Recognition of Hybrid Peptidyl Carrier Proteins/Acyl Carrier Proteins in Nonribosomal Peptide Synthetase Modules by the 4′-Phophopantetheinyl Transferases AcpS and Sfp

Mohammad Reza Mofid,Robert Finking,Mohamed A Marahiel

doi:10.1074/jbc.m200120200

Mohammad Reza Mofid, Robert Finking + Show 1 more

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https://doi.org/10.1074/jbc.m200120200

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Abstract

The acyl carrier proteins (ACPs) of fatty acid synthase and polyketide synthase as well as peptidyl carrier proteins (PCPs) of nonribosomal peptide synthetases are modified by 4'-phosphopantetheinyl transferases from inactive apo-enzymes to their active holo forms by transferring the 4'-phosphopantetheinyl moiety of coenzyme A to a conserved serine residue of the carrier protein. 4'-Phosphopantetheinyl transferases have been classified into two types; the AcpS type accepts ACPs of fatty acid synthase and some ACPs of type II polyketide synthase as substrates, whereas the Sfp type exhibits an extraordinarily broad substrate specificity. Based on the previously published co-crystal structure of Bacillus subtilis AcpS and ACP that provided detailed information about the interacting residues of the two proteins, we designed a novel hybrid PCP by replacing the Bacillus brevis TycC3-PCP helix 2 with the corresponding helix of B. subtilis ACP that contains the interacting residues. This was performed for the PCP domain as a single protein as well as for the TycA-PCP domain within the nonribosomal peptide synthetase module TycA from B. brevis. Both resulting proteins, designated hybrid PCP (hPCP) and hybrid TycA (hTycA), were modified in vivo during heterologous expression in Escherichia coli (hPCP, 51%; hTycA, 75%) and in vitro with AcpS as well as Sfp to 100%. The designated hTycA module contains two other domains: an adenylation domain (activating phenylalanine to Phe-AMP and afterward transferring the Phe to the PCP domain) and an epimerization domain (converting the PCP-bound l-Phe to d-Phe). We show here that the modified PCP domain of hTycA communicates with the adenylation domain and that the co-factor of holo-hPCP is loaded with Phe. However, communication between the hybrid PCP and the epimerization domain seems to be disabled. Nevertheless, hTycA is recognized by the next proline-activating elongation module TycB1 in vitro, and the dipeptide is formed and released as diketopiperazine.

Highlights

A large number of natural bioactive peptides are produced by bacteria and fungi via a template-directed, nucleic acid-inde
The acyl carrier proteins (ACPs) of fatty acid synthase and polyketide synthase as well as peptidyl carrier proteins (PCPs) of nonribosomal peptide synthetases are modified by 4؅-phosphopantetheinyl transferases from inactive apo-enzymes to their active holo forms by transferring the 4؅-phosphopantetheinyl moiety of coenzyme A to a conserved serine residue of the carrier protein. 4؅-Phosphopantetheinyl transferases have been classified into two types; the AcpS type accepts ACPs of fatty acid synthase and some ACPs of type II polyketide synthase as substrates, whereas the Sfp type exhibits an extraordinarily broad substrate specificity
Using the newly created PCP domain within the nonribosomal peptide synthetases (NRPSs) module hybrid TycA (hTycA), we show that hybrid PCP (hPCP) is able to perform the necessary communication with the A and C domains

Summary

Introduction

A large number of natural bioactive peptides (e.g. vancomycin, cyclosporine, tyrocidine, and surfactin) are produced by bacteria and fungi via a template-directed, nucleic acid-inde-. The peptide bond formation between covalently bound intermediates on two consecutive modules is catalyzed by a condensation (C) domain [9, 10] (Fig. 1). During this peptide assembly, optional modifications of the substrates can occur, depending on the presence of modifying domains within the corresponding module. NRPSs, FASs, and PKSs contain one or more small PCP or ACP domains that are 80 –100 amino acids in length that carry the building blocks and elongated intermediates to the different catalytic centers. The PCP domains in NRPSs and the acyl carrier proteins (ACPs) in FASs and PKSs share a conserved sequence motif (GX(D/H)S(L/I)(D/K)) containing an invariable serine residue [11]. During peptide or polyketide synthesis, the activated substrates and elongation intermediates are covalently linked as thioesters to the thiol moiety of the 4Ј-Ppant

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