Comprehensive Structural and Biochemical Analysis of the Terminal Myxalamid Reductase Domain for the Engineered Production of Primary Alcohols

Abstract

The terminal reductase (R) domain from the non-ribosomal peptide synthetase (NRPS) module MxaA in Stigmatella aurantiaca Sga15 catalyzes a non-processive four-electron reduction to produce the myxalamide family of secondary metabolites. Despite widespread use in nature, a lack of structural and mechanistic information concerning reductive release from polyketide synthase (PKS) and NRPS assembly lines principally limits our ability to redesign R domains with altered or improved activity. Here we report crystal structures for MxaA R, both in the absence and, for the first time, in the presence of the NADPH cofactor. Molecular dynamics simulations were employed to provide a deeper understanding of this domain and further identify residues critical for structural integrity, substrate binding, and catalysis. Aggregate computational and structural findings provided a basis for mechanistic investigations and, in the process, delivered a rationally altered variant with improved activity toward highly reduced substrates.

Thioester reduction and aldehyde transamination are universal steps in actinobacterial polyketide alkaloid biosynthesis

“…4,24–26 TR-catalysed reductive chain release has also been proposed to occur in several fungal PKS systems, although direct biochemical evidence for this is currently lacking. 27–31 In contrast, relatively few bacterial modular PKSs have been reported, prior to this work, to contain a TR domain and to the best of our knowledge CpkC-TR is the first of these to be biochemically characterized.…”

“…The three residues that interact with the phosphate group of NADPH in the TR domain from the myxalamid NRPS are completely conserved in CpkC-TR and Zmn14. 26 Thus the structural basis for the cofactor preference of CpkC-TR and Zmn14 is unclear.…”

Evidence of a sequestered imine intermediate during reduction of nitrile to amine by the nitrile reductase QueF from Escherichia coli

“…While in terms of reaction catalyzed, QueF nitrile reductases share commonalities with other four-electron reductases, such as 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (29)(30)(31) and the non-ribosomal peptide synthetase module myxalamid reductase (32,33), that use NAD(P)H to convert thioester-activated forms of carboxylic acids into primary alcohols via an aldehyde intermediate, they are individuated strongly from these other enzymes in having to deal with a highly decomposition-prone intermediate. The central role of intermediate sequestration in the QueF mechanism seems to be reflected in the fact that structural devices used to keep a firm grip on the imine intermediate are likewise involved directly in the catalytic events of the enzymatic reaction.…”

Structural insight into the necessary conformational changes of modular nonribosomal peptide synthetases

“…As the PCP domain is positioned immediately downstream of the adenylation domain, this change of orientation of the C-terminal subdomain influences the location of the PCP domain and, as described below, is a critical feature to shuffle the peptide intermediates between catalytic domains. Finally, structures of the terminal thioesterase [26,27] or reductase [28–30] domains demonstrate that these enzymes belong to larger families of enzymes that catalyze similar chemistry.…”