Subtle Control of Carbon Chain Length in Polyunsaturated Fatty Acid Synthases.

Some marine bacteria synthesize docosahexaenoic acid (DHA; C22) and eicosapentaenoic acid (EPA; C20) by enzyme complexes composed of four subunits (A-D). We recently revealed that ß-ketoacyl synthase (KSC)/chain length factor (CLF)-like domains in the "C" subunit of DHA synthase catalyzed the last elongation step (C20 to C22) even though their amino acid sequences are very similar to those of EPA synthase. To investigate the amino acid residues controlling the product chain length, conserved residues in the KSC/CLF-like domains in DHA synthase were replaced with corresponding EPA synthase residues. Among 12 mutants, two CLF-like domain-mutated genes completely lost DHA productivity and produced trace amounts of EPA when coexpressed with dha-ABD in Escherichia coli, whereas when coexpressed with epa-ABD, they produced the same amounts of EPA as epa-ABCD. These results suggest that the product profiles were subtly controlled by several amino acid residues.

Control Mechanism for Carbon-Chain Length in Polyunsaturated Fatty-Acid Synthases.

Polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are essential fatty acids. PUFA synthases are composed of three to four subunits and each create a specific PUFA without undesirable byproducts. However, detailed biosynthetic mechanisms for controlling final product profiles have been obscure. Here, the bacterial DHA and EPA synthases were carefully dissected by in vivo and in vitro experiments. In vitro analysis with two KS domains (KSA and KSC ) and acyl-acyl carrier protein (ACP) substrates showed that KSA accepted short- to medium-chain substrates while KSC accepted medium- to long-chain substrates. Unexpectedly, condensation from C18 to C20 , the last elongation step in EPA biosynthesis, was catalyzed by KSA domains in both EPA and DHA synthases. Conversely, condensation from C20 to C22 , the last elongation step for DHA biosynthesis, was catalyzed by the KSC domain in DHA synthase. KSC domains therefore determine the chain lengths.