Alteration of Chain Length Substrate Specificity of Aeromonas caviae R -Enantiomer-Specific Enoyl-Coenzyme A Hydratase through Site-Directed Mutagenesis

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Abstract
Aeromonas caviae R -specific enoyl-coenzyme A (enoyl-CoA) hydratase (PhaJ Ac ) is capable of providing ( R )-3-hydroxyacyl-CoA with a chain length of four to six carbon atoms from the fatty acid β-oxidation pathway for polyhydroxyalkanoate (PHA) synthesis. In this study, amino acid substitutions were introduced into PhaJ Ac by site-directed mutagenesis to investigate the feasibility of altering the specificity for the acyl chain length of the substrate. A crystallographic structure analysis of PhaJ Ac revealed that Ser-62, Leu-65, and Val-130 define the width and depth of the acyl-chain-binding pocket. Accordingly, we targeted these three residues for amino acid substitution. Nine single-mutation enzymes and two double-mutation enzymes were generated, and their hydratase activities were assayed in vitro by using trans -2-octenoyl-CoA (C 8 ) as a substrate. Three of these mutant enzymes, L65A, L65G, and V130G, exhibited significantly high activities toward octenoyl-CoA than the wild-type enzyme exhibited. PHA formation from dodecanoate (C 12 ) was examined by using the mutated PhaJ Ac as a monomer supplier in recombinant Escherichia coli LS5218 harboring a PHA synthase gene from Pseudomonas sp. strain 61-3 ( phaC1 Ps ). When L65A, L65G, or V130G was used individually, increased molar fractions of 3-hydroxyoctanoate (C 8 ) and 3-hydroxydecanoate (C 10 ) units were incorporated into PHA. These results revealed that Leu-65 and Val-130 affect the acyl chain length substrate specificity. Furthermore, comparative kinetic analyses of the wild-type enzyme and the L65A and V130G mutants were performed, and the mechanisms underlying changes in substrate specificity are discussed.

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