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Applied and Environmental Microbiology, August 2003, p. 4830-4836, Vol. 69, No. 8
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.8.4830-4836.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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

Takeharu Tsuge,1,2* Tamao Hisano,3 Seiichi Taguchi,2,{dagger} and Yoshiharu Doi1,2

Department of Innovative and Engineered Materials, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8502,1 Polymer Chemistry Laboratory, RIKEN Institute, Wako-shi, Saitama 351-0198,2 Theoretical Structural Biology Laboratory, RIKEN Harima Institute, Mikadukicho, Sayo, Hyogo 679-5148, Japan3

Received 30 December 2002/ Accepted 16 May 2003

Aeromonas caviae R-specific enoyl-coenzyme A (enoyl-CoA) hydratase (PhaJAc) is capable of providing (R)-3-hydroxyacyl-CoA with a chain length of four to six carbon atoms from the fatty acid ß-oxidation pathway for polyhydroxyalkanoate (PHA) synthesis. In this study, amino acid substitutions were introduced into PhaJAc 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 PhaJAc 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 (C8) 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 (C12) was examined by using the mutated PhaJAc as a monomer supplier in recombinant Escherichia coli LS5218 harboring a PHA synthase gene from Pseudomonas sp. strain 61-3 (phaC1Ps). When L65A, L65G, or V130G was used individually, increased molar fractions of 3-hydroxyoctanoate (C8) and 3-hydroxydecanoate (C10) 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.

* Corresponding author. Mailing address: Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8502, Japan. Phone: 81-45-924-5286. Fax: 81-45-924-5426. E-mail: ttsuge{at}iem.titech.ac.jp.

{dagger} Present address: Department of Agricultural Chemistry, School of Agriculture, Meiji University, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan.

Applied and Environmental Microbiology, August 2003, p. 4830-4836, Vol. 69, No. 8
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.8.4830-4836.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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