©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Functional Residues in a 2-Hydroxymuconic Semialdehyde Hydrolase
A NEW MEMBER OF THE alpha/beta HYDROLASE-FOLD FAMILY OF ENZYMES WHICH CLEAVES CARBON-CARBON BONDS (*)

(Received for publication, November 22, 1994; and in revised form, January 12, 1995)

Eduardo Díaz (§) Kenneth N. Timmis (¶)

From the Department of Microbiology, GBF-National Research Centre for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 alpha/beta 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 alpha/beta 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.


INTRODUCTION

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, (^1)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(3) 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(3): 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 alpha/beta hydrolase-fold family of enzymes(14, 17, 18, 19) .

The alpha/beta 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 alpha/beta 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 alpha/beta 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 alpha/beta 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 alpha/beta hydrolase-fold family. We have also generated information on the potential role of other conserved residues in XylF structure and function.


EXPERIMENTAL PROCEDURES

Bacterial Strains, Phages, Plasmids, and Growth Conditions

Escherichia coli K-12 strains used were: HB101 (FhsdS20 (r(B)m(B)) recA13 ara14 proA2 leuB lacY1 galK2 rpsL20 xyl5 mtl1supE44; (20) ), C600 (FthiI leuB6 thrI lacY6 tonA21 supE44; (20) ), and TG1 (Delta(lac-pro) supE thi hsdD5/F` traD36 proABlacI^qlacZ DeltaM15; Amersham Corp.). The latter was used as a host for phage M13tg131 (Amersham Corp.). The plasmids used were pGP1-2 (21) and pJH102, a derivative of pT7-6 containing a 2.1-kilobase pair HindIII-EcoRI fragment bearing the xylF gene(6) . Phage M13tg131-xylF containing the xylF gene was prepared by subcloning the 2.1-kilobase pair HindIII-EcoRI fragment of pJH102 in the HindIII + EcoRI doubly-digested replicative form of phage M13tg131. E. coli was grown in LB medium at 37 °C, except the strain C600 containing pGP1-2 and pJH102, or any of its derivatives, which were grown at 30 °C. 50 µg/ml ampicillin (for cells harboring pJH102 and derivatives) and kanamycin (for cells harboring pGP1-2) were added to the culture medium where appropriate.

Plasmid Isolation and Transformation

Plasmid DNA was prepared by the rapid alkaline method(20) . Standard recombinant DNA techniques (20) were used. Transformation of E. coli was carried out using the RbCl method(20) .

Oligonucleotide Site-directed Mutagenesis

Oligonucleotides were synthesized on an Applied Biosystems model 394 nucleotide synthesizer. Mutagenesis was performed using the mutagenesis system of Amersham International according to the procedure recommended by the supplier using single-stranded M13tg131-xylF DNA and the appropriate oligonucleotides. The following oligonucleotides were used for mutagenesis of the codons to replace: H36A, 5`-CGGGGCCGGATCCGGCGATCAGCAGAG-3`, BamHI(+); D65V, 5`-GCCGAGCATGACCGGTGCGATCACCC-3`, AgeI(+); S107A, 5`-CCGCCGAACGCGTTGCCGACG-3`, MluI(+); S107C, 5`-CCGCCGCCGAAGCAGTTCCCGACGATGTC-3`, XmnI(+); F108M, 5`-GCCCGCCGCCCATGGAGTTGCCG-3`, NcoI(+); S152A, 5`-GCATGTTGGCTAGCGCCGGCGTGTAG-3`, NheI(+); D228A, 5`-GCGGGATGATGCGCGCCTCGCGGCC-3`, BssHII(+); H249A, 5`-CCGAACACGGCTAGCTGGGCG-3`, PmlI(-); C254S, 5`-GGGTCCAGTGTCCGGACTGGCCGAAC-3`, BspEI(+); H256A, [5`-CTGGGTCCAGGCGCCGCACTGG-3`; NarI (+)) (altered bases are represented in boldface letters; created (+) or destroyed(-) restriction sites are underlined; amino acids are shown in one-letter code and their positions in the primary sequence of XylF are specified). Mutations were screened by restriction digestion and confirmed by sequence analysis of the corresponding single stranded phage DNA using an appropriate sequencing primer and the dideoxy chain termination method(22) . Standard protocols of the manufacturer for Taq DNA polymerase-initiated cycle sequencing reactions with fluorescently-labeled dideoxynucleotide terminators (Applied Biosystems Inc., Foster City, CA) were used with the modification that primer annealing times were increased to 15 s. The sequencing reactions were analyzed using a 373A automated DNA Sequencer (Applied Biosystems Inc.). Sequenced DNA fragments containing the mutated region of the xylF gene were then excised from the relevant M13tg131 replicative form DNA and inserted in pJH102 in place of the analogous DNA fragment carrying the wild-type xylF gene (Fig. 3). The mutant XylFT3 protein was constructed starting from a spontaneous mutant xylF gene containing an EcoRI restriction site at its 3` end. After replacing the wild-type xylF in pJH102 by the mutated xylF gene, the 10 COOH-terminal amino acids of XylF (FLAEADALHS) were substituted by the 7 COOH-terminal amino acids of XylFT3 (SMISCQT). All final constructions were checked by restriction analysis.


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. Delta, means a deleted gene. Ap^r, 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.



Identification of Wild-type and Mutant XylF Enzymes

E. coli C600 (pGP1-2) harboring pJH102 or its derivatives was cultured at 30 °C in LB medium supplemented with kanamycin (50 µg/ml) and ampicillin (50 µg/ml) to an optical density at 600 nm of about 0.5. Cells (1 ml) were washed with M9 minimal medium (20) and resuspended in 1 ml of M9 medium supplemented with thiamine (20 µg/ml) and 0.01% of all amino acids except methionine(21) . Cultures were grown at 30 °C for 90 min prior to shifting the temperature to 42 °C for 15 min to induce synthesis of XylF or mutant XylF enzymes. Rifampicin (400 µg/ml) was then added to halt normal cellular transcription, and after 10 additional min at 42 °C the cultures were shifted down to 30 °C for 20 min. 10 µCi of [S]methionine (10 µCi/µl) were added, and cultures were incubated for 4 min at 30 °C, centrifuged, and the pelleted cells resuspended in 120 µl of cracking buffer (21) and heated at 95 °C for 5 min prior to loading onto a SDS-polyacrylamide gel(23) . After electrophoresis, gels were dried at 80 °C and exposed to x-ray films (Kodak X-Omat AR).

Preparation of Crude Extracts from E. coli C600 Overexpressing Wild-type and Mutant XylF Enzymes

E. coli C600 (pGP1-2) harboring pJH102 or its derivatives was cultured at 30 °C in LB medium supplemented with kanamycin (50 µg/ml) and ampicillin (50 µg/ml) to an optical density at 600 nm of about 1.0. The cultures were then induced for T7 RNA polymerase expression by incubation at 42 °C for 30 min, followed by incubation at 37 °C for an additional 2 h. Cells were harvested by centrifugation and washed and resuspended in 0.1 volumes of 50 mM sodium phosphate buffer, pH 7.5, containing 5 mM 2-mercaptoethanol, prior to disruption by a single passage through a French Press (Aminco Corp.) operated at a pressure of 20,000 p.s.i. The cell debris was removed by centrifugation at 18,000 rpm for 45 min in a SS-34 rotor (Sorvall Instruments). The clear supernatant fluid was carefully decanted and used as crude extract. Protein concentration was determined by the method of Bradford (24) using bovine serum albumin as standard.

Enzyme Assays

The hydrolytic activity of XylF was determined by following the formation of reaction products using a Beckman DU-70 spectrophotometer equipped with a thermojacketed cuvette holder and a Haake circulating water bath. Assays of XylF activity in crude extracts were performed at 25 °C in 50 mM sodium phosphate buffer, pH 7.5, with 42 µM substrate (ring-fission products of catechol or 3-methylcatechol). Substrate was prepared fresh daily in a 1-ml volume reaction, containing 600 µM catechol or 3-methylcatechol from a 10 mM stock solution, 0.1 M sodium phosphate buffer, pH 7.5, and 0.5 µl of a pure enzyme preparation of catechol 2,3-dioxygenase, that was allowed to proceed to completion. The reactions performed with the ring-fission products of catechol or 3-methylcatechol were followed by monitoring the decrease in absorbance at 375 or 386 nm, respectively. One unit of enzymatic activity was defined as the amount of enzyme converting 1 µmol of substrate/min, using the extinction coefficients reported by Duggleby and Williams (5) for the ring-fission products. K(m) and V(max) were calculated from Lineweaver-Burk plots.

Chemical Modifications by Site-specific Reagents

Stock solutions of inhibitors (purchased from Sigma, except diisopropyl fluorophosphate (DPF) which was obtained from Aldrich Chemical Co.) were made up fresh daily. N-Ethylmaleimide and iodoacetamide were prepared at a concentration of 0.2 M by dissolving in water. Diethyl pyrocarbonate (DEPC) was diluted to 0.5 M in acetonitrile. Stock solutions of tosylphenylalanine chloromethyl ketone (TPCK), DPF, and 3,4-dichloroisocoumarin (3,4-DCI) were prepared at concentrations of 0.1 M, 0.5 M, and 6 mM, respectively, by dissolving in dimethyl sulfoxide. Phenylmethylsulfonyl fluoride (PMSF) was diluted to 0.1 M in isopropyl alcohol. Inactivation of wild-type XylF enzyme in crude extracts was performed at 25 °C by incubation in 50 mM sodium phosphate buffer, pH 7.5, containing the inhibitor (Table 1). The ring-fission product of 3-methylcatechol was added to the mixtures to a final concentration of 42 µM and the decrease in absorbance at 386 nm was monitored. The solvents used to prepare the stock solutions of inhibitors were included in control incubations at the appropriate concentrations. Inactivation with TPCK and 3,4-DCI was performed in the presence of 10% (v/v) dimethyl sulfoxide, but a 25-fold dilution in 50 mM sodium phosphate buffer, pH 7.5, was carried out before adding the substrate in case of 3,4-DCI. For studies with 3,4-DCI, crude extracts containing XylF were prepared in the absence of 2-mercaptoethanol which inactivates the inhibitor.




RESULTS

Amino Acid Modification Studies on XylF

Based on amino acid sequence comparisons with the P. putida atropinesterase, a serine hydrolase catalyzing the ester hydrolysis of atropine(12) , it was suggested that XylF may also possess a serine residue as nucleophile within the classical catalytic triad (Ser, Asp, His) of serine hydrolases(6, 10) . To analyze more directly the importance of specific residues in the mechanism of action of XylF, we studied the effects on the activity of this enzyme of different reagents that specifically modify serines and histidines.

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.

XylF and Other Bacterial Hydrolases Share a Similar Organization around Their Presumptive Catalytic Triad

Previous amino acid sequence comparisons with Halo, an enzyme whose three-dimensional structure and mechanism of action are known(18, 19) , have suggested XylF and other hydrolases cleaving C-C bonds from aromatic compounds are members of the alpha/beta hydrolase-fold family of enzymes(14, 15) . Fig. 2shows a multiple sequence alignment among XylF and 10 other hydrolases including the 2-hydroxymuconic semialdehyde hydrolases involved in the degradation of phenol (DmpD) and toluene (TodF), and the 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolases (BphD) involved in the catabolism of polychlorinated biphenyls. Inspection of the alignment reveals the existence of discrete regions that display high similarity and that correspond with those bearing the catalytic amino acids in Halo. As previously suggested(6, 10, 15, 16) , the serine in position 107 of XylF is a very good candidate for the nucleophile. First, this residue is the only serine residue conserved in all hydrolases compared (in Halo the nucleophile is the equivalent Asp) and has been shown to be part of the P. putida atropinesterase catalytic site(12) ; and second, it occurs within a sequence pattern, Gly-Asn-Ser-Phe-Gly-Gly, that fits very well in the proposed consensus motif for the nucleophile of alpha/beta hydrolase-fold enzymes(14) .


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; bullet, 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 alpha-helices and beta-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 alpha/beta 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 alpha/beta 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 alpha/beta 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 alpha/beta hydrolase-fold enzymes, i.e. a central beta-pleated sheet surrounded by alpha-helices(14) , could also be present in XylF (Fig. 2). The presumptive catalytic triad residues of XylF have the same sequence order than in alpha/beta hydrolase-fold enzymes and could occupy similar steric positions on the loops following the corresponding beta-strands. A typical nucleophilic elbow could also be present in XylF since Ser is predicted to be located in a turn between a beta-strand and an alpha-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(2)-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 alpha/beta 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.

Site-directed Mutagenesis of XylF and Biochemical Properties of the Mutant Enzymes

To assess the proposed model for the XylF catalytic triad, specific mutant enzymes using site-directed mutagenesis have been constructed. Fig. 3shows the positions of the exchanged amino acids, the altered codons, the altered amino acids, and the names of the mutants. The choice of amino acid replacements was on the basis that the change results in the insertion of a residue that could not be ionized (Ala or Val). In the case of the presumptive nucleophile Ser, the hydroxyl group was also replaced by a sulfhydryl group in the mutant XylFS107C. His, His, and Ser were also replaced because although they are not present in all hydrolases compared in Fig. 2, they are conserved among the hydrolases cleaving C-C bonds (Fig. 2), and thus could be also candidates for the catalytic triad. Since the residue following the putative nucleophilic Ser is a Phe in hydroxymuconic semialdehyde hydrolases and a Met in BphD hydrolases (Fig. 2), mutant XylFF108M was constructed to test whether substitution of Phe to Met would result in an active XylF with a different substrate specificity. The inhibition of XylF activity with N-ethylmaleimide (Table 1) suggested the influence of sulfhydryl groups in the catalytic reaction. For this reason, a XylFC254S mutant was generated replacing the only Cys of the molecule, also unique in the other C-C bond hydrolases (Fig. 2), by a Ser residue. Finally, a mutant (XylFT3) that has the 10 COOH-terminal amino acids replaced by a different sequence of 7 residues, was engineered to analyze the effect of mutations in the COOH-terminal region of XylF.

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 [^14C]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 beta-turn conformation, and the mutant XyFT3, whose change was predicted to change an alpha-helix to a beta-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 [^14C]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(m) 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(max) and K(m) values almost twice those of the wild-type XylF. Mutant XylFH36A again showed the lowest relative V(max). Interestingly, the substitution of Phe by Met increased more than 4-fold the K(m) value for the catechol ring-fission product, the relative V(max)/K(m) 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).


DISCUSSION

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 alpha/beta 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 alpha/beta 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 alpha/beta 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 alpha/beta 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 beta-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 alpha/beta 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 alpha/beta 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 alpha-helix has been truncated and replaced by a beta-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 alpha/beta 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 beta-turn which also contains the catalytic His, and that replacement of this cysteinyl residue by a serine would change the predicted beta-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 alpha/beta hydrolase-fold enzymes (14) .

The catalytic triad positions in the alpha/beta 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 alpha/beta 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(m) 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 alpha/beta 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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by long-term European Molecular Biology postdoctoral fellowship ALTF 336-1991 and by a post-doctoral BRIDGE-EC fellowship during 1993 and 1994. To whom all correspondence should be addressed. Tel.: 49-531-6181405/441; Fax: 49-531-6181411.

Acknowledges support for this work from the Fonds der Chemischen Industrie.

(^1)
The abbreviations used are: XylF, 2-hydroxymuconic semialdehyde hydrolase of the Pseudomonas putida TOL pathway; C-C, carbon-carbon; DEPC, diethyl pyrocarbonate; DPF, diisopropyl fluorophosphate; 3,4-DCI, 3,4-dichloroisocoumarin; Halo, haloalkane dehalogenase from Xanthobacter autotrophicus; PMSF, phenylmethylsulfonyl fluoride; TPCK, tosylphenylalanine chloromethyl ketone.


ACKNOWLEDGEMENTS

We thank E. Moore, A. Arnscheidt, and A. Krüger for valuable help with the sequencing. We are grateful to L. Eltis and J. M. Horn for generously providing purified catechol 2,3-dioxygenase and the plasmid pJH102, respectively.


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