Structures of the Bacillus subtilis Glutamine Synthetase Dodecamer Reveal Large Intersubunit Catalytic Conformational Changes Linked to a Unique Feedback Inhibition Mechanism |
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Authors: | David S. Murray Nagababu Chinnam Nam Ky Tonthat Travis Whitfill Lewis V. Wray Jr. Susan H. Fisher Maria A. Schumacher |
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Affiliation: | From the ‡Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239.;the §Department of Biochemistry, Duke University School of Medicine, Durham, North Carolina 27710, and ;the ¶Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts 02118 |
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Abstract: | Glutamine synthetase (GS), which catalyzes the production of glutamine, plays essential roles in nitrogen metabolism. There are two main bacterial GS isoenzymes, GSI-α and GSI-β. GSI-α enzymes, which have not been structurally characterized, are uniquely feedback-inhibited by Gln. To gain insight into GSI-α function, we performed biochemical and cellular studies and obtained structures for all GSI-α catalytic and regulatory states. GSI-α 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-α-specific residue, Arg62, from an adjacent subunit. Notably, Arg62 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-α-specific regulatory network could be exploited for inhibitor design against Gram-positive pathogens. |
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Keywords: | Cooperativity Enzyme Catalysis Enzyme Inactivation Enzyme Mechanisms Enzyme Structure Glutamine |
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