Peptide Bond Formation in Nonribosomal Peptide Biosynthesis

Abstract

Recently, considerable insight has been gained into the modular organization and catalytic properties of nonribosomal peptide synthetases. However, molecular and biochemical aspects of the condensation of two aminoacyl substrates or a peptidyl and an aminoacyl substrate, leading to the formation of a peptide bond, have remained essentially impenetrable. To investigate this crucial part of nonribosomal peptide synthesis, an in vitro assay for a dipeptide formation was developed. Two recombinant holomodules, GrsA (PheATE), providing D-Phe, and a C-terminally truncated TycB, corresponding to the first, L-Pro-incorporating module (Pro-CAT), were investigated. Upon combination of the two aminoacylated modules, a fast reaction is observed, due to the formation of the linear dipeptide D-Phe-L-Pro-Senzyme on ProCAT, followed by a noncatalyzed release of the dipeptide from the enzyme. The liberated product was identified by TLC, high pressure liquid chromatography-mass spectrometry, 1 H and 13 C NMR, and comparison with a chemically synthesized standard to be the expected D-Phe-L-Pro diketopiperazine. Further minimization of the two modules was not possible without a loss of transfer activity. Likewise, a mutation in a proposed active-site motif (HHXXXDG) of the condensation domain giving ProCAT(H147V), abolished the condensation reaction. These results strongly suggest the condensation domain to be involved in the catalysis of nonribosomal peptide bond formation with the histidine 147 playing a catalytic role.Microorganisms use the nonribosomal pathway to synthesize a large group of structurally diverse and often complex secondary metabolites with biological activities, including antibiotics, siderophores, biosurfactants, immunosuppressants, and antitumor and antiviral agents. These low molecular weight peptide-based compounds are synthesized by means of multifunctional enzymes, the peptide synthetases, which can recruit not only proteogenic amino acids but also a large number of unusual amino acids and hydroxy acids that form peptide and ester bonds. The incorporated constituents can be further altered by epimerization or N-methylation, and the peptide backbone can be acylated, glycosylated, or cyclized (1-3

Roles of VioG and VioQ in the Incorporation and Modification of the Capreomycidine Residue in the Peptide Antibiotic Viomycin

“…13 Analysis of the VioG A domain substrate recognition elements indicated it had a unique substrate-binding pocket that was most similar to A domains activating L-Arg or L-Orn, suggesting VioG may activate L-Cap. [14][15][16] In order to confirm this prediction, full-length vioG and a fragment coding for the A domain were amplified by PCR from cosmid pTOV106, which contains a portion of the viomycin gene cluster. 8 The fragments were then sequenced and cloned into the pET28a vector for expression as N-terminal His 6 -tagged proteins in E. coli.…”

Excision of the Epothilone Synthetase B Cyclization Domain and Demonstration of in Trans Condensation/Cyclodehydration Activity

“…The second histidine residue, based on the similarity of C domains to the acetyltransferase superfamily, is thought to be the catalytic base required for deprotonation of the amine of the downstream aminoacyl-S-PCP to facilitate nucleophilic attack on the thioesterified upstream acyl donor (33). While mutation of this residue to alanine generally leads to diminished activity, the impact of this varies from negligible (22,30) to drastic (8,22,31,32), depending on the context of the specific C domain and the corresponding condensation reaction.…”

Deep sequencing of non-ribosomal peptide synthetases and polyketide synthases from the microbiomes of Australian marine sponges

“…This is likely to reflect either the presence of a single superproducing organism, or indicate that many of these C domains are present in a small number of clusters. The phylogenetic inference of the C domains is enabled by the presence of two binding pockets adjacent to the catalytic centre (Stachelhaus et al, 1998). The C-terminus binding pocket, considered the acceptor site, exhibits specific selectivity for the activated substrate, which is conferred within the amino-acid sequence.…”

“…The KS and condensation (C) domains, that catalyse the condensation of two activated subunits, contain motifs that are conserved at an amino-acid level (Stachelhaus et al, 1998;Walsh and Fischbach, 2010). Between these motifs, variation is observed, typically within catalytic centres, giving rise to domains with functional specificity, reflecting the nature of the subunits being condensed (Moffitt and Neilan, 2003;Rausch et al, 2007), the arrangement of other domains within the module and the expected product (Moffitt and Neilan, 2003;Piel et al, 2004a;Schmitt et al, 2008).…”