Structural and Biochemical Studies of the Hedamycin Type II Polyketide Ketoreductase (HedKR): Molecular Basis of Stereo- and Regiospecificities

Abstract

Bacterial aromatic polyketides that include many antibiotic and antitumor therapeutics are biosynthesized by the type II polyketide synthase (PKS), which consists of 5 – 10 stand-alone enzymatic domains. Hedamycin, an antitumor antibiotic polyketide, is uniquely primed with a hexadienyl group generated by a type I PKS followed by coupling to a downstream type II PKS to biosynthesize a 24-carbon polyketide, whose C9 position is reduced by hedamycin type II ketoreductase (hedKR). HedKR is homologous to the actinorhodin KR (actKR), for which we have conducted extensive structural studies previously. How hedKR can accommodate a longer polyketide substrate than the actKR, and the molecular basis of its regio- and stereospecificities, is not well understood. Here we present a detailed study of hedKR that sheds light on its specificity. Sequence alignment of KRs predicts that hedKR is less active than actKR, with significant differences in substrate/inhibitor recognition. In vitro and in vivo assays of hedKR confirmed this hypothesis. The hedKR crystal structure further provides the molecular basis for the observed differences between hedKR and actKR in the recognition of substrates and inhibitors. Instead of the 94-PGG-96 motif observed in actKR, hedKR has the 92-NGG-94 motif, leading to S-dominant stereospecificity, whose molecular basis can be explained by the crystal structure. Together with mutations, assay results, docking simulations, and the hedKR crystal structure, a model for the observed regio- and stereospecificities is presented herein that elucidates how different type II KRs recognize substrates with different chain lengths, yet precisely reduce only the C9-carbonyl group. The molecular features of hedKR important for regio- and stereospecificities can potentially be applied to biosynthesize new polyketides via protein engineering that rationally controls polyketide ketoreduction.

Engineering polyketide synthases and nonribosomal peptide synthetases

“…Aside from inactivation studies [47], stereospecificity of reductive domains has also been altered to produce analogs with different stereochemistry [48,49]. However, a complete understanding of the molecular basis for stereocontrol is still missing [50,51]. Directed evolution approaches that use small molecule surrogates of the usually ACP-tethered intermediates [52] may be better suited to identify stereochemistry-determining residues both proximal and distal to the ketoreductase (KR) active site.…”

Insights into the programmed ketoreduction of partially reducing polyketide synthases: stereo- and substrate-specificity of the ketoreductase domain

“…[21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38] Enzymatically active KR domains are classified into A-type and B-type according to the stereochemistry of the β-hydroxyl group generated by ketoreduction. 26,[31][32][33][34][35][39][40][41] Signature motifs of the A-or B-type KR domains have been identified. However, because the signature motifs found in the KR domains of modular PKSs are usually absent in the KR domains of most PR-PKSs (Fig.…”

Reconstitution of a Type II Polyketide Synthase that Catalyzes Polyene Formation

“…Alignment of Iga13 with other KRs revealed that the canonical catalytic tetrad (Ser‐Tyr‐Lys‐Asn) of KR and the NAD(P)‐binding site are conserved in Iga13 (Figure S7). However, Iga13 lacks any motifs that have been reported to determine the stereospecificity of KRs (LDD motif in type I PKSs and PGG motif in type II PKSs) . We examined the activity of Iga13 toward 3‐oxooctanoyl‐ N ‐acetylcysteamine (NAC, 1 ; Figure a).…”