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Journal of Bacteriology, November 2005, p. 7543-7545, Vol. 187, No. 21
0021-9193/05/$08.00+0     doi:10.1128/JB.187.21.7543-7545.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Chew Ling Tan,1 Chew Chieng Yeo,3 Hoon Eng Khoo,4 and Chit Laa Poh1,2*

Programme in Environmental Microbiology, Department of Microbiology, Faculty of Medicine, National University of Singapore, 5 Science Drive 2, Singapore 117597, Singapore,1 School of Biomedical Science, Curtin University of Technology, Bentley, Western Australia 6102, Australia,2 Department of Biotechnology, Malaysia University of Science and Technology, Block C, Dataran Usahawan Kelana, 17 Jalan SS7/26, 47301 Petaling Jaya, Malaysia,3 Department of Biochemistry, Faculty of Medicine, National University of Singapore, 5 Science Drive 2, Singapore 117597, Singapore4

Received 10 May 2005/ Accepted 6 August 2005


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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 (kcat/Km) on Y181F towards 3-methylgentisate than that of the wild-type enzyme.


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TEXT
 
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 Fe2+ 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 double-stranded ß-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 ß-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-ß-D-thiogalactopyranoside (IPTG). A one-step purification of GST-tagged GDO and its mutants was performed using GSTrap Fast Flow columns (Amersham Biosciences, NJ) via affinity purification. The formation of substituted maleylpyruvates from various substrates was monitored with purified enzymes in the presence of 0.1 mM ferrous ammonium sulfate, 2 mM L-cysteine, and glycerol at 10% (vol/vol) at the following wavelengths: for gentisate, 330 nm; for 3-methylgentisate, 327 nm; for 4-methylgentisate, 316 nm; for 3-bromogentisate, 335 nm; for 3-fluorogentisate, 331 nm; for 3-isopropylgentisate, 325 nm; and for 4-chlorogentisate, 335 nm. The reaction mixture reported by Feng et al. (4) was employed, and GDO-specific activities were calculated using various molar extinction coefficients based on the reaction products derived from unsubstituted and substituted gentisates. A value of 12,500 M–1 cm–1 was used for gentisate, 11,900 M–1 cm–1 was used for 3-methylgentisate, 11,400 M–1 cm–1 was used for 4-methylgentisate, 11,200 M–1 cm–1 was used for 3-isopropylgentisate, 13,000 M–1 cm–1 was used for 3-bromogentisate, 11,700 M–1 cm–1 was used for 3-fluorogentisate, and 12,400 M–1 cm–1 was used for 4-chlorogentisate (9). For the determination of kinetic parameters, Km values were calibrated from Hanes plots with substrate concentrations ranging from 50 µM to 600 µM. Protein concentrations were determined by the Bradford assay (2), employing bovine serum albumin as the standard. Relative activities of GDO were calculated with respect to the activity of recombinant GST-GDO expressed in E. coli. One enzyme unit is the amount of enzyme required to produce 1 µmol of maleylpyruvate per min at 23°C.


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  		TABLE 1. Amino acid substitutions in mutant enzymes generated by SDM and their resultant activities

The GST-GDO fusion protein expressed in E. coli exhibited a similar specific activity and substrate affinity towards gentisate to those of the previously reported purified GDO enzyme from P25X (wild type [WT]) (4). WT GDO I exhibited specific activity towards gentisate at 0.63 U/mg, whereas recombinant GST-GDO I displayed a specific activity at 0.58 U/mg. The apparent Km values for WT GDO I and GST-GDO I differed slightly, at 92.0 µM and 86.02 µM, respectively. This showed that the purified GST-GDO I enzyme did not differ markedly in catalytic properties from purified WT GDO I.

Eight of the 18 mutants, namely, V36A, S113P, D120K, M146T, G164T, M169T, T260C, and V326Q, exhibited a total loss of enzyme activity, whereas the mutant enzyme G123N showed a 25% reduced GDO activity (Table 1). These amino acid substitutions were found to be mainly located within the periphery of the highly conserved region and could have resulted in an alteration of the quaternary structure of the enzyme that led to a nonfunctional enzyme. However, several mutant enzymes exhibited higher relative activities, ranging from 103% to 464% of the WT activity. The mutant enzyme Y181F was selected for further characterization of its substrate affinities (Km) and catalytic efficiencies (kcat/Km) towards gentisate and substituted gentisates.

The mutant enzyme Y181F showed a 464% increase in relative activity towards gentisate compared to that of the recombinant wild-type GST-GDO protein. Significantly higher relative activities towards 3-methyl- and 4-methylgentisates, at 1,638% and 667%, respectively, of the WT activities, were observed. Relative activities towards 3-bromo- and 3-fluorogentisates were also found to be higher than the WT GDO activities, at 254% and 373%, respectively. Replacement of the tyrosine at position 181 of GDO by phenylalanine had altered the catalytic properties significantly.

To further characterize the importance of tyrosine (Tyr) at position 181 in the xlnE gene product, tyrosine was replaced with either an acidic or a basic amino acid by SDM. When an acidic (aspartic acid) or a basic (histidine) amino acid was introduced to generate two new variant enzymes, Y181D and Y181H, respectively, there was no improvement in specific activity towards gentisate compared to that of the WT. These observations demonstrated that a single amino acid exchange in the mutant enzyme Y181F was responsible for the significantly altered catalytic properties observed. The substitution at position 181 favors catalysis, and the absence of a hydroxyl group might have contributed to favorable hydrophobic interactions between the polypeptide chains and aromatic hydrocarbons such as gentisate and substituted gentisates.

All enzymes displayed Michaelis-Menten kinetics, and Hanes plots of the enzyme activities yielded apparent Km values towards gentisate of 86.02 µM for the WT and 68.43 µM for the mutant enzyme (Table 2). Remarkably, the catalytic efficiency of the mutant enzyme towards 3-methylgentisate was 13-fold higher than that of the WT. The mutant enzyme Y181F also exhibited a 4.8-fold increase in the turnover rate of gentisate compared to that of the WT.


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  		TABLE 2. Specific activities and catalytic efficiencies of recombinant P25X GST-GDO and the mutant enzyme Y181F

The Km value of the WT towards gentisate was close to the Km values reported for Pseudomonas testosteroni (85.0 µM) and Pseudomonas acidovorans (74.0 µM), but this value was considerably higher than those reported for Moraxella osloensis (7.1 µM) and Sphingomonas sp. strain RW5 (15.0 µM) (3, 6, 13). The Km values towards gentisates for the mutant enzyme Y181F showed that the alkylated and halogenated gentisates were well tolerated. The mutant enzyme Y181F displayed a 17-fold higher catalytic efficiency towards 3-methylgentisate than the enzyme from Pseudomonas testosteroni (6). The higher catalytic efficiencies observed for the mutant enzyme towards alkylated and halogenated gentisates could be explained either by the favorable steric effects encountered in the active sites of the mutant enzyme or by the interactions with the electron attracting/donating alkyl/halogen groups present in the substituted gentisates.

Several extradiol dioxygenases have been studied extensively, while the GDOs are relatively undercharacterized (12, 15). Enzymes with broader substrate specificities or enhanced levels of production are important in industrial applications and could be employed in the bioremediation of toxic aromatic hydrocarbons. Since the three-dimensional crystal structure of the enzyme remains to be elucidated, our results indicated that GDOs with improved catalytic properties could be generated by SDM of randomly selected amino acid residues in the absence of a three-dimensional structure of GDO.


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ACKNOWLEDGMENTS
 
This investigation was supported by University Research Council (URC) grant R-182-000-069-112 from the National University of Singapore awarded to C. L. Poh. C. L. Tan acknowledges the support of a postgraduate research scholarship from Faculty of Medicine, National University of Singapore.


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FOOTNOTES
 
* Corresponding author. Present address: Programme in Environmental Microbiology, Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, 5 Science Drive 2, Singapore 117597. Phone: (65) 6874-3674. Fax: (65) 6776-6872. E-mail: micpohcl{at}nus.edu.sg. Back


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Journal of Bacteriology, November 2005, p. 7543-7545, Vol. 187, No. 21
0021-9193/05/$08.00+0     doi:10.1128/JB.187.21.7543-7545.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.




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