Mutational analysis of the epimerization domain in the initiation module PheATE of gramicidin S synthetase.
The epimerase (E) domain of the three-domain (ATE) initiation module of Bacillus brevis gramicidin S synthetase equilibrates the Calpha 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 Calpha-[(3)H] 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 Calpha-(3)H 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.
Stachelhaus
T
T
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.
Walsh
C T
CT
D000591:Amino Acid Isomerases / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology; D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000235:genetics*; D002384:Catalysis; D004252:DNA Mutational Analysis; D054659:Diketopiperazines; D004579:Electron Transport; D006096:Gramicidin / Q000737:chemistry*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010204:Pantetheine / Q000031:analogs & derivatives / Q000737:chemistry; D010442:Peptide Chain Initiation, Translational / Q000235:genetics*; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics*; D010879:Piperazines / Q000737:chemistry; D017434:Protein Structure, Tertiary / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D054659:Diketopiperazines; D010879:Piperazines; D006096:Gramicidin; D010649:Phenylalanine; D010204:Pantetheine; D000591:Amino Acid Isomerases; C022343:phenylalanine racemase (ATP-hydrolyzing); D002238:Carbohydrate Epimerases; C003129:4'-phosphopantetheine