Structural Basis of a Broadly Selective Acyltransferase from the Polyketide Synthase of Splenocin

Abstract

Polyketides are a large family of pharmaceutically important natural products, and the structural modification of their scaffolds is significant for drug development. Herein, we report high-resolution X-ray crystal structures of the broadly selective acyltransferase (AT) from the splenocin polyketide synthase (SpnD-AT) in the apo form and in complex with benzylmalonyl and pentynylmalonyl extender unit mimics. These structures revealed the molecular basis for the stereoselectivity and substrate specificity of SpnD-AT, and enabled the engineering of the industrially important Ery-AT6 to broaden its substrate scope to include three new types of extender units.

Structural and Biochemical Insight into the Recruitment of Acyl Carrier Protein‐Linked Extender Units in Ansamitocin Biosynthesis

“…If the serine‐histidine catalytic dyad functions as in related ATs, S180 acts as a nucleophile to attack the thioester bond of the acyl donor to form a covalent acyl‐ O ‐ester intermediate while H285 enhances the nucleophilicity of S180 by the deprotonation of the hydroxy group. A hydrogen bond is formed between the side chain hydroxy group of the catalytic serine and the imidazole of the catalytic histidine in previously reported PKS AT structures . Interestingly, the side chain hydroxy group of the catalytic S180 forms a hydrogen bond with the imidazole of H179 instead of that of the catalytic H285 (Figure S3).…”

“…The purified AsmAT3 was crystallized and the structure was solved by molecular replacement using EryAT5 (PDB ID: 2HG4) as a search model and refined to 1.79 Å resolution (Table S1). Its overall fold closely resembles those of CoA specific ATs (PDB ID: 2QO3, 1.0 Å RMSD; PDB ID: 2HG4, 0.7 Å RMSD; PDB ID: 6IYO, 1.9 Å RMSD; PDB ID: 6IYR, 1.4 Å RMSD; PDB ID: 6IYT, 1.0 Å RMSD; PDB ID: 5YDA, 2.1 Å RMSD; PDB ID: 2G2Z, 2.2 Å RMSD; PDB ID: 4AMP, 3.5 Å RMSD) and that of ZmaAT (PDB ID: 4QBU, RMSD: 2.5 Å) recruiting HM‐ACP. The AsmAT3 structure contains a KS‐AT linker, an α/β‐hydrolase subdomain and a ferredoxin‐like subdomain (Figure ).…”

“…The S preference of CoA‐specific ATs has been confirmed by biochemical assays of erythromycin PKSs . Although the C2 carbon of the MOM‐ACP is predicted to have the opposite 2 R conformation in keeping with the biosynthetic derivation from the d ‐glycolytic intermediate, the catalytic residues of AsmAT3 are oriented as observed in ATs specific for (2 S )‐MM‐CoA and (2 S )‐EM‐CoA (Figure S3) . To understand the substrate binding mode in the AsmAT3 active site, (2 RS )‐MOM‐SNAC ( N ‐acetyl cysteamine, “ S ” indicates the thioester linkage) was used as a substrate mimic in soaking experiments.…”

“…Herein, we report the crystal structures of the apo and acylated AsmAT3. The importance of a conserved tryptophan at the entrance of the substrate binding tunnel in discriminating between ACP and CoA carriers was revealed by structural comparisons of AsmAT3 with previously reported CoA specific ATs . Mutation of the single tryptophan residue altered the carrier specificity of AsmAT3 from ACP to CoA.…”

“…In the MOM‐AsmAT3 complex structure, the side chain of catalytic S180 rotates about 97° relative to the apo structure and adopts the orientation observed in previously reported ATs (Figure S3), consistent with the role of H285 as a general base to deprotonate S180 for nucleophilic attack on the carbonyl carbon of the substrate. In the FabD and SpnD‐AT complex structures, the guanidine group of the conserved arginine at the bottom of the acyl binding pocket stabilizes the C3 carboxyl group of the acylated malonate derivate through a bidentate salt bridge (Figure S10 A). In the MOM‐AsmAT3 complex structure, both the C3 carboxylate group and the conserved R205 are differently oriented.…”