Mutations of the ATP-Grasp Enzyme TabS Alter Substrate Specificity for Tripeptide and Nonpeptide Amide Formation

Abstract

ATP-grasp enzymes, such as l-amino acid ligases (Lals), catalyze the coupling of carboxylate and amino acid acyl donor substrates to amino acid acyl acceptor substrates via an acyl phosphate intermediate. In addition to their roles in the biosynthesis of natural products, ATP-grasp enzymes have generated significant interest as preparative biocatalysts for the selective synthesis of peptides and amides. TabS is a Lal from Pseudomonas syringae NBRC14081 that couples l-threonine and tabtoxinine-β-lactam to give tabtoxin, which, when hydrolyzed, yields tabtoxinine-β-lactam as a glutamine synthetase inhibitor. However, TabS also has a broad substrate specificity encompassing the coupling of many pairs of l-amino acids, including l-leucine and l-phenylalanine, to form a range of homo- and heterodipeptides. Here we report the structure of TabS, in an apo- form and also in complex with the noncleavable ATP analog AMP-PNP, refined to resolutions of 2.17 and 2.90 Å, respectively. The structures shed light on the broad substrate specificity of TabS and have also informed the exploration, through modeling, mutation, and activity assays, of the possibilities of engineering these enzymes for the coupling of simple carboxylates and amines to form nonpeptide amide products. Rational mutation of hydrophilic residues thought to recognize the amino group of acyl donors and the carboxylate of acyl acceptors has given variants, including S16A/Y44F/Y45F/Y235F/L382F/Q337L/Q339L, which successfully convert the non-amino-acid analogs of the l-leucine donor and acceptor, i.e., 4-methyl pentanoate and 3-methyl butylamine, respectively, to the nonpeptide amide product, where the wild-type TabS displayed negligible activity.