1m64
Crystal structure of Q363F mutant flavocytochrome c3Crystal structure of Q363F mutant flavocytochrome c3
Structural highlights
FunctionFRDA_SHEFR Catalyzes fumarate reduction using artificial electron donors such as methyl viologen. The physiological reductant is unknown, but evidence indicates that flavocytochrome c participates in electron transfer from formate to fumarate and possibly also to trimethylamine oxide (TMAO). This enzyme is essentially unidirectional. Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMedThe ability of an arginine residue to function as the active site acid catalyst in the fumarate reductase family of enzymes is now well-established. Recently, a dual role for the arginine during fumarate reduction has been proposed [Mowat, C. G., Moysey, R., Miles, C. S., Leys, D., Doherty, M. K., Taylor, P., Walkinshaw, M. D., Reid, G. A., and Chapman, S. K. (2001) Biochemistry 40, 12292-12298] in which it acts both as a Lewis acid in transition-state stabilization and as a Bronsted acid in proton delivery. This proposal has led to the prediction that, if appropriately positioned, a water molecule would be capable of functioning as the active site Bronsted acid. In this paper, we describe the construction and kinetic and crystallographic analysis of the Q363F single mutant and Q363F/R402A double mutant forms of flavocytochrome c(3), the soluble fumarate reductase from Shewanella frigidimarina. Although replacement of the active site acid, Arg402, with alanine has been shown to eliminate fumarate reductase activity, this phenomenon is partially reversed by the additional substitution of Gln363 with phenylalanine. This Gln --> Phe substitution in the inactive R402A mutant enzyme was designed to "push" a water molecule close enough to the substrate C3 atom to allow it to act as a Bronsted acid. The 2.0 A resolution crystal structure of the Q363F/R402A mutant enzyme does indeed reveal the introduction of a water molecule at the correct position in the active site to allow it to act as the catalytic proton donor. The 1.8 A resolution crystal structure of the Q363F mutant enzyme shows a water molecule similarly positioned, which can account for its measured fumarate reductase activity. However, in this mutant enzyme Michaelis complex formation is impaired due to significant and unpredicted structural changes at the active site. Engineering water to act as an active site acid catalyst in a soluble fumarate reductase.,Mowat CG, Pankhurst KL, Miles CS, Leys D, Walkinshaw MD, Reid GA, Chapman SK Biochemistry. 2002 Oct 8;41(40):11990-6. PMID:12356299[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences |
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