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.

Keywords: acyl carrier protein; biosynthesis; ketosynthases; polyketide synthases; protein-protein interactions.

Figure 1

Proposed reaction mechanism of the type I PKS KS domain.

Figure 2

Transacylation reaction between the VinP2 NDD₄KS₄ domain and VinP1 ACP₃CDD₃. A) The substrate of the VinP2 KS₄ domain. B) Tiglyl‐ACP₃CDD₃, which is a substrate mimic for the VinP2 KS₄ domain. C) The transacylation reaction between tiglyl‐VinP1 ACP₃CDD₃ and VinP2 NDD₄KS₄AT₄ S684G. D) HPLC analysis of the transacylation reaction between the VinP2 NDD₄KS₄ domain and VinP1 ACP₃CDD₃.

Figure 3

A) Crosslinking between VinP2 NDD₄KS₄AT₄ S684G and crypto‐VinP1 ACP₃CDD₃. B) SDS‐PAGE analysis of the crosslinking reaction with Cl‐acetyl pantetheinamide‐ACP₃CDD₃. Lane M: Protein Marker. Lanes 1–4: crosslinking reaction of VinP2 NDD₄KS₄AT₄ S684G at 0, 15, 120 and 360 min reaction times, respectively. Lanes 5–8: crosslinking reaction of VinP2 NDD₄KS₄AT₄ C206G/S684G at 0, 15, 120 and 360 min reaction times, respectively. C) Crosslinking reaction of VinP2 NDD₄KS₄AT₄ S684G with various crypto‐ACP proteins. Lane M: Protein Marker. Lane 1: without crypto‐ACP. Lane 2: with crypto‐ACP₃CDD₃. Lane 3: with crypto‐ACP₃. Lane 4: with crypto‐ACP₄CDD₄. Lane 5: with crypto‐ACP₆CDD₆. Lane 6: with crypto‐ACP₄CDD₃. Lane 7: with crypto‐ACP₆CDD₃. Lane 8: with crypto‐ACP₇CDD₃.