In vitro reconstitution of the myxochelin biosynthetic machinery of Stigmatella aurantiaca Sg a15: Biochemical characterization of a reductive release mechanism from nonribosomal peptide synthetases
N. Gaitatzis et al., In vitro reconstitution of the myxochelin biosynthetic machinery of Stigmatella aurantiaca Sg a15: Biochemical characterization of a reductive release mechanism from nonribosomal peptide synthetases, P NAS US, 98(20), 2001, pp. 11136-11141
Citations number
32
Categorie Soggetti
Multidisciplinary
Journal title
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
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.