron
monooxygenases and also the
dmpM gene product (P2) of the
three-component
phenol hydroxylase from
Pseudomonas sp.
strain CF600 (
19);
aamE encodes a polypeptide of
341 amino acids (38,188 Da) which is
homologous to the oxygenase
-subunits of
222
hexameric monooxygenases
and also the
-subunit of the
R. rhodochrous B276 AMO, which is
probably tetrameric (
18,
25). As is the case with most other
multicomponent mono- and
dioxygenases, the final ORF (
aamF) encodes
a reductase
homolog (327 amino acids [34,171 Da]).
Gene order.
On the basis of the comparisons above, it is
reasonable to conclude that aamA through aamF
encode the oxygenase -subunit, oxygenase -subunit, ferredoxin,
coupling or effector protein, oxygenase -subunit, and reductase,
respectively (Fig. 2). This gene order is
identical to that of the four-component benzene and toluene
monooxygenases (4, 39, 40) and the Ralstonia eutropha JMP134 phenol hydroxylase, to which the individual
subunits show the highest homology. The extent of sequence identity or similarity between the component polypeptides of the hexameric monooxygenase subunits of these enzymes also shows a clear grouping, separating them from MMO (5, 30) and the almost identical pair of phenol hydroxylases from Pseudomonas sp. strain
CF600 (20) and Pseudomonas putida P35X
(19), all of which have hexameric oxygenases, and the
tetrameric R. rhodochrous B276 AMO (25). All of
the latter appear to be three-component systems, lacking the separate
ferredoxin component, and have a gene order different from that of the
Xamo-aromatic ring monooxygenase family. In MMO the genes encoding the
- and -subunits are in reverse order with respect to that for the
"coupling" protein, while in the R. rhodochrous B276 AMO
and the pseudomonad phenol hydroxylases, the first structural gene
encodes the oxygenase -subunit, and the -subunit is actually the
third structural gene in the sequence.
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FIG. 2.
Gene orders of the Xamo and related gene clusters. The
monooxygenase components encoded are indicated at the top, as follows:
, , and , oxygenase -, -, and -subunits; Fd,
ferredoxin; C/E, coupling or effector protein; Red, reductase. The
arrow indicates the direction of transcription. Diagram 1, Xamo and the
benzene (bmo), toluene 3- (tbh), toluene 4- (tmo), toluene/benzene (tbu), and phenol
(phl) monooxygenases; diagram 2, MMO (an additional ORF
[not shown] lies between the genes encoding the -subunit and the
reductase); diagram 3, phenol hydroxylase (dmp and
phh); diagram 4, R. rhodochrous B276 AMO.
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Regulatory features and codon usage.
Potential ribosome
binding sites precede the ATG or GTG translational initiation sites by
4 to 13 bp in all six ORFs, namely AGGA for aamA,
aamB, aamE, and aamF and GAGG for
aamC and aamD (Fig.
3). The translational stop codon is TAA
for aamA and aamD and TGA for the other genes. No
homolog of the 
35 and 10 consensus sequence of
70-dependent promoters was found immediately upstream of
the aamA gene, which might explain the lack of expression of
the cosmid pNY2 in E. coli. However, a possible
54 (RpoN)-dependent promoter sequence
(GGGCACGCCATGCGCT) is present approximately 300 bp upstream
of the translational start site of aamA.
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FIG. 3.
Regulatory features of the aam gene cluster,
showing the putative 54 (RpoN)-dependent promoter
sequence (lowercase letters in the top line) and ribosome binding sites
(underlined).
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|
Xanthobacter spp. have genomes which are typically composed
of 65 to 70% G+C (
38). While this feature is evident when
the
combined frequencies of all six ORFs (67% GC) are considered,
there is significant variation, from 62.4% (
aamC) to 71.9%
(
aamF,
among the different coding regions. A high GC content
implies
that codons in which the final base in the triplet is G or C
should
be more frequently used, and this is also evident (90.5% G/C).
The codon usage is similar to that described for other genes in
Xanthobacter spp. (
31,
33).
Intriguingly, the codons UUA (Leu), CUA (Leu), GUA (Val), UCA (Ser),
and AGA (Arg) are not used once either in the 1,476 codons
characterized here or in the 1,469 codons described by Swaving
et al.
(
31) from the same
organism.
Further analysis of aamC and aamD. (i)
aamC.
Alignment of the amino acid sequence of the
aamC gene product with that of the homologous monooxygenase
and dioxygenase ferredoxins (Fig. 4)
clearly shows that a number of key residues are absolutely conserved,
including the pairs of cysteine and histidine residues which coordinate
the 2Fe-2S cluster. The latter are diagnostic of Rieske-type
iron-sulfur proteins and confirm the spectroscopic assignments
(14, 29). Although it is clear that the aamC gene product is most similar to the ferredoxins present in the
four-component aromatic ring monooxygenases, XamoC contains an
additional 12 amino acids at the N terminus which are not seen in any
of the near homologs. This enzyme contains a high proportion of acidic residues and could well be important in protein-protein interactions.
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FIG. 4.
Homology between the putative ferredoxin of Xamo
(XAMOC), encoded by aamC, and ferredoxins from
four-component aromatic ring monooxygenases. Dark highlighting
indicates residues which are identical or functionally conserved in at
least three sequences. The two cysteines and two histidines which form
the metal binding sites are shown. TBUB, TBHC, and TMOC, ferredoxins
from the Ralstonia pickettii PKO1 toluene 3-monooxygenase
(4), the Burkholderia cepacia AA1 toluene
3-monooxygenase, and the Pseudomonas mendocina KR1 toluene
4-monooxygenase (39), respectively.
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(ii) aamD.
Alignments of the predicted amino acid
sequence of the aamD gene product (Fig.
5) show that it clearly falls into the
aromatic ring monooxygenase family and is more distantly related to the coupling protein in MMO (not shown). The role of this small protein, which does not appear to contain any cofactors, in the catalytic cycle
of monooxygenases is still unclear. Small and Ensign (29) reported that this small protein was obligately required for
steady-state alkene epoxidation. In MMO the coupling protein is
necessary to couple electron transfer to substrate oxidation, and it is
also known to affect the regioselectivity of substrate oxidation and the redox potential of the oxygenase binuclear nonheme iron center. dmpM encodes the homologous coupling protein (P2) in the
phenol hydroxylase from Pseudomonas sp. strain CF600. A
solution structure for this protein has recently been determined by
nuclear magnetic resonance (23), and although the structure
was poorly defined in places, it is evident that this small protein has
a hydrophobic cavity which could well act to bind substrate, either to
deliver it to the oxygenase, or to act as a substrate-dependent
regulator of electron transport. The conserved residues indicated in
Fig. 5 are all either within, or at the ends of, regions of secondary structure evident from the P2 structure (23).
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FIG. 5.
Homology between the putative small coupling or effector
protein of Xamo (XAMOD), encoded by aamD, and the coupling
or effector proteins of aromatic ring monooxygenases, MMO, and the
R. rhodochrous B276 AMO. Dark highlighting indicates
residues which are identical or functionally conserved in at least six
sequences. DMPM, TBUV, TBHD, TMOD, MEMC, and AMOB, small coupling or
effector proteins from the Pseudomonas sp. strain CF600
phenol hydroxylase (20), the R. pickettii PKO1
toluene 3-monooxygenase (4), the B. cepacia AA1
toluene 3-monooxygenase, the P. mendocina KR1 toluene
4-monooxygenase (39), the Methylosinus
trichosporium OB3b MMO (5), and the R. rhodochrous B276 AMO (25), respectively.
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Does Xamo function as an aromatic hydrocarbon monooxygenase?
Despite the similarity of the primary sequence of the Xamo oxygenase
-subunit to that of the aromatic monooxygenases, there are certain
features which clearly link it to the R. rhodochrous B276
AMO and also to MMO. In particular, it was noted previously that at the
position equivalent to Cys151 in the memA gene product, the
AMO enzymes have an acid residue, whereas the aromatic hydroxylases have a Gln residue (Fig. 6).
Additionally, residues 206 and 208 in the memA protein
sequence are small aliphatic amino acids, whereas in all of the
aromatic ring monooxygenases they are Phe's. This is particularly
significant because Glu209 in the memA-encoded gene product
is one of the acidic residues which coordinate the binuclear nonheme
iron center, and it is probable that the residue at position 208 (and
its equivalent in homologous polypeptides) has a significant effect on
the approach and orientation of the substrate. Aromatic residues at
positions equivalent to positions 206 and 208 in the
memA-encoded protein sequence should therefore facilitate
the approach of aromatic substrates.
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FIG. 6.
Homology between the putative oxygenase iron binding
subunit of Xamo (XAMOA), encoded by aamA, and the iron
binding subunits of aromatic ring monooxygenases, MMO, and the R. rhodochrous B276 AMO. Only the sequence around the iron binding
sites is shown. Dark highlighting indicates residues which are
identical or functionally conserved in at least four sequences. The
glutamate and histidine ligands that coordinate the binuclear iron
center (24) in MMO (and are completely conserved in the
other homologs) are shown as subscripts in capital letters, and the
residues referred to in the text are indicated by triangles. TMOA,
BMOA, AMOB, and MEMA, iron binding subunits from the P. mendocina KR1 toluene 4-monooxygenase (39), the
Pseudomonas aeruginosa JI 104 benzene monooxygenase
(16), the R. rhodochrous B276 AMO
(25), and the M. trichosporium OB3b MMO
(5), respectively.
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Despite these differences, the evident homology between all of the Xamo
components and those of aromatic ring monooxygenases
suggested that
Xamo might hydroxylate aromatic substrates. By
using propene-grown
resting cells,
Xanthobacter strain Py2 was
shown to convert
benzene to phenol and to convert toluene to a
mixture of cresols and a
trace of benzyl alcohol (Table
2). It
should be noted that, although the rates of aromatic hydrocarbon
oxidation are similar to those cited for propene oxidation, the
latter
was carried out at pH 9 to limit the further metabolism
of
epoxypropane. Propene oxidation assays measured by substrate
disappearance at pH 7.2 give rates at least 5 times higher
(
10)
than those observed here for aromatic hydrocarbon
oxidation at
a similar pH. Confirmation that oxidation was due to the
AMO was
obtained by using the Amo
mutants NZ1 and NZ2,
which cannot oxidize propene but retain
the ability to grow on propene
oxide. Additionally, resting cells
of noninduced control cultures of
the wild type grown on AMS fructose
were also unable to oxidize propene
or the aromatic substrates.
In the biotransformation of benzene, it was
noticed that phenol
accumulation was transient and that phenol was
metabolized as
soon as the benzene had been converted. By repetition of
the same
assays with phenol as a substrate, it was evident that phenol
was also oxidized by the AMO, and thus, benzene was being oxidized
consecutively by the same enzyme. However, tests with pure cresols
indicated that these were not good substrates for Xamo. Therefore,
in
the biotransformation of toluene, the resulting cresols remained
at
their final concentrations once the toluene had been completely
oxidized.
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TABLE 2.
Whole cell propene, benzene, and toluene monooxygenase
activities, and phenol hydroxylase and propene oxide degradation
activities, of Xanthobacter strain Py2 and the
Amo mutants NZ1 and NZ2a
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Biotransformation of a range of substrates is a common feature of
iron-containing monooxygenases, but it is generally observed
that the
range of substrates used for growth is much more restricted,
usually
because of the greater inducer specificity or inability
to further
metabolize the hydroxylated product. Given the evidence
for metabolism
of propene oxide via isomerization and carboxylation,
it seemed
unlikely that aromatic hydrocarbons would act as growth
substrates.
Thus we were surprised to find that although benzene
and toluene were
not growth substrates (even at nontoxic concentrations),
growth of Py2
was observed on 2 and 4 mM phenol, and this required
a functional Xamo,
as seen by the fact that the Amo
mutants failed to grow
at any concentration. Cells grown on phenol
were able to oxidize
propene and metabolize propene oxide in resting
cell assays, but at
rates lower than those obtained with propene-grown
cells, confirming
that phenol induces both Xamo and the epoxide
carboxylase. Growth on
phenol presumably occurred as a result
of oxidation to catechol and
subsequent ring cleavage reactions;
certainly
Xanthobacter
strain Py2 (and the Amo
mutants) can grow on catechol as
a sole carbon and energy source.
However, although the sequencing is
not complete, we have found
no evidence for genes encoding ring
cleavage enzymes in the vicinity
of the
aamA-through-
aamF cluster. At this stage we can
only speculate
that the broad range of inducers previously described
for Xamo
also includes phenol and that the resulting catechol can
induce
the necessary ring cleavage pathway. The ability to grow on
phenol
via the hybrid route may well have provided selective pressure
for the evolution of inducer specificity to include phenol.
Interestingly,
we have been unable to detect induction with the
nongrowth substrates,
benzene and toluene (2 mM), in cells grown on
either glucose or
isopropanol, although at this stage we cannot exclude
the possibility
that they are weak
inducers.
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ACKNOWLEDGMENT |
We are grateful to BBSRC for financial support to C.K.N.C.K.C.
(grant 28/T07300).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry, Imperial College of Science, Technology and Medicine,
London SW7 2AZ, United Kingdom. Phone: 44 171 5945227. Fax: 44 171 5945207. E-mail: d.leak{at}bc.ic.ac.uk.
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REFERENCES |
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1997.
Purification to homogeneity and reconstitution of the individual components of the epoxide carboxylase multiprotein enzyme complex from Xanthobacter strain Py2.
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Butler, C. S., and J. R. Mason.
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Applied and Environmental Microbiology, April 1999, p. 1589-1595, Vol. 65, No. 4
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
