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J. Biol. Chem., Vol. 283, Issue 5, 2835-2845, February 1, 2008
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¶23
From the
Department of Biochemistry and Biomedical Sciences, DeGroote School of Medicine, McMaster University, Hamilton, Ontario L8N 3Z5, the Ontario Center for Structural Proteomics, Best Institute, Toronto, Ontario M5G 1L6, the ¶Structural Genomics Consortium, University of Toronto, Toronto, Ontario M5G 1L5, and the ||Infectious Diseases Research Group, Siebens Drake Research Institute, Department of Microbiology and Immunology, University of Western Ontario, London, Ontario N6A 5C1, Canada
The barrier imposed by lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria presents a significant challenge in treatment of these organisms with otherwise effective hydrophobic antibiotics. The absence of L-glycero-D-manno-heptose in the LPS molecule is associated with a dramatically increased bacterial susceptibility to hydrophobic antibiotics and thus enzymes in the ADP-heptose biosynthesis pathway are of significant interest. GmhA catalyzes the isomerization of D-sedoheptulose 7-phosphate into D-glycero-D-manno-heptose 7-phosphate, the first committed step in the formation of ADP-heptose. Here we report structures of GmhA from Escherichia coli and Pseudomonas aeruginosa in apo, substrate, and product-bound forms, which together suggest that GmhA adopts two distinct conformations during isomerization through reorganization of quaternary structure. Biochemical characterization of GmhA mutants, combined with in vivo analysis of LPS biosynthesis and novobiocin susceptibility, identifies key catalytic residues. We postulate GmhA acts through an enediol-intermediate isomerase mechanism.
Received for publication, July 26, 2007 , and in revised form, November 30, 2007.
The atomic coordinates and structure factors (code 212W and 3BJZ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
* This work was supported in part by grants from the National Science and Engineering Research Council (to M. A. V.), Special Program Grant Initiative "In Memory of Michael O'Reilly" funded by the Canadian Cystic Fibrosis Foundation and the Cardiovascular and Respiratory Health Institute of the Canadian Institutes of Health Research (to M. A. V. and G. D. W.), Canadian Cystic Fibrosis Foundation studentship (to P. L. T.), and the Canadian Institutes of Health Research (to G. D. W., and M. S. J., respectively). Use of the Advanced Photon Source was supported by the U. S. Dept. of Energy, Office of Science, Office of Basic Energy Sciences, under Contract DE-AC02-06CH11357. 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-S4, Tables S1 and S2, and a movie.
1 Holds a Canada Research Chair in Infectious Diseases and Microbial Pathogenesis.
2 Holds a Canada Research Chair in Molecular Studies of Antibiotics.
3 To whom correspondence should be addressed: 1200 Main St. West, Dept. of Biochemistry and Biomedical Sciences, 1200 Main St. West, Health Sciences Center, Rm. 4N20A, Hamilton, Ontario L8N 3Z5, Canada. Tel.: 905-525-9140 (ext. 22912); Fax: 905-522-9033; E-mail: junopm{at}mcmaster.ca.
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