On the domain construction of the multienzyme gramicidin S synthetase 2

Abstract

The multienzyme gramicidin S synthetase 2, composed of one polypeptide chain, was treated with trypsin and chymotrypsin to give fragments retaining partial enzyme activities. Previously, a tryptic fragment of this multi‐enzyme has been identified as a structural and functional domain. In this study two more fragments, activating Leu and Val, respectively, are shown to represent domains. Careful inspection of the data on limited proteolysis, from this study as well as from previous work, suggests that domains are not simply connected like pearls on a string, and a model for the structure of gramicidin S synthetase, with implications for other peptide synthetase multienzymes, is presented. It is suggested that gramicidin S synthetase 2 is constructed from core catalytic domains and intervening framework. Such an interpretation is in accordance with all published data on limited proteolysis of peptide synthetases, but needs an interplay with gene structural studies in order to be validated and refined.

Expression of an active adenylate‐forming domain of peptide synthetases corresponding to acyl‐CoA‐synthetases

“…More reliable evidence can be obtained from limited proteolysis data, as this method is generally used to prepare stable domains. Tryptic digestion of gramicidin S synthetase 2, a 512 kDa multienzyme integrating 4 modules, led to amino acidactivating domains of I 10-115 kDa [35][36][37]. The Pro-activating domain has been analysed in detail, subcloned, and can be concluded to be truncated before the J-motif [38].…”

Modular Peptide Synthetases Involved in Nonribosomal Peptide Synthesis

“…Protein chemical studies and the recent progress in cloning and sequencing of genes encoding peptide synthetases of bacterial and fungal origin provide valuable insights into the molecular architecture of these enzymes. − A modular structure for these multienzyme complexes has emerged, in which the substrate activating/modifying units are aligned in a sequence that is colinear with the amino acid sequence of the assembled peptide. − , These units have been designated as modules according to a definition originally applied by L. Katz and co-workers to the arrangement of genes encoding type I polyketide synthases . On the basis of comparison of DNA sequences encoding several peptide synthetases and recent studies on heterologous expression of DNA fragments ,− that encode proteins that activate individual amino acids, modules were defined as semiautonomous units within peptide synthetases that carry all information needed for recognition, activation, and modification of one substrate. This means that the number of modules and their order within a peptide synthetase define the sequence and the length of the synthesized peptide (Figure A, B).…”

“…While the A domain of peptide synthetases is an integrated part of a multifunctional enzyme, the homologous acyl-CoA synthases are distinct proteins. , Following the idea of the modular architecture of peptide synthetases, the question arose whether such a single A domain could function independently from the adjacent domains. The first insights were obtained through biochemical characterization of proteolytic and gene encoded fragments, expressed in E. coli , of the multimodular gramicidin S and tyrocidine synthetases, which exhibited activation of one specific amino acid residue only. ,− ,− The catalytic independence of the integrated A domain itself was demonstrated for the first time by deletion studies on the starter synthetases GrsA and TycA (Figure ). , The A domains, located in the N-terminal region, were proven to catalyze amino acid activation (Figure ) with the same specificity as the wild-type enzymes. Very recently, it has also been shown that internal A domains of the multimodular enzymes TycB and TycC can be expressed in E. coli as soluble, functional proteins …”

The Pipecolate‐Incorporating Enzyme for the Biosynthesis of the Immunosuppressant Rapamycin — Nucleotide Sequence Analysis, Disruption and Heterologous Expression of Rap P from Streptomyces Hygroscopicus

“…PIE is then thought to dock onto the C-terminal ACP domain of R A P S , for the transfer of the polyketide chain to the amino group of pipecolate (3), where cyclisation takes place (4). The domain structure shown fur PIE is inferred from the sequence analysis reported here and analyses of the protein structure of peptide synthetases [9,[52][53][54] of the macrolide polyketide synthases, for example the multienzyines for the biosynthesis of erythromycin [3], avermectin 1201, and oleandomycin [45]. However, neither PIE nor the rapamycin polyketide synthase contain a thioesterase motif, nor is there an activity with similarities to discrete thioesterase enzymes in the rup cluster.…”