Coordinate Transcription and Physical Linkage of Domains in Surfactin Synthetase Are Not Essential for Proper Assembly and Activity of the Multienzyme Complex

Eric Guenzi, Giuliano Galli, Ingeborg Grgurina, Emanuela Pace, Pasquale Ferranti, Guido Grandi
1998 Journal of Biological Chemistry  
Bacterial peptide synthetases have two common features that appear to be strictly conserved. 1) The enzyme subunits are co-regulated at both transcriptional and translational level. 2) The organization of the different enzymatic domains constituting the enzyme fulfills the "colinearity rule" according to which the order of the domains along the chromosome parallels their functional hierarchy. Considering the high degree of conservation of these features, one would expect that mutations such as
more » ... ranscription uncoupling and domain dissociations, deletions, duplications, and reshuffling would result in profound effects on the quality and quantity of synthesized peptides. To start testing this hypothesis, we designed two mutants. In one mutant, the operon structure of surfactin synthetase was destroyed, thus altering the concerted expression of the enzyme subunits. In the other mutant, the thioesterase domain naturally fused to the last amino acid binding domain of surfactin was physically dissociated and independently expressed. When the lipopeptides secreted by the mutant Bacillus subtilis strains were purified and characterized, they appeared to be expressed approximately at the same level of the wild type surfactin and to be identical to it, indicating that specific domaindomain interactions rather than coordinated transcription and translation play the major role in determining the correct assembly and activity of peptide synthetases. A number of peptides with highly variable structures and a broad range of biological activities are produced in bacteria and fungi as secondary metabolites via a nonribosomal mechanism. The synthesis involves large multienzyme complexes called peptide synthetases that are organized in structural domains and utilize the thiotemplate mechanism first described by Lipmann (1) and recently revised (2). According to this mechanism, each domain recognizes a specific amino acid that, after activation to the corresponding acyladenylate derivative, is covalently bound via a thioester bond to the phosphopantetheine cofactor present on each do-
doi:10.1074/jbc.273.23.14403 pmid:9603952 fatcat:i5fy7zlvjbabpbosuayoc7pzg4