Substrate-bound structures of a ketoreductase from amphotericin modular polyketide synthase

Abstract

Ketoreductase (KR) domains of modular polyketide synthases (PKSs) control the stereochemistry of C2 methyl and C3 hydroxyl substituents of polyketide intermediates. To understand the molecular basis of stereocontrol exerted by KRs, the crystal structure of a KR from the second module of the amphotericin PKS (AmpKR2) complexed with NADP+ and 2-methyl-3-oxopentanoyl-pantetheine was solved. This first ternary structure provides direct evidence to the hypothesis that a substrate enters into the active site of an A-type KR from the side opposite the coenzyme to generate an L-hydroxyl substituent. A comparison with the ternary complex of a G355T/Q364H mutant sheds light on the structural basis for stereospecificity toward the substrate C2 methyl substituent. Functional assays suggest the pantetheine handle shows obvious influence on the catalytic efficiency and the stereochemical outcome. Together, these findings extend our current understanding of the stereo-chemical control of PKS KR domains.

Biosynthesis of depsipeptides, or Depsi: The peptides with varied generations

“…(1) α-hydroxy acids are not common in primary metabolism, so in fungal systems that use α-hydroxy acids as NRPS substrates, the BGC often contains standalone ketoreductase (KR) proteins which convert α-keto acids to α-hydroxy acids in an NADPH-dependent reduction reaction [130][131][132][133][134] (Figure 4), with keto acids arising from branched-chain amino acid biosynthesis pathways 135 or from glycolysis. These KR proteins are not closely related to PKS KR domains, 128,136,137 but belong to the ApbA_C superfamily, which includes ketopantoate reductases. 130,133,138 Deletion of the KR protein abolishes production of the depsipeptide, which can be rescued by supplementing the media with α-hydroxy acids.…”

“…[161][162][163] They can be classified according to the stereochemistry of their product, A-type producing L-β-hydroxy acids and B-type producing D-β-hydroxy acids. 136,137 The type is distinguishable from sequence in PKSs, with a highly conserved Trp in type A and a Leu-Asp-Asp in type B KR. 164 In both types, the 4 0 -pro-S hydrogen faces the active site Tyr.…”

“…These KR domains are pseudo‐dimeric with the N‐terminal Rossmann‐like fold having a structural role and the C‐terminal one containing the active site with its Tyr‐Ser‐Lys catalytic triad 161–163 . They can be classified according to the stereochemistry of their product, A‐type producing l ‐β‐hydroxy acids and B‐type producing d ‐β‐hydroxy acids 136,137 . The type is distinguishable from sequence in PKSs, with a highly conserved Trp in type A and a Leu‐Asp‐Asp in type B KR 164 .…”

“…The chirality of the product is determined by the approach of the β‐ketoacyl‐ S ‐ACP, with the conserved motifs directing the β‐ketoacyl‐ACP to one or another ACP binding site with grooves reaching the active site from two opposite directions 137,165 . Depsipeptide KS domains have no identifiable sequence motifs 34,128 analogous to those found in PKS KR domains, 137 and both putative PCP binding sites and grooves to the active site appear feasible in the KR domain of StrA 34 . Despite being present in the crystals of StrA A‐KR‐PCP, the PCP was disordered.…”

Site‐Directed Mutagenesis of Modular Polyketide Synthase Ketoreductase Domains for Altered Stereochemical Control

“…Regarding structural characterization by X‐ray crystallography, AmpKR2 is the most thoroughly investigated KR, and as such has been a major target of structure‐guided mutagenesis. To date, AmpKR2 is the only KR that has been co‐crystalized with a substrate (2‐methyl‐3‐oxopentaonyl‐ S ‐pantetheine [59] ) in addition to both apo and NADP + ‐bound structures [18] . Additionally, multiple structures of mutants exist including W359F, Q367H, [18] and Q367H/G358T [27,59] .…”