Semi-rational approach to Expand the Acyl-CoA Chain Length Tolerance of Sphingomonas paucimobilis Serine Palmitoyltransferase

Publication date: Available online 21 January 2020Source: Enzyme and Microbial TechnologyAuthor(s): Hyunjun Choe, Minsun Cha, Jon D. StewartAbstractSerine palmitoyltransferase (SPTase), the first enzyme of the sphingolipid biosynthesis pathway, produces 3-ketodihydrosphingosine by a Claisen-like condensation / decarboxylation reaction of L-Ser and palmitoyl-CoA (n-C16-CoA). Previous structural analysis of Sphingomonas paucimobilis SPTase (SpSPTase) revealed a dynamic active site loop (RPPATP; amino acids 378 - 383) in which R378 (underlined) forms a salt bridge with the carboxylic acid group of the PLP : L-Ser external aldimine. We hypothesized that this interaction might play a key role in acyl group substrate selectivity and therefore performed site-saturation mutagenesis at position 378 based on semi-rational design to expand tolerance for shorter acyl-CoA’s. The resulting library was initially screened for the reaction between L-Ser and dodecanoyl-CoA (n-C12-CoA). The most interesting mutant (R378 K) was then purified and compared to wild-type SpSPTase against a panel of acyl-CoA’s. These data showed that the R378 K substitution shifted the acyl group preference to shorter chain lengths, opening the possibility of using this and other engineered variants for biocatalytic C-C bond-forming reactions.
Source: Enzyme and Microbial Technology - Category: Biotechnology Source Type: research