(Received for publication, November 22, 1994; and in revised form, January 12, 1995)
From the
The 2-hydroxymuconic semialdehyde hydrolase, XylF, of the Pseudomonas putida TOL plasmid-encoded pathway for the
catabolism of toluene and xylenes, catalyzes one of the rarest types of
enzyme reaction (EC 3.7.1.9), the hydrolysis of a carbon-carbon bond in
its substrate, the ring-fission product of 3-alkyl-substituted
catechols. In this study, amino acid sequence comparisons between XylF
and other hydrolases, and analysis of the similarity between the
predicted secondary structure of XylF and the known secondary structure
of the haloalkane dehalogenase from Xanthobacter autotrophicus strain GJ10, led us to identify several conserved residues likely
to have a functional role in the catalytic center of XylF. Three amino
acids, Ser, Asp
, and His
,
were found to be arranged in a sequential order similar to that in
/
hydrolase-fold enzymes. Investigations of the potential
functional role of these and other residues through amino acid
modification and in vitro site-directed mutagenesis
experiments provided evidence in support of the hypothesis that XylF is
a serine hydrolase of the
/
hydrolase-fold family of enzymes,
and pointed to the residues identified above as the catalytic triad of
XylF. These studies also provided information on other conserved
residues in XylF-related enzymes. Interestingly, the substitution of
Phe by Met in position 108 of XylF created an enzyme with increased
thermostability and altered substrate specificity.
Biotechnological treatment of waste materials and bioremediation
of polluted environments is based on the capacity of microorganisms to
transform pollutants to non-toxic products. Attention focused on
enzymes catalyzing the transformation of synthetic compounds has
steadily increased since it became clear that some xenobiotics are
ubiquitous in the environment due to their slow rates of biodegradation (1) . Some of the most environmentally relevant xenobiotics are
aromatic compounds. Although bacteria employ a range of enzymes for the
initial attack on different aromatic substrates, the aerobic catabolic
pathways tend to converge on just a few key dihydroxylated
intermediates subsequently cleaved and further metabolized to Krebs
cycle intermediates by two distinct sets of enzymes, those of the ortho- and meta-cleavage pathways(2) . The
principal genetic and biochemical determinants governing the TOL (pWW0)
plasmid-specified meta-cleavage pathway for the oxidation of
catechol and alkyl-substituted catechols by Pseudomonas putida have been elucidated(3) . The initial step involves the meta-cleavage of catechol or its alkyl-substituted derivatives
by the enzyme catechol 2,3-dioxygenase. The ring-cleavage product thus
formed is processed through either a hydrolytic or a dehydrogenative
route (4-oxalocrotonate branch)(4) . The hydrolytic branch
preferentially converts the ring-fission products of 3-substituted
catechols, which are ketones, directly to 2-hydroxypent-2,4-dienoate
and acetic acid through the activity of a 2-hydroxymuconic semialdehyde
hydrolase, XylF, ()the product of the xylF gene (Fig. 1).
Figure 1:
Initial steps in the hydrolytic branch
of the meta-cleavage pathway for the catabolism of catechol
and alkyl-substituted catechols encoded by the P. putida TOL
plasmid pWW0. The enzyme abbreviation is: XylE, catechol
2,3-dioxygenase. R1 and R2 indicate the positions of
the alkyl-substituents. When R1 and R2 are H: compound 1 is catechol,
compound 2 is 2-hydroxymuconic semialdehyde, and compound 3 is
2-hydroxypent-2,4-dienoate. When R1 is CH and R2 is H:
compound 1 is 3-methylcatechol, compound 2 is
2-hydroxy-6-oxohepta-2,4-dienoate, and compound 3 is
2-hydroxypent-2,4-dienoate. When R1 is H and R2 is CH
:
compound 1 is 4-methylcatechol, compound 2 is
2-hydroxy-5-methyl-6-oxohexa-2,4-dienoate, and compound 3 is
2-hydroxy-cis-hex-2,4-dienoate.
XylF is a TOL (pWW0) plasmid-encoded 282-amino acid
length protein that catalyzes one of the rarest of all enzyme reaction
types, the hydrolytic cleavage of a carbon-carbon (C-C) bond (EC
3.7.1.-)(5, 6) . Whereas there is an increasing
understanding of meta-cleavage enzymes much less is known
about the hydrolytic cleavage of C-C bonds in the degradation of
aromatic compounds. Analogous hydrolases cleaving C-C bonds in
biodegradative pathways have been cloned and their primary structure
determined (7, 8, 9, 10) . In some
cases, these hydrolases have been shown to be determinants of substrate
specificity of the catabolic pathways(11) . Although
structure-function relationships in XylF-related proteins have not thus
far been elucidated, comparison of their amino acid sequences with that
of the chromosomally-encoded atropinesterase of P. putida, a
member of the serine hydrolase family(12, 13) ,
revealed significant similarity(6, 10) . Recently,
sequence comparisons have revealed a significant relationship of XylF
to other hydrolases (14, 15, 16) , in
particular to the haloalkane dehalogenase (Halo) from Xanthobacter
autotrophicus GJ10, a member of the /
hydrolase-fold
family of enzymes(14, 17, 18, 19) .
The /
hydrolase-fold family of enzymes comprises a group
of proteins that share a three-dimensional core structure although no
significant primary structure similarity has been detected. While the
catalytic specificities of the members of the
/
hydrolase-fold family are radically different from one another, their
enzymatic mechanisms appear to be rather similar(14) . They all
contain a catalytic triad with the configuration
nucleophile-acid-histidine in order of amino acid sequence. These three
amino acids, although separated from each other by a variable number of
other residues in the primary structure of each enzyme, are in similar
topological locations in the correctly folded proteins. In all
representatives of this family of hydrolases the nucleophile, in most
of cases a serine residue, is located in a conserved motif (the
``nucleophilic elbow'') with a proposed consensus sequence
Sm-X-Nu-X-Sm-Sm, where Sm indicates a small amino
acid (generally glycine), X is any amino acid, and Nu is the
nucleophile, which constitutes a structural link relating
/
hydrolase-fold enzymes(14) .
As a first step toward the
analysis of the structure/function relationships in XylF, we have
compared the primary sequences of members of the XylF-related family
with one another and with members of the /
hydrolase-fold
family, in order to identify putative catalytic residues of this
enzyme. Based on this, we performed amino acid modification studies and in vitro site-directed mutagenesis and obtained evidence for
involvement of Ser
, Asp
, and His
as the potential active amino acids within the catalytic triad of
XylF, and for the previous hypothesis that hydrolases cleaving C-C
bonds of the meta-cleavage products of aromatic compounds
belong to the
/
hydrolase-fold family. We have also generated
information on the potential role of other conserved residues in XylF
structure and function.
Figure 3:
Plasmid constructs used for expression of
wild-type and mutant XylF proteins, and enzymatic activities in
cellular extracts. A, the wild-type XylF expression vector
(pJH102) (6) contains a 2.1-kilobase pair DNA fragment (black arrow) bearing the gene encoding the XylF protein (xylF), placed under the control of the phage T7 promoter (PT7). xylG and xylJ are the genes flanking xylF in the meta-operon of the TOL plasmid. ,
means a deleted gene. Ap
, indicates the gene that
confers ampicillin resistance. Restriction sites are: E, EcoRI; H, HindIII; P, PstI; S, ScaI. A schematic representation of
XylF (black box) is detailed in the lower part of the
figure. The relative positions of the residues which were substituted
by site-directed mutagenesis and their corresponding codons (in brackets) are shown. The new residues and the altered codons
are indicated in boldface letters. Amino acids from position
273 at the COOH-terminal end of the protein are shown in one-letter
code. B, relative activities of the mutant enzymes in crude
extracts from E. coli C600 (pGP1-2) harboring the
corresponding derivatives of pJH102. 100% represents the specific
activity of wild-type XylF using the 3-methylcatechol ring-fission
product as substrate. The amount of mutated protein in the extract was
estimated by comparison to the amount of wild-type XylF. aa.,
amino acids; b.d., below detection limits (more than 500-fold
decreased). Values are the mean of three independent
experiments.
PMSF, a well-known transition state inhibitor for serine hydrolases that sulfonylates the catalytic serine residue(25) , inhibited XylF activity (Table 1) in a concentration- and time-dependent manner (data not shown). This inactivation was irreversible, as evidenced by the fact that the inactivated enzyme regained no activity when diluted. Another serine-specific reagent, DPF, also inhibited XylF irreversibly but to a lesser extent than PMSF (Table 1). 3,4-DCI, a reversible active-site blocking group for serine hydrolases(26) , significantly inhibited the activity of XylF (Table 1), although upon standing the enzyme regained activity (data not shown).
A histidine residue(s) appears to be part of the active site of XylF, as indicated both by the pH-dependent kinetics (5) and inhibition experiments with the histidine-specific reagents DEPC and TPCK. Thus, the activity of XylF was 85% inhibited by exposure to DEPC (Table 1) and this time-dependent loss of enzyme activity was even higher at pH 6.0, a pH value at which the reaction of DEPC with proteins is relatively specific for histidyl residues(27) . TPCK suppressed enzyme activity to 60% of the activity of the untreated XylF (Table 1). Inhibition by DEPC and TPCK was reversible by dilution of the inactivated enzyme.
XylF was also reversibly inhibited by some bulky thiol-modifying reagents such as N-ethylmaleimide, although the smaller iodoacetamide molecule which also alkylates SH groups did not significantly inhibit the enzyme activity in crude extracts (Table 1).
In conclusion, the evidence obtained with inhibitors is consistent with XylF being a serine hydrolase.
Figure 2:
Multiple amino acid sequence alignment of
XylF and other hydrolases. Sequences: 1, P. putida XylF (2-hydroxymuconic semialdehyde hydrolase; 6, 10); 2, Pseudomonas CF600 DmpD (2-hydroxymuconic semialdehyde
hydrolase; 7); 3, P. putida F1 TodF
(2-hydroxy-6-oxo-2,4-heptadienoate hydrolase; 8); 4, Pseudomonas sp. LB400 BphD
(2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase; 10); 5, P. putida KF715 BphD(9) ; 6, Pseudomonas sp. KKS102 BphD(10) ; 7, Moraxella sp.
Lipase 3(28) ; 8, Pseudomonas sp. KWI-56
esterase V(29) ; 9, P. putida atropinesterase(12) ; 10, Xanthobacter
autotrophicus GJ10 Halo (haloalkane dehalogenase; 17); 11, Lactobacillus delbrueckii sub sp. bulgaricus proline iminopeptidase(16) . The amino acid residues of
each sequence are numbered on the right. The sequences were
aligned using the multiple alignment program CLUSTAL (30) , and
manually realigned in the regions of the putative catalytic aspartic
acid and histidine residues. Amino acids are indicated by their
standard one-letter code. Symbols used are as follows: * ,
identical residues; , conservative amino acid replacements;
⇓, residue replaced by site-directed mutagenesis in the XylF
sequence. The serine, aspartic acid, and histidine residues that may
form the catalytic triad are indicated in bold-face letters.
The first line at the bottom of the sequences shows the predicted
secondary structure elements of XylF, while the second line names and
indicates the secondary structure elements of Halo(19) . Dotted and striped bars represent
-helices and
-strands, respectively, as determined by the Garnier
method(31) . The cap domain of Halo is boxed with continuous line; the predicted substrate binding domain of
XylF is boxed with discontinuous
line.
Another important
constituent of the catalytic triad in /
hydrolase-fold
enzymes is a histidine residue. It was suggested previously that
His
could be the catalytic histidine of XylF since, like
the catalytic histidine of Halo and other members of the
/
hydrolase-fold family, it is close to the COOH terminus of the enzyme
and is situated within a highly conserved stretch of amino acid
residues(15) . Supporting this hypothesis is the fact that
histidine 256 of XylF is the only histidine residue conserved through
all the enzymes compared in Fig. 2.
Asp of XylF
is a good candidate for the third member of the catalytic triad since
it is located in the equivalent position of the catalytic aspartic of
Halo. Asp
of XylF, the other aspartic acid residue
residing in a conserved part of the sequences analyzed, the
RVIAPD-XX-GXGXS motif (X, any amino
acid)(15) , should be excluded from the catalytic triad since
it is preceding the putative nucleophilic Ser
instead of
following the order Ser-Asp-His described for
/
hydrolase-fold enzymes.
Another criterion used to place XylF in a family of related hydrolases was the spacing between active-site residues and other relevant motifs. The distances in XylF between the putative catalytic histidine and aspartic acid, 27 residues; between the dipeptide His-Gly of the putatives oxyanion hole and the nucleophilic serine, 69 residues; and between the RVIAPDXXGXGXS motif and the putative catalytic serine, 34 residues, fit very well with those found in Halo and the other hydrolases of Fig. 2.
When the predicted
secondary structure of XylF was compared to the known secondary
structure of Halo, it seemed that the characteristic scaffold of
/
hydrolase-fold enzymes, i.e. a central
-pleated sheet surrounded by
-helices(14) , could
also be present in XylF (Fig. 2). The presumptive catalytic
triad residues of XylF have the same sequence order than in
/
hydrolase-fold enzymes and could occupy similar steric positions on the
loops following the corresponding
-strands. A typical nucleophilic
elbow could also be present in XylF since Ser
is
predicted to be located in a turn between a
-strand and an
-helix (Fig. 2). The nucleophile of Halo
(Asp
) is placed in an internal, predominantly
hydrophobic, cavity; similarly, hydropathicity plots indicated a
hydrophobic character for the amino acid sequence surrounding
Ser
of XylF (data not shown).
The regions of the
proteins compared in Fig. 2exhibiting lower similarity are the
NH-terminal end and a central segment. Since the substrates
of these enzymes have different structures, the substrate binding
domains may correspond to protein regions displaying low similarity. In
Halo, the central segment of the protein, the cap domain, has been
shown to be an excursion on the
/
hydrolase-fold structure
that plays an important role in substrate specificity(33) .
Interestingly, the central regions of XylF, DmpD, and TodF, the three
hydroxymuconic semialdehyde hydrolases that cleave the fission products
of one-ring aromatic compounds (catechol and derivatives), are similar
to each other and different to the central regions of the three BphD
hydrolases that cleave the fission products of two-ring aromatic
compounds (dihydroxybiphenyl and derivatives). These considerations
suggest that the central region of XylF (Fig. 2) may be involved
in substrate specificity.
The enzymatic activity of the mutant
XylF molecules was assayed using the 3-methylcatechol ring-fission
product as substrate. Results summarized in Fig. 3B show that substitution of the proposed catalytic triad residues,
Ser, Asp
, and His
, by Ala,
abolished all detectable enzymatic activity. Also mutants XylFD65V and
XylFT3 did not exhibit activity above background levels. Mutants
XylFS107C and XylFC254S showed an extremely low activity, 500- and
100-fold lower than that of the wild-type enzyme, respectively. In
contrast, substitution at Ser
, which we assumed not to
participate in the catalytic triad of XylF, exhibited significant
levels of enzymatic activity. However, replacement of the other two
conserved histidines, His
and His
, by Ala
residues, and substitution of Phe
by Met, resulted in a
reduction of activity to 15, 26, and 18%, respectively, of that of the
wild-type XylF, thus indicating some influence of these residues on
catalysis.
An absence of enzymatic activity in most of the mutants
could be caused either by the production of inactive XylF molecules or
by a lack of production of XylF protein. To examine XylF production in
the recombinant strains, we analyzed incorporation of
[S]methionine into the wild-type or mutant
enzymes expressed from the corresponding genes cloned into the T7
expression system (Fig. 3A). SDS-polyacrylamide gel
electrophoresis analysis of cell extracts prepared from the different
mutants revealed that all the different XylF molecules were produced
and migrated as a band with molecular mass of 32 kDa (Fig. 4),
which corresponds to the subunit molecular mass of XylF(5) .
Therefore, lack of production of XylF was not the reason for the
absence of enzyme activity in any of the inactive mutants. It should be
noted, however, that XylFH249A and XylFC254S were expressed at lower
levels than the other mutants (Fig. 4).
Figure 4:
Visualization of wild-type and mutant XylF
enzymes in E. coli extracts using the T7 gene expression
system. Autoradiogram of a 12.5% SDS-polyacrylamide gel of
[S]methionine-labeled cell extracts from E.
coli C600 (pGP1-2) harboring also plasmid pJH102 expressing
wild-type XylF (lane 10), or derivatives expressing XylFT3 (lane 2), XylFC254S (lane 3), XylFS107C (lane
4), XylFS107A (lane 5), XylFS152A (lane 6),
XylFD228A (lane 7), XylFD65V (lane 8), XylFF108M (lane 9), XylFH256A (lane 11), XylFH249A (lane
12), and XylFH36A (lane 13). Lane 1 shows
[
C]methylated molecular mass markers (Amersham):
carbonic anhydrase and ovalbumin. Molecular mass on the left is in kilodaltons.
To discriminate
whether changes in the enzymatic activity of the mutants might result
from global changes in protein structure and not from specific effects
of the side chains of the new amino acids, three different aspects were
considered. First, the exchange of the residues in the mutants did not
significantly alter the hydropathicity plots (34) . Also, the
secondary structure predictions, as determined by the
Garnier(31) , Robson(35) , and Chou-Fasman (36) methods, for most of the mutant proteins were similar to
that of wild-type XylF (data not shown). Only the mutants XylFS152A and
XylFC254S, whose changes were predicted to destroy a -turn
conformation, and the mutant XyFT3, whose change was predicted to
change an
-helix to a
-sheet, showed different secondary
structure predictions from the wild-type XylF. Second, the modified
enzymes migrated during nondenaturing polyacrylamide gel
electrophoresis like the wild-type enzyme (data not shown), which makes
extensive conformational changes unlikely and suggests that the dimeric
quaternary structure of XylF (5) is also characteristic of the
mutant proteins. Third, the stability of the mutant proteins in
vivo was measured by pulse-chase experiments. Nine of the 11
mutant enzymes were found to behave similarly to the unmodified enzyme,
being highly stable even after 90 min of chase with unlabeled
methionine. The other two, XylFD65V and XylFT3, were unstable and
decreased in cellular extracts after 10 min of chase; after 30 min
these proteins were hardly visible (Fig. 5). This indicates
that, with the exception of XylFD65V and XylFT3, the mutants and
wild-type XylF have similar sensitivities to cellular proteases, and
suggests that no drastic changes in the folding of the mutant molecules
have occurred.
Figure 5:
Stability of XylF, XylFD65V, and XylFT3
enzymes by in vivo pulse-chase experiments. Autoradiogram of a
10% SDS-polyacrylamide gel of
[S]methionine-labeled cell extracts from E.
coli C600 (pGP1-2) harboring also the plasmid pJH102
expressing wild-type XylF (lanes 1 and 2), or its derivatives
expressing XylFD65V (lanes 3-6) and XylFT3 (lanes
7-10). Pulse-chase experiments were carried out at 30
°C. Cells were incubated 2.5 min with
[
S]methionine as described under
``Experimental Procedures'' and incorporation was terminated
by resuspending the cells in fresh M9 medium supplemented with 20
µg/ml thiamine and the 20 unlabeled amino acids (0.5 mM).
Cell samples were incubated further and 500-µl aliquots were
removed and centrifuged at zero time (lanes 1, 3, and
7), 10 min (lanes 4 and 8), 20 min (lanes 5 and
9), 30 min (lanes 6 and 10), and 90 min (lane 2)
later. The cell pellets were resuspended in 80 µl of cracking
buffer (21) and heated to 95 °C for 5 min prior to loading
15 µl onto the SDS-polyacrylamide gel. The molecular mass of the
[
C]methylated protein standards (Amersham),
lysozyme, carbonic anhydrase, ovalbumin, and bovine serum albumin, is
indicated on the left in
kilodaltons.
Some kinetic parameters of the active XylF mutants
were analyzed (Table 2). When the 3-methylcatechol ring-fission
product was used as substrate, the K values of the
mutants differed little from that of the wild-type enzyme, indicating
minor or no perturbation in substrate binding. Therefore, the lower
specific activity of XylFH36A, XylFH249A, and XylFF108M mutant proteins
with respect to that of the wild-type enzyme should be the result of
partially impaired catalysis. The S152A substitution resulted in an
enzyme which did not markedly differ kinetically from wild-type XylF (Table 2). With the catechol ring-fission product as substrate,
the XylFS152A mutant exhibited relative V
and K
values almost twice those of the wild-type XylF.
Mutant XylFH36A again showed the lowest relative V
. Interestingly, the substitution of
Phe
by Met increased more than 4-fold the K
value for the catechol ring-fission product, the
relative V
/K
value being
nearly 20-fold lower than that of the unmodified enzyme. XylFF108M
exhibited no activity with the fission product of dihydroxybiphenyl as
substrate, a behavior similar to that of the wild-type XylF (data not
shown).
Thermal stability of the active mutants was also analyzed. XylFH36A, XylFS152A, and XylFH249A mutants showed a severe decrease in protein stability at 42 °C over that of the wild-type enzyme (Table 2), indicating that the residues substituted in the mutants may be important for maintaining secondary-tertiary structure. In contrast, the XylFF108M mutant displayed greater thermostability than the unmodified enzyme (Table 2).
XylF, an enzyme encoded by the TOL plasmid pWW0 of P.
putida which hydrolyzes the aromatic ring-fission products of
preferably 3-substituted alkyl catechols(4, 5) , is a
member of a small group (includes only 10 representatives) of
hydrolytic enzymes which cleave C-C bonds (EC 3.7.1.-)(37) .
Although the structure-function relationships of XylF are not yet
known, previous studies based on primary sequence comparisons have
suggested XylF and other hydrolases cleaving C-C bonds of the meta-cleavage products of aromatic compounds as serine
hydrolases (6, 10) and as members of the /
hydrolase-fold family(14, 15, 16) . Here we
provide experimental evidence in support of this proposal.
Results obtained through amino acid modification studies on XylF activity using either serine-specific (PMSF, DPF, 3,4-DCI) or histidine-specific (DEPC, TPCK) modifying reagents, suggest the participation of serine and histidine residues in the catalytic mechanism. Thus, XylF could be a serine hydrolase having the serine residue of its active center linked in a charge-relay system with the imidazole ring of a histidine and probably the carboxylate anion of an acidic residue enhancing the nucleophilicity of the serine hydroxyl.
The catalytic triad we have
proposed for XylF, i.e. Ser, Asp
,
and His
, was directly evaluated by in vitro site-directed mutagenesis. The complete inactivation of XylF after
substitution by alanine of any of these three putative catalytic
residues was consistent with our proposal. None of these amino acid
replacements affected XylF protein production, stability, or migration
in nondenaturing polyacrylamide gel electrophoresis. Furthermore, the
hydropathicity plots and secondary structure predictions of the
XylFS107A, XylFD228A, and XylFH256A mutants did not significantly
differ from those of the XylF protein. Taken together, these data point
to a proper folding and common three-dimensional structure for the
wild-type and mutant enzymes, suggesting that loss of activity in the
mutants is the functional consequence on catalysis of changing the side
chains of essential residues, and not the result of unspecific global
structural alterations. Two other lines of evidence agree with the
involvement of Ser
, Asp
, and His
in the catalytic triad of an
/
hydrolase-fold enzyme:
the order and distances between these amino acids in the primary
sequence of XylF, and the secondary structure predictions suggesting
that the folding of XylF is in accordance with the overall fold for
members of the
/
hydrolase-fold family. Interestingly, the
sequence LVGNS-X-GG (X is Phe in XylF) around the
active-site Ser of XylF is also present in other XylF-related
hydrolases cleaving C-C bonds and fits very well in the nucleophilic
motif present in all members of the
/
hydrolase-fold
family(14) .
There are other Ser, Asp, and His residues
conserved in most of the enzymes compared in Fig. 2that in
principle could be also candidates for the catalytic triad of XylF.
Ser of XylF is conserved in other hydrolases cleaving C-C
bonds and also in lipase 3 of Moraxella sp. Substitution of
this Ser residue by Ala produced a XylF mutant with a level of activity
similar to that of the wild-type enzyme, but with a significantly lower
thermal stability that could reflect the change in its secondary
structure predicted by the Chou-Fasman method. These data indicate that
Ser
of XylF is not essential for catalysis and are
consistent with the proposal that this residue is not within a
nucleophilic motif and that it occurs in a region of the molecule not
highly conserved which may be involved in substrate specificity.
In
the alignment of Fig. 2, His and His
of XylF are conserved in all enzymes (except for esterase V from Pseudomonas sp.), and in hydrolases cleaving C-C bonds,
respectively. When these amino acids were substituted by Ala residues,
a significant reduction in specific activity and thermostability was
observed. It seems, however, that although His
and
His
have some influence in catalysis, only His
can be clearly defined as essential for catalytic activity. The
impaired activity of XylFH36A, the conserved sequence of hydrophobic
residues surrounding His
, and the location of this residue
at the end of a
-sheet and before a coil conformation within the
predicted secondary structure of XylF, point to an important functional
role for this residue, and allow us to tentatively assign the dipeptide
His
-Gly
of XylF as the central dipeptide that
characterizes the oxyanion hole in members of the
/
hydrolase-fold family(14, 18, 32) .
Substitution of the conserved Asp by a valine residue
resulted in a total loss of XylF activity and a drastic reduction in in vivo stability. This suggests that although Asp
may be not essential for catalysis, substitution at this position
may result in aberrant folding which in turn may lead to intracellular
degradation of the mutant polypeptide. Interestingly, Asp
is located in a sequence that matches perfectly the conserved
RVIAPD motif found between the oxyanion hole region and the nucleophile
in the primary structure of Halo and putative
/
hydrolase-fold enzymes(15) . These findings, and the
observation that no conserved glutamic acid residue could be found
between the nucleophile and the catalytic His
of XylF,
reinforce the model that considers Asp
as the residue
providing the acidic group in the catalytic triad of this hydrolase.
The COOH-terminal region of XylF seems to be indispensable for
enzyme activity since the XylFT3 mutant protein was inactive. A similar
finding has been reported with the analogous BphD hydrolase from Pseudomonas sp. LB400(38) . The mobility of the XylFT3
molecule during nondenaturing polyacrylamide gel electrophoresis was
similar to that of the wild-type XylF, suggesting no changes in the
homodimeric conformation of this hydrolase. However, the fact that the
mutant protein was degraded much faster in vivo than the
unmodified enzyme, together with the prediction that in XylFT3 the long
terminal -helix has been truncated and replaced by a
-sheet,
suggest an important role for the COOH-terminal region of XylF in
maintaining secondary-tertiary structure. In human deamidase and wheat
carboxypeptidase (another member of the
/
hydrolase-fold
family), the COOH-terminal region has been shown to be important in the
formation of homodimers, and a single residue mutation in this region
of the human deamidase results also in synthesis of an inactive protein
which is rapidly degraded(39) .
The inhibition of XylF by
some thiol-specific reagents as N-ethylmaleimide is consistent
with previous results with other serine
hydrolases(17, 40, 41, 42) . The
observation that the only cysteine of XylF, Cys, is not
involved in disulfide bridges between subunits(5) , and the
fact that this amino acid is located one residue away from the
catalytic His
in the primary sequence of XylF, suggest
that bulky reagents which react with cysteinyl residues, i.e.N-ethylmaleimide, may disturb the arrangement of
catalytic residues or exclude the substrate from the active center,
even if the susceptible group is not part of the catalytic mechanism.
If this were the case, a small reagent attached to the thiol group
should exert no steric hindrance, which would be consistent with the
absence of XylF inhibition by the small iodoacetamide molecule. A
similar pattern of inhibition has been described with another C-C bond
hydrolase, the phloretin hydrolase from Aspergillus niger, which is very susceptible to the bulky p-chloromercuribenzoate but not to the smaller iodoacetate (43) . Secondary structure predictions suggest that Cys
of XylF forms part of a
-turn which also contains the
catalytic His
, and that replacement of this cysteinyl
residue by a serine would change the predicted
-turn into a random
coil structure. The activity of the XylFC254S mutant enzyme is 100-fold
lower than that of XylF, which reinforces the notion that Cys
of XylF participates in the structure of the active site,
probably placing the catalytic His residue on top of a turn as already
described for
/
hydrolase-fold enzymes (14) .
The
catalytic triad positions in the /
hydrolase-fold family
might in principle accommodate different amino acids(14) . In
an attempt to engineer a modified hydrolase triad into the XylF
backbone, the nucleophilic hydroxyl of Ser
was replaced
by a sulfhydryl group. Although the XylFS107C mutant protein was
produced and did not show any significant changes in stability,
secondary structure prediction, and electrophoretic mobility, its
activity was decreased more than 500-fold. Similar results were
obtained during some previous attempts to engineer efficient thiol
active-site enzymes from naturally occurring serine active-site
hydrolases, although an equivalent mutant of the rat mammary gland
thioesterase II did show normal activity(44) . This latter
report supports an earlier hypothesis that consider all serine
active-site hydrolases to be derived from cysteine active-site
enzymes(13) , and agrees with the observation that
thioesterases utilize both types of serine codons, TCN and AGY, that
could have evolved from cysteine codons (TGY)(44) . The fact
that hydrolases cleaving C-C bonds of the meta-cleavage
products of aromatic compounds utilize the same type of codon (TCN) for
the presumptive active-site serine, and the finding that the XylFS107C
mutant has a very poor activity, might indicate during evolution of
XylF a shift toward a catalytic mechanism of higher stringency than is
the case for other members of the
/
hydrolase-fold family
like thioesterase II and dienelactone
hydrolase(44, 45) .
In serine hydrolases residues
immediately preceding and following the nucleophile are not
conserved(13) , suggesting that these are not essential for
catalytic activity. Sequence comparison analysis revealed that whereas
hydrolases cleaving fission products of one-ring aromatic compounds
have a phenylalanine residue following the presumptive nucleophile,
hydrolases cleaving fission products of two-ring aromatic compounds
have a methionine. To test whether substitution of Phe to Met at
position 108 in the XylF molecule would result in an active mutant
enzyme exhibiting different substrate specificity, we generated the
XylFF108M protein. This mutant was, as expected, active, but its
activity was reduced and restricted to the fission products of
3-methylcatechol and catechol; no activity was detected with the
fission product of dihydroxybiphenyl. However, the XylFF108M mutant
enzyme did exhibit changes in two of its properties: its K value for the catechol ring-fission product was
more than 4-fold higher than that of XylF, and surprisingly it
exhibited a greater thermal stability. These results suggest that the
phenylalanine residue following the catalytic serine in XylF is
involved in substrate recognition, endowing upon the enzyme some
substrate specificity. Since the secondary structure predictions for
XylFF108M and wild-type XylF were similar, the increased stability of
the mutant enzyme to thermal inactivation may result from an
improvement of the overall tertiary packing; this would in turn suggest
a participation of Phe
in the hydrophobic core of the
protein. This possibility must, however, be assessed by determination
of the three-dimensional structure of the enzyme.
It has been proposed that the 4-oxalocrotonate branch of the meta-TOL pathway, the one that acts on the aldehyde ring-fission products of catechol and 4-substituted catechols, may have preceded the appearance of the hydrolytic branch, with recruitment of a cellular hydrolase (XylF at present) to deal with the keto ring-fission products of 3-substituted catechols at some later stage in the evolution of the catabolic pathway(6, 46) . If this is the case, the XylFF108M mutant enzyme may represent an example of what could be a further step in the adaptation of the hydrolytic route toward the cleavage exclusively of ring-fission products of 3-alkyl substituted catechols.
The present observations strongly suggest
Ser, Asp
, and His
as the
active residues within the catalytic triad of XylF. The
/
hydrolase-fold family of enzymes therefore appears to be a rapidly
growing group of proteins that share the same scaffolding for an
extremely versatile catalytic triad able to hydrolyze esters, amides,
carbon-halogen, and now C-C bonds, a clear example of divergent
evolution of catalytic sites(14) . The results presented in
this work provide insights into the structure-function relationships of
XylF and constitute a framework for further investigations to obtain
more knowledge on the structural and biochemical properties of this and
other aromatic hydrolases, and to engineer more stable enzymes with
improved catalytic efficiencies and altered substrate specificities
that could find applications in the development of new and better
biological catalysts for the degradation of xenobiotics.