Mutational Analysis of the Epimerization Domain in the Initiation Module PheATE of Gramicidin S Synthetase

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Abstract
The epimerase (E) domain of the three-domain (ATE) initiation module of Bacillus brevis gramicidin S synthetase equilibrates the Cα configuration of the phenylalanyl moiety presented as Phe-S-4‘-phosphopantetheine-modified (Ppant) acyl enzyme. Mutants at 22 residues of this E domain that are conserved across the approximately 450 residue E domains of nonribosomal peptide synthetases were constructed, and the PheATE* derivatives expressed in Escherichia coli as C-terminal His tag fusions and then purified and assayed for three activities: (1) the l-Phe Cα-[3H] exchange to solvent, (2) the rate of approach to d-Phe/l-Phe-S-Ppant acyl enzyme equilibrium from either l- or d-Phe, and (3) the rate of Phe-Pro dipeptidyl-S-Ppant enzyme formation with the downstream ProCAT module. We found that for wild-type PheATE epimerization is much faster than subsequent condensation, leading to a 1.9:1 ratio of d-Phe-S-Ppant/l-Phe-S-Ppant acyl enzyme. Only d-Phe is then transferred to yield d-Phe-l-Pro-S-Ppant ProCAT acyl enzyme. Among the mutants generated, three PheATE* constructs, H753A, D757S, and Y976A, showed no detectable Cα-3H washout, while E892A and R896A were among a larger set partially impaired. All these mutants were dramatically impaired in approach to d-Phe/l-Phe-S-Ppant equilibrium from either d- or l-Phe, while another construct, D767S, was asymmetrically impaired only for d-to-l-Phe direction. In the d-Phe-l-Pro dipeptidyl-S-Ppant condensation assay, the H753A and E892A forms of PheATE* were only slightly active from l-Phe but unimpaired from d-Phe; N975A epimerizes faster than Y976A from l-Phe. When the chirality of the Phe-Pro-diketopiperazine released product was analyzed the d,l/l,l ratio from wild-type PheATE and ProCAT was 98:2. From E892A and N975A it was comparably 95:5 and 92:8, but H753A and Y976A yielded 56% of the l,l-product, reflecting a gain of function to transfer l-Phe. The 98:2 preference of wild-type PheATE for d-Phe transfer reflects the kinetically controlled stereopreference of the condensation (C) domain of ProCAT for the d-Phe-S-Ppant donor substrate. It may be that other NRPS C domains immediately downstream of E domains will likewise be d-selective.