Structural insight into the tetramerization of an iterative ketoreductase siam through aromatic residues in the interfaces.

Hua Wang,Zhixiong Xie,Yi Zou,Huaidong Zhang,Yanling Mi,Shuangjun Lin,Yunjun Yan,Houjin Zhang,Petri Kursula

doi:10.1371/journal.pone.0097996

Hua Wang, Zhixiong Xie + Show 7 more

Open Access

https://doi.org/10.1371/journal.pone.0097996

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Abstract

In the biosynthesis of polyketides, ketoreductases (KRs) are an important group of enzymes that determine the chiralities of the carbon backbones. SiaM is a special member of this group that can recognize substrates with different lengths and can be used iteratively. Here we report the crystal structure of SiaM. Structural analysis indicates that the overall structure resembles those of other KRs. However, significant disparity can be found in the conserved LDD motif that is replaced with IRD motif in SiaM. The isoleucine and aspartic acid residues take similar orientations as leucine and aspartic acid in the conserved LDD motif, while the arginine residue points out towards the solvent. PISA analysis shows that SiaM forms a tetramer. Several aromatic residues are found in the interfaces, which have aromatic stacking interactions with the aromatic residues in the neighboring protomers. Mutagenesis studies performed on the aromatic residues show that these sites are important for maintaining the structural integrity of SiaM. However, the aromatic residues contribute differently to the enzymatic activity. In the N-terminal interface, the aromatic residues can be replaced with leucine without affecting the enzymatic activity while, in the other interface, such mutations abolish the enzymatic activity.

Highlights

Many clinically important natural products are synthesized by large modular polyketide synthases (PKSs) [1,2]
A lot of research has been conducted to study their structures, functions as well as catalytic mechanisms [11,13,38]. In this SiaM structure, a water molecule is found next to the catalytic lysine (K163)
In the other crystal form of SiaM, three more water molecules are found in the active site that form a water chain (Data not shown)

Summary

Introduction

Many clinically important natural products are synthesized by large modular polyketide synthases (PKSs) [1,2]. As the 4-pro-S hydride of cofactor NADPH is commonly used in ketoreduction reactions, it was proposed that the orientations of the polyketide substrates in the active site are the determining factors for the b-hydroxyl stereochemistry [8]. The comparison of A-type and B-type KR domain structures has shown that the LDD motif and the conserved tryptophan are located at the opposite sides of the active sites [12,13]. In the closed form, the lid helix makes extensive contacts with the residues in the active site, which provides the closing power for the lid structure [13] Some ketoreductases, such as tylosin ketoreductase (TylKR1) and ketoreductase from the erythromycin synthase (EryKR1), have two subdomains. Mutagenesis studies performed on the aromatic residues show that these sites are important for maintaining the protein structure

Materials and Methods

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Discussion