Structure and mechanism of a cysteine sulfinate desulfinase engineered on the aspartate aminotransferase scaffold.
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.
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ABSTRACT: Enzyme design is an important area of ongoing research with a broad range of applications in protein therapeutics, biocatalysis, bioengineering, and other biomedical areas; however, significant challenges exist in the design of enzymes to catalyze specific reactions of interest. Here, we develop a computational protocol using an approach that combines molecular dynamics, docking, and MM-GBSA scoring to predict the catalytic activity of enzyme variants. Our primary focuses are to understand the molecular basis of substrate recognition and binding in an S stereoselective ω-aminotransferase (ω-AT), which naturally catalyzes the transamination of pyruvate into alanine, and to predict mutations that enhance the catalytic efficiency of the enzyme. The conversion of (R)-ethyl 5-methyl-3-oxooctanoate to (3S, 5R)-ethyl 3-amino-5-methyloctanoate in the context of several ω-AT mutants was evaluated using the computational protocol developed in this work. We correctly identify the mutations that yield the greatest improvements in enzyme activity (20-60-fold improvement over wild type) and confirm that the computationally predicted structure of a highly active mutant reproduces key structural aspects of the variant, including side chain conformational changes, as determined by X-ray crystallography. Overall, the protocol developed here yields encouraging results and suggests that computational approaches can aid in the redesign of enzymes with improved catalytic efficiency.Journal of Chemical Information and Modeling 07/2014; · 4.30 Impact Factor
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ABSTRACT: The sulfur atoms of sulfur-containing cofactors that are essential for numerous cellular functions in living organisms originate from L-cysteine via cysteine desulfurase (CSD) activity. However, many (hyper)thermophilic archaea, which thrive in solfataric fields and are positioned near the root of the evolutionary tree of life, lack CSD orthologs. The existence of CSD orthologs in a subset of (hyper)thermophilic archaea is of interest with respect to the evolution of sulfur-trafficking systems for the cofactors. This study demonstrates that the disruption of the csd gene of Thermococcus kodakarensis, a facultative elemental sulfur (S0)-reducing hyperthermophilic archaeon, encoding Tk-CSD, conferred a growth defect evident only in the absence of S0, and that growth can be restored by the addition of S0, but not sulfide. We show that the csd gene is not required for biosynthesis of thiamine pyrophosphate or molybdopterin, irrespective of the presence or absence of S0, but is necessary for iron-sulfur cluster biosynthesis in the absence of S0. Recombinant form of Tk-CSD expressed in Escherichia coli was obtained and it was found to catalyze the desulfuration of L-cysteine. The obtained data suggest that hyperthermophiles might benefit from a capacity for CSD-dependent iron-sulfur cluster biogenesis, which allows them to thrive outside solfataric environments.Molecular Microbiology 06/2014; 93(2). · 5.03 Impact Factor
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ABSTRACT: In order to maintain proper cellular function, the metabolism of the bacterial microbiota presents several mechanisms oriented to keep a correctly balanced amino acid pool. Central components of these mechanisms are enzymes with alanine transaminase activity, pyridoxal 5'-phosphate-dependent enzymes that interconvert alanine and pyruvate, thereby allowing the precise control of alanine and glutamate concentrations, two of the most abundant amino acids in the cellular amino acid pool. Here we report the 2.11-Å crystal structure of full-length AlaA from the model organism Escherichia coli, a major bacterial alanine aminotransferase, and compare its overall structure and active site composition with detailed atomic models of two other bacterial enzymes capable of catalyzing this reaction in vivo, AlaC and valine-pyruvate aminotransferase (AvtA). Apart from a narrow entry channel to the active site, a feature of this new crystal structure is the role of an active site loop that closes in upon binding of substrate-mimicking molecules, and which has only been previously reported in a plant enzyme. Comparison of the available structures indicates that beyond superficial differences, alanine aminotransferases of diverse phylogenetic origins share a universal reaction mechanism that depends on an array of highly conserved amino acid residues and is similarly regulated by various unrelated motifs. Despite this unifying mechanism and regulation, growth competition experiments demonstrate that AlaA, AlaC and AvtA are not freely exchangeable in vivo, suggesting that their functional repertoire is not completely redundant thus providing an explanation for their independent evolutionary conservation.PLoS ONE 01/2014; 9(7):e102139. · 3.53 Impact Factor