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Originally published In Press as doi:10.1074/jbc.M202882200 on May 9, 2002

J. Biol. Chem., Vol. 277, Issue 30, 26769-26778, July 26, 2002
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Structure-Function Studies of Two Novel UDP-GlcNAc C6 Dehydratases/C4 Reductases
VARIATION FROM THE SYK DOGMA*

Carole CreuzenetDagger §, Robert V. Urbanic§, and Joseph S. Lam§||

From the Dagger  Department of Microbiology and Immunology, University of Western Ontario, London, Ontario N6A 5C1, Canada and the § Department of Microbiology, University of Guelph, Guelph, Ontario N1G 2W1, Canada

Two subfamilies of UDP-GlcNAc C6 dehydratases were recently identified. FlaA1, a short soluble protein that exhibits a typical SYK catalytic triad, characterizes one of these subfamilies, and WbpM, a large membrane protein that harbors an altered SMK triad that was not predicted to sustain activity, represents the other subfamily. This study focuses on investigating the structure and function of these C6 dehydratases and the role of the altered triad as well as additional amino acid residues involved in catalysis. The significant activity retained by the FlaA1 Y141M triad mutant and the low activity of the WbpM M438Y mutant indicated that the methionine residue was involved in catalysis. A Glu589 residue, which is conserved only within the large homologues, was shown to be essential for activity in WbpM. Introduction of this residue in FlaA1 enhanced the activity of the corresponding V266E mutant. Hence, this glutamate residue might be responsible for the retention of catalytic efficiency in the large homologues despite alteration of their catalytic triad. Mutations of residues specific for the short homologues (Asp70, Asp149-Lys150, Cys103) abolished the activity of FlaA1. Among them, C103M prevented dimerization but did not significantly affect the secondary structure. The fact that we could identify subfamily-specific residues that are essential for catalysis suggested an independent evolution for each subfamily of C6 dehydratases. Finally, the loss of activity of the FlaA1 G20A mutant provided evidence that a cofactor is involved in catalysis, and kinetic study of the FlaA1 H86A mutant revealed that this conserved histidine is involved in substrate binding. None of the mutations investigated altered the substrate, product, and function specificity of these enzymes.

* This work was supported by operating grants from the Canadian Institute for Health Research (CIHR) (Grant MOP-14687) (to J. S. L.) and from the Canadian Bacterial Diseases Network, a consortium of the Federal Networks of Centres of Excellence program. This work was also supported by the Collaborative Health Research Projects program of the Natural Sciences and Engineering Research Council Grant CHRPJ-251007 (to J. S. L. and C. C.) and by CIHR Grant MMA-41558 (to J. S. L.) for the purchase of the capillary electrophoresis instrument.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a postdoctoral fellowship from the Canadian Cystic Fibrosis Foundation (CCFF).

|| A Zellers Senior Scientist and a recipient of a Marsha Morton Scholarship from CCFF. To whom correspondence should be addressed. Tel.: 519-824-4120 (ext. 3823); Fax: 519-837-1802; E-mail: jlam@ uoguelph.ca.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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