In vitro reconstitution of the myxochelin biosynthetic machinery of Stigmatella aurantiaca Sg a15: Biochemical characterization of a reductive release mechanism from nonribosomal peptide synthetases

Microorganisms produce iron-chelating compounds to sequester the iron essen tial for growth from the environment. Many of these compounds are biosynthe sized by nonribosomal peptide synthetases, some in cooperation with polyket ide synthases. Myxochelins are produced by the myxobacterium Stigmatella au rantiaca Sg a15, and the corresponding gene cluster was cloned recently. We have undertaken to express heterologously the myxochelin biosynthetic mach inery in Escherichia coli. To activate the involved proteins posttranslatio nally, they were coexpressed with the phospho-pantetheinyltransferase MtaA from the myxothiazol biosynthetic gene cluster. Phosphopantetheinylation of the carrier proteins could be verified by protein mass analysis. Six activ e domains in proteins MxcE, MxcF, and MxcG are capable of assembling myxoch elin from ATP, NAD(P)H, lysine, and 2,3-dihydroxybenzoic acid in vitro. Thi s fact demonstrates that the condensation domain of MxcG performs two conde nsation reactions, creating the aryl-capped alpha -amide and the aryl-cappe d gamma -amide of the molecule. A previously unknown type of reductive rele ase is performed by the reduction domain of MxcG, which alternatively uses NADPH and NADH to set free the peptidyl-carrier protein-bound thioester as an aldehyde and further reduces it to the alcohol structure that can be fou nd in myxochelin A. This type of reductive release seems to be a general me chanism in polyketide and nonribosomal peptide biosynthesis, because severa l systems with C-terminal similarity to the reductase domain of MxcG can be found in the databases. Alternatively, the aldehyde can be transaminated, giving rise to a terminal amine.