Structure and mechanism of a cysteine sulfinate desulfinase engineered on the aspartate aminotransferase scaffold

ArticleinBiochimica et Biophysica Acta 1824(2):339-49 · November 2011with61 Reads
DOI: 10.1016/j.bbapap.2011.10.016 · Source: PubMed
Abstract
The joint substitution of three active-site residues in Escherichia coli (L)-aspartate aminotransferase increases the ratio of l-cysteine sulfinate desulfinase to transaminase activity 10(5)-fold. This change in reaction specificity results from combining a tyrosine-shift double mutation (Y214Q/R280Y) with a non-conservative substitution of a substrate-binding residue (I33Q). Tyr214 hydrogen bonds with O3 of the cofactor and is close to Arg374 which binds the α-carboxylate group of the substrate; Arg280 interacts with the distal carboxylate group of the substrate; and Ile33 is part of the hydrophobic patch near the entrance to the active site, presumably participating in the domain closure essential for the transamination reaction. In the triple-mutant enzyme, k(cat)' for desulfination of l-cysteine sulfinate increased to 0.5s(-1) (from 0.05s(-1) in wild-type enzyme), whereas k(cat)' for transamination of the same substrate was reduced from 510s(-1) to 0.05s(-1). Similarly, k(cat)' for β-decarboxylation of l-aspartate increased from<0.0001s(-1) to 0.07s(-1), whereas k(cat)' for transamination was reduced from 530s(-1) to 0.13s(-1). l-Aspartate aminotransferase had thus been converted into an l-cysteine sulfinate desulfinase that catalyzes transamination and l-aspartate β-decarboxylation as side reactions. The X-ray structures of the engineered l-cysteine sulfinate desulfinase in its pyridoxal-5'-phosphate and pyridoxamine-5'-phosphate form or liganded with a covalent coenzyme-substrate adduct identified the subtle structural changes that suffice for generating desulfinase activity and concomitantly abolishing transaminase activity toward dicarboxylic amino acids. Apparently, the triple mutation impairs the domain closure thus favoring reprotonation of alternative acceptor sites in coenzyme-substrate intermediates by bulk water.
    • "As such, PLPdependent aminotransferases are assumed to share a common enzymatic mechanism for alanine transamination that implies cofactor recycling from a covalently linked internal aldimine with an enzyme's key catalytic lysine residue (Lys-PLP) to covalent substrate adduct intermediates termed external aldimines (Figure S1 in File S1) [4,101112. Factors such as the orientation of the scissile bond and the electron repartition within the resonance system of the covalent adduct are heavily influenced by the array of interactions established between the various cofactor forms and the active site residues, which ultimately determine the course of the reaction pathway toward transamination, decarboxylation, desulfination, elimination or aldol cleavage [4,11,13]. The degree of versatility afforded by the chemistry of PLP-dependent enzyme catalyzed reactions has indeed motivated the use of these catalysts as scaffolds for enzyme redesign and protein engineering studies of aspartate and aromatic amino acid transaminases [10,141516. "
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