gramicidin-a and 4--phosphopantetheine

Nonribosomal peptide synthetases (NRPSs) are very large proteins that produce small peptide molecules with wide-ranging biological activities, including environmentally friendly chemicals and many widely used therapeutics. NRPSs are macromolecular machines, with modular assembly-line logic, a complex catalytic cycle, moving parts and many active sites. In addition to the core domains required to link the substrates, they often include specialized tailoring domains, which introduce chemical modifications and allow the product to access a large expanse of chemical space. It is still unknown how the NRPS tailoring domains are structurally accommodated into megaenzymes or how they have adapted to function in nonribosomal peptide synthesis. Here we present a series of crystal structures of the initiation module of an antibiotic-producing NRPS, linear gramicidin synthetase. This module includes the specialized tailoring formylation domain, and states are captured that represent every major step of the assembly-line synthesis in the initiation module. The transitions between conformations are large in scale, with both the peptidyl carrier protein domain and the adenylation subdomain undergoing huge movements to transport substrate between distal active sites. The structures highlight the great versatility of NRPSs, as small domains repurpose and recycle their limited interfaces to interact with their various binding partners. Understanding tailoring domains is important if NRPSs are to be utilized in the production of novel therapeutics.

Topics: Amino Acid Isomerases; Anti-Bacterial Agents; Binding Sites; Biocatalysis; Brevibacillus; Carbohydrate Metabolism; Carrier Proteins; Catalytic Domain; Coenzymes; Crystallography, X-Ray; Gramicidin; Hydroxymethyl and Formyl Transferases; Models, Molecular; Multienzyme Complexes; Pantetheine; Peptide Synthases; Protein Binding; Protein Structure, Tertiary; RNA, Transfer

The three-domain initiation module PheATE (GrsA) of Bacillus brevis gramicidin S synthetase catalyzes the activation, thiolation and epimerization of L-phenylalanine (L-Phe), the first amino acid incorporated into the decapeptide antibiotic gramicidin S. There are three activated intermediates in the PheATE catalyzed chemical pathway: L-phenylalanyl-adenosine-5'-monophosphate diester (L-Phe-AMP), L-Phe-S-4'-phosphopantetheine(Ppant)- and D-Phe-S-4'-Ppant-acyl enzyme. In this study, we examined PheATE in single-turnover catalysis using rapid chemical quench techniques. Kinetic modeling of the process of disappearance of the substrate L-Phe, transient appearance and disappearance of L-Phe-AMP and the ad seriatim formation and equilibration of the L- and D-Phe-S-Ppant-acyl enzyme adducts allowed evaluation of the microscopic rate constants for the three chemical reactions in the initiation module PheATE. This study provides the first transient-state kinetic analysis of a nonribosomal peptide synthetase (NRPS) module.

Topics: Adenosine Monophosphate; Alanine; Amino Acid Isomerases; Amino Acid Motifs; Aminoacylation; Apoenzymes; Bacillus; Carbon Radioisotopes; Catalysis; Gramicidin; Histidine; Holoenzymes; Kinetics; Mutagenesis, Site-Directed; Pantetheine; Peptide Chain Initiation, Translational; Phenylalanine; Protein Structure, Tertiary

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.

Topics: Amino Acid Isomerases; Amino Acid Sequence; Bacillus; Carbohydrate Epimerases; Catalysis; Diketopiperazines; DNA Mutational Analysis; Electron Transport; Gramicidin; Hydrolysis; Molecular Sequence Data; Mutagenesis, Site-Directed; Pantetheine; Peptide Chain Initiation, Translational; Phenylalanine; Piperazines; Protein Structure, Tertiary; Sequence Homology, Amino Acid