Structural and functional studies of a trans -acyltransferase polyketide assembly line enzyme that catalyzes stereoselective α- and β-ketoreduction

Abstract

While the cis-acyltransferase modular polyketide synthase assembly lines have largely been structurally dissected, enzymes from within the recently discovered trans-acyltransferase polyketide synthase assembly lines are just starting to be observed crystallographically. Here we examine the ketoreductase from the first polyketide synthase module of the bacillaene nonribosomal peptide synthetase/polyketide synthase at 2.35-Å resolution. This ketoreductase naturally reduces both α- and β-keto groups and is the only ketoreductase known to do so during the biosynthesis of a polyketide. The isolated ketoreductase not only reduced an N-acetylcysteamine-bound β-keto substrate to a D-β-hydroxy product, but also an N-acetylcysteamine- bound α-keto substrate to an L-α-hydroxy product. That the substrates must enter the active site from opposite directions to generate these stereochemistries suggests that the acyl-phosphopantetheine moiety is capable of accessing very different conformations despite being anchored to a serine residue of a docked acyl carrier protein. The features enabling stereocontrolled α-ketoreduction may not be extensive since a β-ketoreductase from a cis-acyltransferase polyketide synthase was identified that performs a completely stereoselective reduction of the same α-keto substrate to generate the D-α-hydroxy product. A sequence analysis of trans-acyltransferase ketoreductases reveals that a single residue, rather than a three-residue motif found in cis-acyltransferase ketoreductases, is predictive of the orientation of the resulting β-hydroxyl group.

Structural and Functional Trends in Dehydrating Bimodules from trans-Acyltransferase Polyketide Synthases

“…The structure of PksKR3 was solved to 1.98-Å resolution by molecular replacement using PksKR2 as a search model (PDB: 4J1Q)(Piasecki et al, 2014)(Figures 2 and S2a, Table 1). Like previously structurally-characterized KRs from cis -AT PKSs (Keatinge-Clay & Stroud, 2006; Keatinge-Clay, 2007; Zheng et al, 2010; Zheng et al, 2013a, Zheng et al, 2013b, Bonnett et al, 2013) and trans -AT PKSs (Piasecki et al, 2014; Zeng et al, 2016), PksKR3 is comprised of an N-terminal structural subdomain (KR s ) and a C-terminal catalytic subdomain (KR c ), each possessing a Rossmann fold (Figure 2a).…”

Portability and Structure of the Four-Helix Bundle Docking Domains of trans-Acyltransferase Modular Polyketide Synthases

“…33,34 The structure was solved by molecular replacement with PhaserMR using another KR from a trans -AT PKS (PDB 4J1S) as the search model. 35 The solution contains one monomer per asymmetric unit. The model was refined with Coot and Refmac5 (Table 1).…”

The LINKS motif zippers trans-acyltransferase polyketide synthase assembly lines into a biosynthetic megacomplex

“…While cis -AT PKSs harbor ATs that are integrated into the multi-domain polypeptide, trans -AT systems rely on discretely-encoded AT domains that noncovalently interact with the megasynthase (Figure 1). Each of the independently-folded domains from cis -AT PKSs has been structurally characterized 4 , and although structural information has recently become available for domains from trans -AT PKSs, if and how the eponymous trans -AT domains dock to the megasynthase to charge ACPs with extender units remains to be determined 8–10 . A conserved ~100-residue region C-terminal to KS was hypothesized to facilitate the docking of trans -ATs to megasynthases and was named the AT-docking domain, or ATd 11 .…”