Mutation of the Nucleophilic Elbow of the Lux-Specific Thioesterase from Vibrio harveyi

Abstract

Myristoyl-ACP thioesterase (LuxD) from Vibrio harveyi causes the slow release of fatty acids for reduction into the aldehyde substrate required for the bacterial bioluminescence reaction. The active site Ser nucleophile (S114) of the LuxD thioesterase is in a γ-turn with a sequence (AXS114XS) quite different from the standard motif of GXSXG found in almost all (thio) esterases and lipases. The presence of an Arg residue (R118) in the first turn of the helix after the γ-turn also distinguishes LuxD from other enzymes. Mutation of R118 to Leu inactivated the enzyme and prevented acylation of the Ser114 nucleophile, while even a conservative replacement with Lys resulted in over 75% loss of the same functions, suggesting that R118 helps maintain the configuration of the active site. In contrast, replacement of S116 with Gly but not Ala stimulated the esterase and deacylation rates by over threefold. Purification of the S116G mutant to homogeneity and analyses of its intrinsic fluorescence on acylation with myristoyl-CoA clearly demonstrated that this mutant was much more active than wild-type LuxD. The presence of S116 rather than the expected Gly residue in the γ-turn containing the Ser nucleophile may function so that release of fatty acids from LuxD is restricted allowing a more efficient delivery of fatty acids to the luminescent system.