Biocatalytic engineering of phenylalanine aminomutase

PhD ceremony: Mr. B. Wu, 13.15 uur, Academiegebouw, Broerstraat 5, Groningen

Thesis: Biocatalytic engineering of phenylalanine aminomutase

Promotor(s): prof. D.B. Janssen

Faculty: Mathematics and Natural Sciences

This thesis covers the mechanistic studies, biocatalytic applications and protein engineering of a MIO-based phenylalanine aminomutase (PAM). PAM from Taxus chinensis catalyzes the conversion of α-phenylalanine to β-phenylalanine, an important step in the biosynthesis of the N-benzoyl phenylisoserinoyl side chain of the anticancer drug taxol. PAM exhibits high enantioselectivity towards β-amino acids and its broad substrate scope has been exploited for the production of non-natural enantiopure β-amino acids. We describe a novel synthetic strategy for the biocatalytic production of several (R)-β-amino acids which is based on the finding that ring-substituted (E)-cinnamic acids can serve as a substrate in PAM-catalyzed ammonia addition reactions. Studies on the scope of the system are reported, together with the efforts to determine the factors governing the kinetic parameters and product distribution. In addition, we exploited the use of PAM for the preparation of the (S)-β-amino acids from the corresponding racemates. In this process, PAM catalyzes the stereoselective isomerization of (R)-β-phenylalanines to (S)-α-phenylalanines, which are in situ transformed to cinnamic acids by phenylalanine ammonia lyase (PAL). Furthermore, we have identified a single active-site residue important for substrate selectivity in the MIO-based aminomutases. By replacing the active site residue Cys107 with Ser in PAM, the enzyme gained tyrosine aminomutase activity while retaining PAM activity and high enantioselectivity. This engineered enantioselective tyrosine aminomutase may prove to be useful for the synthesis of enantiopure β-tyrosine and its derivatives. While still challenges lie ahead, PAM has proven to be a promising tool for the preparation of enantiomerically pure β-amino acids.