Active-Site Engineering of ω-Transaminase for Production of Unnatural Amino Acids Carrying a Side Chain Bulkier than an Ethyl Substituent

ABSTRACTω-Transaminase (ω-TA) is a promising enzyme for use in the production of unnatural amino acids from keto acids using cheap amino donors such as isopropylamine. The small substrate-binding pocket of most ω-TAs permits entry of substituents no larger than an ethyl group, which presents a significant challenge to the preparation of structurally diverse unnatural amino acids. Here we report on the engineering of an (S)-selective ω-TA fromOchrobactrum anthropi(OATA) to reduce the steric constraint and thereby allow the small pocket to readily accept bulky substituents. On the basis of a docking model in whichl-alanine was used as a ligand, nine active-site residues were selected for alanine scanning mutagenesis. Among the resulting variants, an L57A variant showed dramatic activity improvements in activity for α-keto acids and α-amino acids carrying substituents whose bulk is up to that of ann-butyl substituent (e.g., 48- and 56-fold increases in activity for 2-oxopentanoic acid andl-norvaline, respectively). An L57G mutation also relieved the steric constraint but did so much less than the L57A mutation did. In contrast, an L57V substitution failed to induce the improvements in activity for bulky substrates. Molecular modeling suggested that the alanine substitution of L57, located in a large pocket, induces an altered binding orientation of an α-carboxyl group and thereby provides more room to the small pocket. The synthetic utility of the L57A variant was demonstrated by carrying out the production of optically purel- andd-norvaline (i.e., enantiomeric excess [ee] > 99%) by asymmetric amination of 2-oxopantanoic acid and kinetic resolution of racemic norvaline, respectively.

Effect of Steric Constraint at the γ-Backbone Position on the Conformations and Hybridization Properties of PNAs

Conformationally preorganized peptide nucleic acids (PNAs) have been synthesized through backbone modifications at the γ-position, where R = alanine, valine, isoleucine, and phenylalanine side chains. The effects of these side-chains on the conformations and hybridization properties of PNAs were determined using a combination of CD and UV-Vis spectroscopic techniques. Our results show that the γ-position can accommodate varying degrees of sterically hindered side-chains, reaffirming the bimodal function of PNAs as the true hybrids of “peptides” and “nucleic acids.”