Structural Analysis of Actinorhodin Polyketide Ketoreductase:  Cofactor Binding and Substrate Specificity

Abstract

Aromatic polyketides are a class of natural products that include many pharmaceutically important aromatic compounds. Understanding the structure and function of PKS will provide clues to the molecular basis of polyketide biosynthesis specificity. Polyketide chain reduction by ketoreductase (KR) provides regio- and stereochemical diversity. Two cocrystal structures of actinorhodin polyketide ketoreductase (act KR) were solved to 2.3 A with either the cofactor NADP(+) or NADPH bound. The monomer fold is a highly conserved Rossmann fold. Subtle differences between structures of act KR and fatty acid KRs fine-tune the tetramer interface and substrate binding pocket. Comparisons of the NADP(+)- and NADPH-bound structures indicate that the alpha6-alpha7 loop region is highly flexible. The intricate proton-relay network in the active site leads to the proposed catalytic mechanism involving four waters, NADPH, and the active site tetrad Asn114-Ser144-Tyr157-Lys161. Acyl carrier protein and substrate docking models shed light on the molecular basis of KR regio- and stereoselectivity, as well as the differences between aromatic polyketide and fatty acid biosyntheses. Sequence comparison indicates that the above features are highly conserved among aromatic polyketide KRs. The structures of act KR provide an important step toward understanding aromatic PKS and will enhance our ability to design novel aromatic polyketide natural products with different reduction patterns.

Crystal structure and functional analysis of tetracenomycin ARO/CYC: Implications for cyclization specificity of aromatic polyketides

“…S2B). In the case of didomain ARO/CYCs, the first-ring cyclization has been proposed to occur before ARO/ CYC action, either in upstream KS/CLF or KR domains (15). Therefore, in the di-domain ARO/CYCs, the pocket residues likely orient the intermediate for first-ring aromatization but do not direct the first-ring cyclization.…”

Inhibition Kinetics and Emodin Cocrystal Structure of a Type II Polyketide Ketoreductase,

“…The α6-α7 region also has the highest B-factor in the actKR crystal structure. A comparison of monomers A and B in the published binary actKR-NADPH structure (14,15) or the actKR-NADP(H) -emodin ternary structures (this work) show that there is a significant difference in the loop regions between monomers A and B. In the ternary actKR-NADP-(H)-emodin complex, this difference is highlighted by the fact that clear electron density for the bent emodin is observed in monomer A but not in monomer B.…”

“…Growth conditions for the trigonal crystals (space group P3 2 21) containing actKR in complex with either NADPH or NADP + were previously reported simultaneously by our group and Hadfield et al (14,15). Crystals of actKR wild-type or mutant complexes with cofactor (NADP + or NADPH) and emodin grew within 3 days at room temperature by sitting-drop vapor diffusion in 3.8-4.8 M sodium formate (14,15).…”

“…Crystals of actKR wild-type or mutant complexes with cofactor (NADP + or NADPH) and emodin grew within 3 days at room temperature by sitting-drop vapor diffusion in 3.8-4.8 M sodium formate (14,15). Emodin was added to 10 mg/mL acktKR containing 5 mM NADP(H) to a final concentration of 250 μM, where the final concentration of DMSO was 1% (v/v).…”

Engineered Biosynthesis of a Novel Amidated Polyketide, Using the Malonamyl-Specific Initiation Module from the Oxytetracycline Polyketide Synthase

“…The cyclization regioselectivity of compound 2 was completely unanticipated, considering that C-9-reduced, acetate-primed decaketides cyclize solely between C-7 and C-12 (as observed in RM20b/c) (49). From the crystal structures of act-encoded KR (29) and act-encoded KS-CLF (25), it has been tempting to propose that the C-7/C-12 connectivity is formed within the active site of KS, prior to C-9 reduction by the KR. It is evident from the novel structural features of compound 2 that C-9 ketoreduction must take place independently of C-7/C-12 cyclization.…”