Alteration of chain length substrate specificity of Aeromonas caviae R-enantiomer-specific enoyl-coenzyme A hydratase through site-directed mutagenesis.
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 beta-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.
Tsuge
Takeharu
T
Department of Innovative and Engineered Materials, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8502, Japan. ttsuge@iem.titech.ac.jp
Hisano
Tamao
T
Taguchi
Seiichi
S
Doi
Yoshiharu
Y
D000333:Aeromonas / Q000201:enzymology*; D002942:Circular Dichroism; D004746:Enoyl-CoA Hydratase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011091:Polyesters / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D011091:Polyesters; D004746:Enoyl-CoA Hydratase