The Multiple Carrier Model of Nonribosomal Peptide Biosynthesis at Modular Multienzymatic Templates

Abstract

Gramicidin S synthetase 1 and 2 were affinity-labeled at their thiolation centers either by thioesterification with the amino acid substrate or by specific alkylation with the thiol reagent N-ethylmaleimide in combination with a substrate protection technique. The labeled proteins were digested either chemically by cyanogen bromide or by proteases. An efficient multistep high pressure liquid chromatography methodology was developed and used to isolate the active site peptide fragments of all five thiolation centers of gramicidin S synthetase in pure form. The structures of these fragments are investigated by N-terminal sequencing, mass spectrometry, and amino acid analysis. Each of the active site peptide fragments contains the consensus motif LGG(H/D)S(L/I), which is specific for thioester formation in nonribosomal peptide biosynthesis. It was demonstrated that a 4'-phosphopantetheine cofactor is attached to the central serine of the thiolation motif in each amino acid-activating module of the gramicidin S synthetase multienzyme system forming the thioester binding sites for the amino acid substrates and catalyzing the elongation process. Our data are strong support for a "multiple carrier model" of nonribosomal peptide biosynthesis at multifunctional templates, which is discussed in detail.

Ebony, a Novel Nonribosomal Peptide Synthetase for β-Alanine Conjugation with Biogenic Amines in Drosophila

“…Expression of the corresponding putative P-pant transferase cDNA in E. coli gave rise to a protein that in vitro enhanced low level phosphantetheinylated S2 cell-derived Ebony activity depending on the presence of CoA comparable with the aforementioned Sfp. 2 Our results demonstrate that the higher eucaryote Drosophila has preserved an amino acid activation mechanism that until now was considered to be specific for microbial NRPSs (27). Ebony combines this unique feature with a functional domain that allows peptide bond formation with a structurally constrained group of amines.…”

Activity Screening of Carrier Domains within Nonribosomal Peptide Synthetases Using Complex Substrate Mixtures and Large Molecule Mass Spectrometry

“…Here we describe a mass spectrometry based method to identify substrates based on mass changes that take place during acylation of a phosphopantetheinyl functionality on the carrier domain(s) of an NRPS protein from very complex substrate reaction mixtures. Observing such acylations by mass spectrometry on carrier domains is now becoming routine (5,19,(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36).The general method to identify covalently loaded substrates or intermediates is shown in Figure 1. Overproduced protein that contains a carrier domain is purified and incubated with Sfp, a promiscuous phosphopantetheinyl transferase from B .subtilis, and CoA in order to generate the holo form of the carrier domain (37).…”

The Adenylation Domain of Tyrocidine Synthetase 1

“…Analysis of the sequence data has provided insight into the modular construction of their multifunctional polypeptide chains, the occurrence of the modules being associated with the number and the sequence of the amino acid constituents in the peptide product. The reaction sequence involves carboxyl (amino, imino and hydroxy acid) activation in form of an acyladenylate, followed by transfer of the activated acyl moiety to a cysteamine group of an enzyme bound cofactor, 4'-phosphopantetheine (Stein et al, 1996). Sequence infoimation has been obtained for more than 58 peptide synthetase modules, including tyrocidine synthetase 1, gramicidin S synthetase, and &(L-a-aminoadipyl)-L-cysteinyl-D-vahe synthetase (Pavela-Vrancic et al, 1994 a).…”