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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

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

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.