Computationally Guided Enzymatic Studies on Schizochytrium -Sourced Malonyl-CoA:ACP Transacylase.

The malonyl-CoA:ACP transacylase (MAT) domain is responsible for the selection and incorporation of malonyl building blocks in the biosynthesis of polyunsaturated fatty acids (PUFAs) in eukaryotic microalgae ( Schizochytrium ) and marine bacteria ( Moritella marina , Photobacterium profundum , and Shewanella ). Elucidation of the structural basis underlying the substrate specificity and catalytic mechanism of the MAT will help to improve the yield and quality of PUFAs. Here, a methodology guided by molecular dynamics simulations was carried out to identify and mutate specificity-conferring residues within the MAT domain of Schizochytrium . Combining mutagenesis, cell-free protein synthesis, and in vitro biochemical assay, we dissected nearby interactions and molecular mechanisms relevant for binding and catalysis and found that the reorientation of the Ser154 C β -O γ bond establishes distinctive proton-transfer chains (His153-Ser154 and Asn235-His153-Ser154) for catalysis. Gln66 can be replaced by tyrosine to shorten the distance between His153 (N ε2 ) and Ser154 (O γ ), which facilitates a faster proton-transfer rate, allowing better use of acyl substrates than the wild type. Furthermore, we screened a mutant that displayed an 18.4% increase in PUFA accumulation. These findings provide important insights into the study of MAT through protein engineering and will benefit dissecting the molecular mechanisms of other PUFA-related catalytic domains.