Protein‐Protein Recognition Involved in the Intermodular Transacylation Reaction in Modular Polyketide Synthase in the Biosynthesis of Vicenistatin

Abstract

The ketosynthase (KS) domain is a core domain found in modular polyketide synthases (PKSs). To maintain the polyketide biosynthetic fidelity, the KS domain must only accept an acyl group from the acyl carrier protein (ACP) domain of the immediate upstream module even when they are separated into different polypeptides. Although it was reported that both the docking domain‐based interactions and KS‐ACP compatibility are important for the interpolypeptide transacylation reaction in 6‐deoxyerythronolide B synthase, it is not clear whether these findings are broadly applied to other modular PKSs. Herein, we describe the importance of protein‐protein recognition in the intermodular transacylation between VinP1 module 3 and VinP2 module 4 in vicenistatin biosynthesis. We compared the transacylation activity and crosslinking efficiency of VinP2 KS4 against the cognate VinP1 ACP3 with the noncognate one. As a result, it appeared that VinP2 KS4 distinguishes the cognate ACP3 from other ACPs.

Docking Domain Engineering in a Modular Polyketide Synthase and its Impact on Structure and Function

“…The DD-less VEMS was able to produce venemycin, as confirmed by LC-MS analysis of the reaction mixture (Figure S3), but at just 1% of the activity of the native VEMS (2, Figure 2B). Similar effects were previously observed for other PKSs 25 , underscoring the importance of DDs in colocalizing protein subunits to increase the effective concentration of domain interaction partners within and across modules. We investigated three different interaction domains in their ability to replace the VEMS DDs.…”

Docking Domain Engineering in a Modular Polyketide Synthase and Its Impact on Structure and Function

“…The DD-less VEMS was able to produce venemycin, as confirmed by liquid chromatography−mass spectrometry (LC−MS) analysis of the reaction mixture (Figure S3), but at just 1% of the activity of the native VEMS (2, Figure 2B). Similar effects were previously observed for other PKSs, 27 underscoring the importance of DDs in colocalizing protein subunits to increase the effective concentration of domain interaction partners within and across modules. We investigated three different interaction domains in their ability to replace the VEMS DDs, i.e., DEBS-derived DDs, SYNZIPSs, and the SpyCatcher:Spy-Tag complex, each of them well understood in their properties.…”

Structure-Based Analysis of Transient Interactions between Ketosynthase-like Decarboxylase and Acyl Carrier Protein in a Loading Module of Modular Polyketide Synthase

“…In general, the interactions between the catalytic domain and ACP are transient. To analyze the transient interactions between them, crosslinking methods using pantetheinamide probes have been developed. , Several catalytic domain–ACP interaction modes have been elucidated on the basis of the structural analysis of crosslinked complexes. − We previously reported the structural analysis of crosslinked complexes using α-haloacylpantetheinamides. − Because we previously succeeded in the analysis of the KS domain–ACP interaction with Cl-acetyl pantetheinamide, in this work, we examined the crosslinking reaction between the GfsA KS Q domain and GfsA ACP L with Cl-acetyl pantetheinamide to structurally elucidate the mode of GfsA KS Q domain–GfsA ACP L interactions. For the crosslinking reaction, we first prepared crypto -GfsA ACP L , which is GfsA ACP L modified with Cl-acetyl pantetheinamide using CoA biosynthetic enzymes (CoaA, CoaD, and CoaE) and Sfp (Figures S3 and S6 and Table S1).…”

“…12−15 We previously reported the structural analysis of crosslinked complexes using α-haloacylpantetheinamides. 16−19 Because we previously succeeded in the analysis of the KS domain− ACP interaction with Cl-acetyl pantetheinamide, 20 in this work, we examined the crosslinking reaction between the GfsA KS Q domain and GfsA ACP L with Cl-acetyl pantetheinamide to structurally elucidate the mode of GfsA KS Q domain−GfsA ACP L interactions. For the crosslinking reaction, we first prepared crypto-GfsA ACP L , which is GfsA ACP L modified with Cl-acetyl pantetheinamide using CoA biosynthetic enzymes (CoaA, CoaD, and CoaE) and Sfp (Figures S3 and S6 and Table S1).…”