Structural And Enzymatic Properties Insight Into An Iterative Ketoreductase SiaM

Polyketides are important compounds in nature possessing highly diverse structures and various biological activities. They include highly-efficient antibiotics, which have great medical value including antibacterial, antifungal, and anti-parasites effects, and are considered as potential anticancer candidates. Polyketides are synthesized by large modular polyketide synthases, and each module is responsible for one round of chain extension and subsequent modification. The nascent polyketide is then passed to next module to continue the elongation process. PKSs are responsible for the synthesis of the polyketide backbone, and generally possess three functional domains:a ketosynthase (KS) domain, an acyltransferase (AT) domain and an acyl carrier protein (ACP) domain. In addition to these essential domains, most PKS modules contain one or more enzymes responsible for post-modification of β-keto carbons. The P-keto groups are reduced to P-hydroxyl groups with ketoreductases (KRs). In the biosynthesis of polyketides, the KRs are responsible for the chirality at p-carbons. Several representative structures of polyketide ketoreductases have been solved in recently years. The KR structures feature a Rossmann fold that is responsible for the binding of NADPH cofactor. Next to the N ADPH binding site, at the bottom of a cleft is the catalytic triad consisting of a tyrosine, a serine and a lysine. On the top of the active site are the lid helix and the LDD loop that regulate the transportation of substrates and products.Most KRs target substrates with a certain length. However, a recent study has shown that, in the biosynthesis of a symmetric polyketide dimer SIA7248, a trans-acting KR SiaM iteratively reduces the β-ketoacyl intermediates attached to different modules of the PKS. To illuminate catalytic mechanism of ketoreductase SiaM from Streptomyces sp. A7248and diferent binding patterns of SiaM with diferent length of substrates, the crystal structure of SiaM are reported. Structural analysis indicates that the overall structure resembles those of other KRs. However, significant disparity can be found in the conserved LDD motif. In SiaM, it is replaced with IRD motif. In addition, PISA analysis shows that SiaM forms a tetramer. Several aromatic residues are found in the tetrameric 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 essential for maintaining the structure and function.To obtain the structural models of complexes, autodock was used for docking of SiaM and NADPH, together with C4-SNAC. And the resulting models were tested for their stabilities via molecular danymics simulation by using Amber14, which show that the complexes are stable. Based on the docking structures of SiaM_NADPH and SiaM_NADPH_C4-SNAC, a series of mutants were designed. Enzymatic assay and enzymatic kinetics experiments were carried out to test the binding interection of SiaM and NADPH, as well as C4-SNAC. It is confirmed that hydrogen bonds are formed between NADPH and sidechains of Y159, K163, D67and N94, and play an important role in stabilizing NADPH. While the sidechain of S146forms inderct hydrogen bond to the adenine ring via a water molecule. And S146plays key function in electron transferring during the catalytic reaction. Two hydrophobic residues M196and198stabilize NADPH via hydrophobic interection. Also, M196and198play a crucial role in stabilizing C4-SNAC. As expected, F151and M247play an important role in stabilizing the substrate. It was inferred that M196and198together with other hydrophobic residues such as M247, V147, L191and F151form the hydrophobic binding pocket. The substrate are fastened by all these residues via hydrophobic interections. Apart from hydrophobic interections, hydrogen bonds are formed between the sidechains of Q156, Q198, R206and C4-SNAC, and are important to stabilize the substrate.In addition, it is observed the loop of SiaM from191to200sequences are highly flexible from the results of docking and molecular dynamic simulation with SiaM-ligand complexes (C6-SNAC,C8-SNAC,C10-SNAC). Consequently, the size of binding pocket formed by M196,198, V147, L191and M247, can be altered to bind substrates with different lengths.In this work, binding patterns of SiaM and NADPH together with four dirrerent substrates are illuminated, which provides the structural foundation for the enzyme engineering.