4rm3 | pdb_00004rm3
Crystal structure of a benzoate coenzyme A ligase with 2-Furoic acid
Structural highlights
Publication Abstract from PubMedA benzoate CoA ligase (BadA), isolated from the bacterium Rhodopseudomonas palustris, catalyzes the conversion of benzoate to benzoyl CoA on the catabolic pathway of aromatic carboxylic acids. Herein, Michaelis constants kcat and KM were determined for an expanded array of 31 substrates chosen to systematically probe the active site architecture of the enzyme and provide a baseline for expansion of the wild-type substrate specificity. Acyl CoA products were observed for 25 of the 31 substrates and, in general, BadA converted ortho-substituted substrates better than the corresponding meta- and para-regioisomers, and the turnover number was more affected by sterics than electronic effects. The kinetic data are interpreted in relation to six crystal structures of BadA in complex with several substrates and a benzoyl-AMP reaction intermediate. In contrast to other known natural substrate-bound benzoate ligase structures, all substrate-bound BadA structures adopted the thiolation conformation instead of the adenylation conformation. We also observed all the aryl carboxylates to be uniquely oriented within the active site, relative to the other structures. Together, the kinetics and structural data suggested a mechanism that involves substrate binding in the thiolation conformation, followed by substrate rotation to an active orientation upon the transition to the adenylation conformation. Based on this hypothesis and the structural data, sterically demanding active site residues were mutated, and the substrate specificity was expanded substantially over BadA. Novel activities were seen for substrates with larger substituents, including phenylacetate. Additionally, the mutant Lys427Ala identified this non-conserved residue as essential for the thiolation step of BadA, but not adenylation. These variously acylated CoAs can serve as novel substrates of acyl CoA-dependent acyltransferases in coupled enzyme assays to produce analogs of bioactive natural products. Kinetic- and Crystallographic-Guided Mutations of a Benzoate CoA Ligase (BadA) Elucidate Mechanism and Expand Substrate Permissivity.,Thornburg CK, Wortas-Strom S, Nosrati M, Geiger JH, Walker KD Biochemistry. 2015 Oct 1. PMID:26378464[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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