Crystal structure of the condensation domain from lovastatin polyketide synthase

Abstract

The highly reducing iterative polyketide synthases responsible for lovastatin biosynthesis contains a section homologous to condensation (CON) domain observed in nonribosomal peptide synthetases (NRPSs). In the present study, we expressed the isolated lovastatin CON domain and solved the crystal structure to 1.79 Å resolution. The overall structure shows similarity to canonical condensation domains of NRPSs, containing the N-terminal and C-terminal subdomains that resemble enzymes of chloramphenicol acetyltransferase family, whereas distinct structural features are observed at the active site. The acceptor entry of the substrate channel is blocked by a flexible loop, thereby preventing the loading of substrate for a new round of chain elongation. The mutation of conserved catalytic motif located at the midpoint of substrate channel agrees with the incapability of CON to catalyzed amide-bond formation. The structure helps to understand the function of CON in lovastatin biosynthesis.

Fig. 1

Synthesis of dihydromonacolin L (DML) catalyzed by lovastatin nonaketide synthase (LNKS). (A) Domain organization of LNKS. A trans-acting enoylreductase is required for DML synthesis. (B) The Diels-Alder reaction involved in DML synthesis.

Fig. 2

Structure of CON domain. (A) The V-shaped fold of CON. Two bridge regions are colored grey. The N-terminal and C-terminal residues and the G233 related to swiveling motion are shown as spheres. (B) Interactions stabilizing the floor loop plugged into the N-terminal subdomain.

Fig. 3

Active site of CON. (A) Secondary structure alignment of CON and its homologues shows their similarity. Orange: CON. Yellow: Srf A-C condensation domain (2VSQ). Marine: the C_T domain (5DIJ). Green: the X domain (4TX2). Conserved residues of the HHxxxD motif are shown as sticks in color magenta. The first histidine is replaced by serine in 5DIJ, and the second histidine is replaced by arginine in CON and 4TX2. (B) The substrate channel between two subdomains. The floor loop at the donor entry and the helix α1 at the acceptor entry are colored grey and cyan respectively. The loop blocking the acceptor entry is colored yellow. Residues R188 and D192 corresponding to conserved catalytic motif of condensation domain are shown as sticks. (C) The sequence alignment showing the conserved HHxxxD motif of condensation domains. The second histidine is replaced by arginine in CON and X domain (4TX2). 1L5A, 5DU9, 5U89, 4ZXI, 5T3D, 2JGP and 2VSQ are condensation domains. 5T3E and 5T81 are heterocyclization domain. 5DIJ is a condensation-like domain (C_T) for macrocyclization. 5ISX and 2XHG are epimerization domains. 4TX2 is an X-domain for recruiting another enzyme.

Fig. 4

Structural comparison of CON, the Srf A-C condensation domain, the C_T domain, and the X domain. They all show a similar V shape fold with a substrate channel running through the protein. The surface structural comparison of CON (orange), the Srf A-C condensation domain (2VSQ, yellow), the C_T domain (5DIJ, marine), and the X domain (4TX2, green) shows that conserved resides of HHxxxD motif (white) locate at the midpoint of the substrate channel.

Fig. 5

Possible binding model for the cyclization product to the CON domain obtained from AutoDock Vina. (A) The CON active channel has enough room to accommodate the cyclized product (cyan), residues of the conserved motif is displayed as green sticks. (B) Surface diagram depicting the simulated docking of cyclization product in the CON structure. The cyclization product are shown as cyan sticks. HRxxxD motif is colored in red.