Abstract
The typical reaction catalyzed by type III polyketide synthases (PKSs) is a decarboxylative condensation between acyl-CoA (starter substrate) and malonyl-CoA (extender substrate). In contrast, curcumin synthase 1 (CURS1), which catalyzes curcumin synthesis by condensing feruloyl-CoA with a diketide-CoA, uses a β-keto acid (which is derived from diketide-CoA) as an extender substrate. Here, we determined the crystal structure of CURS1 at 2.32 Å resolution. The overall structure of CURS1 was very similar to the reported structures of type III PKSs and exhibited the αβαβα fold. However, CURS1 had a unique hydrophobic cavity in the CoA-binding tunnel. Replacement of Gly-211 with Phe greatly reduced the enzyme activity. The crystal structure of the G211F mutant (at 2.5 Å resolution) revealed that the side chain of Phe-211 occupied the hydrophobic cavity. Biochemical studies demonstrated that CURS1 catalyzes the decarboxylative condensation of a β-keto acid using a mechanism identical to that for normal decarboxylative condensation of malonyl-CoA by typical type III PKSs. Furthermore, the extender substrate specificity of CURS1 suggested that hydrophobic interaction between CURS1 and a β-keto acid may be important for CURS1 to use an extender substrate lacking the CoA moiety. From these results and a modeling study on substrate binding, we concluded that the hydrophobic cavity is responsible for the hydrophobic interaction between CURS1 and a β-keto acid, and this hydrophobic interaction enables the β-keto acid moiety to access the catalytic center of CURS1 efficiently.
Highlights
Are responsible for the syntheses of various biologically and pharmaceutically important compounds [1, 2]
The universal reactions catalyzed by type III polyketide synthases (PKSs) are as follows: (i) transfer of acyl-CoA to the catalytic Cys, resulting in an acyl-PKS complex; (ii) decarboxylation of malonyl-CoA to form an active anion; (iii) Claisen condensation of the active anion with the acyl moiety attached to the catalytic Cys to generate an acylCoA that has an additional two-carbon unit; (iv) extension of the polyketide chain by repeating reactions i–iii; and (v) cyclization of the resultant polyketide chain and release from the enzyme [1, 2]
We recently discovered a novel class of type III PKSs that uses a -keto acid as an extender substrate; this class catalyzes the decarboxylative condensation between an acyl-CoA and a -keto acid, resulting in unusual head-to-head condensation of polyketide chains (8 –11)
Summary
Materials—Escherichia coli strains JM109 and BLR(DE3), plasmid pUC19, restriction enzymes, T4 DNA ligase, Taq DNA polymerase, PrimeSTAR HS DNA polymerase, and other DNA-modifying enzymes were purchased from Takara. 3-Oxo-5-phenyl-4-pentenoic acid methyl ester was synthesized as described previously [8, 9]. Enzyme Assay—The standard reaction mixture contained 100 M feruloyl-CoA, 100 M extender substrate (cinnamoyldiketide-NAC, or 3-oxo-5-phenyl-4-pentenoic acid), 100 mM potassium phosphate buffer (pH 8.0), and 4.0 g of recombinant CURS1 protein in a total volume of 100 l. Reactions were incubated at 37 °C for 20 min (cinnamoyldiketide-NAC), 5 min (3-oxo-5-phenyl-4-pentenoic acid), and 1 h (others) before being quenched with 20 l of 6 M HCl. The products were extracted with ethyl acetate, and the organic layer was evaporated to dryness. Kinetic Analysis of CURS1 and CURS1 Mutants—The reaction mixture contained 100 mM potassium phosphate buffer (pH 7.5), 1–100 M each of feruloyl-CoA with 200 M cinnamoyldiketide-NAC or 100 M 3-oxo-5-phenyl-4-pentenoic acid, and 0.2– 4.0 g of recombinant CURS1 protein in a total volume of 100 l. After the reaction mixture without substrates was preincubated at 37 °C for 2 min, the reaction was initiated by adding the substrates and was continued for
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