Replacement of Tyrosine 181 by Phenylalanine in Gentisate 1,2-Dioxygenase I from Pseudomonas alcaligenes NCIMB 9867 Enhances Catalytic Activities

Abstract

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...

Identification and Characterization of a Novel Gentisate 1,2-Dioxygenase Gene from a Halophilic Martelella Strain

“…The results suggest that all four highly conserved His residues are crucial for GDO activity. However, in Pseudomonas alcaligenes NCIMB 9867 22 , Silicibacter (Ruegeria) pomeroyi DSS-3 (AAV97252.1) 11 , Klebsiella pneumoniae M5a1, and Ralstonia sp. strain U2 1 , site-directed mutagenesis was performed, changing His residues to Asp residues, based on the results of error-prone PCR mutagenesis of gdo from Pseudomonas alcaligenes NCIB 9867 6 23 .…”

Insights into environmental bioremediation by microorganisms through functional genomics and proteomics

“…A recombinant E. coli carrying a starvation promoter and toluene monooxygenase could selectively enhanced expressions of phenol-and TCE-degrading activities during growth [58]. Degradation of aromatic hydrocarbons can be increased significantly with enzymes having desirable catalytic activities or broad substrate specificities that can attack and degrade halogenated benzoates and gentisates [59,60].…”