Functional analysis of an acyltransferase‐like domain from polyunsaturated fatty acid synthase in thraustochytrium

Abstract

Biosynthesis of very long chain polyunsaturated fatty acids (VLCPUFA) such as do-cosahexaenoic acid (DHA, 22:6‐4,7,10,13,16,19) and docosapentaenoic acid (DPA, 22:5‐4,7,10,13,16) in protist Thraustochytrium is catalyzed by a polyunsaturated fatty acids (PUFA) synthase compris-ing three large subunits, each with multiple catalytic domains. This study used complementation test, in vitro assays, and functional expression to characterize an acyltransferase (AT)‐like domain in Subunit‐B of a PUFA synthase from Thraustochytrium. Complementation test in Escherichia coli showed that the AT‐like domain could not restore the growth phenotype of a temperature‐sensitive mutant (∆fabDts ) defective in malonyl‐CoA:ACP transacylase activity. In vitro assays showed that the AT‐like domain possessed thioesterase activity towards a few acyl‐CoAs tested where do-cosahexaenoyl‐CoA (DHA‐CoA) was the preferred substrate. Expression of this domain in an E. coli mutant (∆fadD) defective in acyl‐CoA synthetase activity resulted in the increased accumulation of free fatty acids. Site‐directed mutagenesis showed that the substitution of two putative active site residues, serine at 96 (S96) and histidine at 220 (H220), in the AT‐like domain significantly reduced its activity towards DHA‐CoA and accumulation of free fatty acids in the ∆fadD mutant. These results indicate that the AT‐like domain of the PUFA synthase does not function as a malonyl‐ CoA:ACP transacylase, rather it functions as a thioesterase. It might catalyze the last step of the VLCPUFA biosynthesis by releasing freshly synthesized VLCPUFAs attached to ACP domains of the PUFA synthase in Thraustochytrium.