Alteration of chain length substrate specificity of Aeromonas caviae R-enantiomer-specific enoyl-coenzyme A hydratase through site-directed mutagenesis

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 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.

FIG. 1.

Schematic diagram of the acyl-chain-binding site of PhaJ_Ac, represented by a space-filling model. The acyl tail of trans-2-hexenoyl-CoA is yellow, the side chains of the Ser-62, Leu-65, and Val-130 residues are magenta, and Asp-31 and His-36 are green. For clarity, residues 70 to 75, 132, and 133, which obscure the binding site, are not shown. Ser-62, Leu-65, and Val-130 were replaced by other amino acids with shorter side chains in this study. Asp-31 and His-36 were identified as a catalytic dyad in a previous study (12). A prime indicates that the amino acid residue is from another polypeptide chain. The atomic coordinates of this enzyme have been deposited in the Protein Data Bank (accession code 1IQ6).

FIG. 2.

Relationship between log(k_cat/K_m) and acyl chain length of the trans-2-enoyl-CoA substrate for the wild-type enzyme (□), L65A (▴), and V130G (⧫). The values for k_cat, K_m, and k_cat/K_m are shown in Table 5.

FIG. 3.

CD spectra of purified wild-type enzyme, L65A, and V130G: far-UV spectra of the wild-type enzyme, L65A, and V130G.