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J. Biol. Chem., Vol. 282, Issue 24, 17738-17748, June 15, 2007

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Comparative Analysis of Two UDP-glucose Dehydrogenases in Pseudomonas aeruginosa PAO1^*

Ruei-Jiun Hung, Han-Sheng Chien, Ruei-Zeng Lin, Ching-Ting Lin, Jaya Vatsyayan, Hwei-Ling Peng, and Hwan-You Chang¹

From the Institute of Molecular Medicine, National Tsing Hua University and the Department of Biological Science and Technology, National Chiao Tung University, Hsin Chu 300, Taiwan, Republic of China

UDP-glucose dehydrogenase (UGDH) catalyzes a two-step NAD⁺-dependent oxidation of UDP-glucose to produce UDP-glucuronic acid, which is a common substrate for the biosynthesis of exopolysaccharide. Searching the Pseudomonas aeruginosa PAO1 genome data base for a UGDH has helped identify two open reading frames, PA2022 and PA3559, which may encode a UGDH. To elucidate their enzymatic identity, the two genes were cloned and overexpressed in Escherichia coli, and the recombinant proteins were purified. Both the gene products are active as dimers and are capable of utilizing UDP-glucose as a substrate to generate UDP-glucuronic acid. The K_m values of PA2022 and PA3559 for UDP-glucose are 0.1 and 0.4 mM, whereas the K_m values for NAD⁺ are 0.5 and 2.0 mM, respectively. Compared with PA3559, PA2022 exhibits broader substrate specificity, utilizing TDP-glucose and UDP-N-acetylglucosamine with one-third the velocity of that with UDP-glucose. The PA2022 mutant and PA2022-PA3559 double mutant, but not the PA3559 mutant, are more susceptible to chloramphenicol, cefotaxime, and ampicillin. The PA3559 mutant, however, shows a reduced resistance to polymyxin B compared with wild type PAO1. Finally, real time PCR analysis indicates that PA3559 is expressed primarily in low concentrations of Mg²⁺, which contrasts with the constitutive expression of PA2022. Although both the enzymes catalyze the same reaction, their enzymatic properties and gene expression profiles indicate that they play distinct physiological roles in P. aeruginosa, as reflected by different phenotypes displayed by the mutants.

Received for publication, March 1, 2007 , and in revised form, April 17, 2007.

^* This work was supported by the National Science Council and the National Research Program of Genome Medicine, Taiwan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3.

¹ To whom correspondence should be addressed: Institute of Molecular Medicine, National Tsing Hua University, 101 Kuang Fu Rd., 2nd Sec., Hsin Chu 300, Taiwan. Tel.: 886-35742909; Fax: 886-35742910; E-mail: hychang{at}life.nthu.edu.tw.

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