Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 288, Issue 50, Pages 35801-35811Publisher
ELSEVIER
DOI: 10.1074/jbc.M113.519496
Keywords
Cooperativity; Enzyme Catalysis; Enzyme Inactivation; Enzyme Mechanisms; Enzyme Structure; Glutamine
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Funding
- National Institutes of Health Grants [GM074815, GM051127]
- M. D. Anderson Trust Fellowship
- Burroughs Wellcome Career Development Award
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Background:B. subtilis GS catalyzes the production of glutamine, a key metabolite in nitrogen assimilation. Results:B. subtilis GS forms a dodecamer that undergoes large intersubunit conformational changes during catalysis. Conclusion:B. subtilis GS structures reveal a heretofore unseen active site restructuring that is linked to a novel feedback regulatory mechanism. Significance: The GS specific regulatory/catalytic mechanism may be used to target Gram-positive pathogens. Glutamine synthetase (GS), which catalyzes the production of glutamine, plays essential roles in nitrogen metabolism. There are two main bacterial GS isoenzymes, GSI- and GSI-. GSI- enzymes, which have not been structurally characterized, are uniquely feedback-inhibited by Gln. To gain insight into GSI- function, we performed biochemical and cellular studies and obtained structures for all GSI- catalytic and regulatory states. GSI- forms a massive 600-kDa dodecameric machine. Unlike other characterized GS, the Bacillus subtilis enzyme undergoes dramatic intersubunit conformational alterations during formation of the transition state. Remarkably, these changes are required for active site construction. Feedback inhibition arises from a hydrogen bond network between Gln, the catalytic glutamate, and the GSI--specific residue, Arg(62), from an adjacent subunit. Notably, Arg(62) must be ejected for proper active site reorganization. Consistent with these findings, an R62A mutation abrogates Gln feedback inhibition but does not affect catalysis. Thus, these data reveal a heretofore unseen restructuring of an enzyme active site that is coupled with an isoenzyme-specific regulatory mechanism. This GSI--specific regulatory network could be exploited for inhibitor design against Gram-positive pathogens.
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