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, Ser107, Asp228, and His256, 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.
Highlights
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
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 possess a serine residue as nucleophile within the classical catalytic triad (Ser, Asp, His) of serine hydrolases [6, 10]
The activity ofXylF was 85% inhibited by exposure to Diethyl pyrocarbonate (DEPC) (Table I) 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]
Summary
XylF, 2-hydroxymuconic semialdehyde hydrolase of the Pseudomonas putida TOL pathway; C-C, carboncarbon; 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. 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 8m-X-Nu-X-8m-8m, 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 a/{3 hydrolase-fold enzymes [14]. We have generated information on the potential role of other conserved residues in XylF structure and function
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
