The xlnD gene from Pseudomonas alcaligenes NCIMB 9867 (strain P25X) was shown to encode 3-hydroxybenzoate 6-hydroxylase I, the enzyme that catalyzes the NADH-dependent conversion of 3-hydroxybenzoate to gentisate. Active recombinant XlnD was purified as a hexahistidine fusion protein from Escherichia coli, had an estimated molecular mass of 130 kDa, and is probably a trimeric protein with a subunit mass of 43 kDa. This is in contrast to the monomeric nature of the few 3-hydroxybenzoate 6-hydroxylases that have been characterized thus far. Like other 3-hydroxybenzoate 6-hydroxylases, XlnD could utilize either NADH or NADPH as the electron donor. P25X harbors a second 3-hydroxybenzoate 6-hydroxylase II that was strictly inducible by specific aromatic substrates. However, the degradation of 2,5-xylenol and 3,5-xylenol in strain P25X was found to be dependent on the xlnD-encoded 6-hydroxylase I and not the second, strictly inducible 6-hydroxylase II.

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Pseudomonas alcaligenes NCIMB 9867 (strain P25X) degrades xylenols and cresols via the gentisate pathway. P25X expresses two isofunctional gentisate 1,2-dioxygenases (GDO I and GDO II). The expression of both GDOs was not detected when P25X cells were grown at 42 degrees C, even in the presence of gentisate. A total of 19 heat shock proteins (Hsps) belonging to the Hsp100, Hsp90, Hsp70, Hsp60, Hsp45, and small heat shock protein (sHsp) families were identified among the protein spots that were either newly detected or were expressed at levels of at least twofold higher when P25X cells were cultured at 32 or 42 degrees C in the presence and absence of gentisate. Among these, 16 Hsps were commonly expressed at 42 degrees C. Two additional Hsps (H5 and H13) from the Hsp90 and Hsp60 families, respectively, were expressed only when P25X cells were grown at 42 degrees C and in the presence of gentisate. A protein of the sHsp (H16) family was expressed only in the presence of gentisate at 32 degrees C but not at 42 degrees C. The GroEL chaperonins of the Hsp60 family comprised the largest group of Hsps identified and exhibited high level of expression at 42 degrees C following gentisate exposure.

xlnE, encoding gentisate 1,2-dioxygenase (EC 1.13.11.4), from Pseudomonas alcaligenes (P25X) was mutagenized by site-directed mutagenesis. The mutant enzyme, Y181F, demonstrated 4-, 3-, 6-, and 16-fold increases in relative activity towards gentisate and 3-fluoro-, 4-methyl-, and 3-methylgentisate, respectively. The specific mutation conferred a 13-fold higher catalytic efficiency (k cat /K m ) on Y181F towards 3-methylgentisate than that of the wild-type enzyme.Pseudomonas alcaligenes NCIMB 9867 (strain P25X) degrades m-cresol, 2,5-xylenol, 3,5-xylenol, and their catabolites via the gentisate pathway (7, 9). A critical step in the gentisate pathway is the fission of the gentisate aromatic ring catalyzed by gentisate 1,2-dioxygenase (GDO I; EC 1.13.11.4) that initiates this reaction by destabilizing the aromatic ring, employing Fe 2ϩ as a cofactor (5) and yielding maleylpyruvate, which is then channeled to the tricarboxylic acid cycle (7). P25X harbored isofunctional GDOs, with one set being constitutively expressed yet further inducible (14) and the other set being strictly inducible (9). Both GDOs were reported to possess broad substrate specificities towards unsubstituted, alkylated, and halogenated gentisate analogs. The constitutive GDO I enzyme was shown to have marked differences in substrate specificities compared to the inducible GDO II enzyme (9).Enhancing the catalytic properties of biodegradative enzymes by site-directed mutagenesis (SDM) represents a potential strategy for improving the efficacy of biodegradation processes (10, 11). Mutations of specific amino acids have been known to alter either the substrate specificity or the kinetic properties of an enzyme (1,8). In this study, site-specific mutations were targeted in the xlnE gene to assess the effects these have on the substrate specificities and catalytic properties of the variant enzymes.A Clustal W alignment of GDOs from P25X (GenBank accession no. AF173167), Pseudomonas aeruginosa (AE004674), Escherichia coli O157:H7 (AE005174), Bacillus halodurans I, II (AP001514), Ralstonia sp. strain U2 (AP001514), Haloferax sp. strain D1227 (AF069949), and Sphingomonas sp. strain RW5 (AJ224977) showed the presence of a highly conserved doublestranded ␤-helix domain (data not shown). To evaluate the influence of specific amino acid residues on the catalytic properties of the enzyme, amino acid residues located outside and within the ␤-helix domain were randomly selected and subjected to SDM (Table 1). Random substitutions with different amino acid residues were constructed, for instance, a polar amino acid was replaced with a nonpolar amino acid and an acidic amino acid was replaced with a basic amino acid. The mutagenized genes were fully sequenced and transformed into E. coli BL21(DE3) for protein expression and purification. The recombinant glutathione S-transferase (GST)-tagged GDO proteins were overexpressed in the respective mutants of E. coli BL21 when induced with 1 mM isopropyl-␤-D-thiogalactopyranoside (IPTG). A one-step purification of GST-t...