Cloning and Structure-Function Analyses of Quinolone- and Acridone-producing Novel Type III Polyketide Synthases from Citrus microcarpa

Abstract

Background: Type III polyketide synthases (PKSs) synthesize various polyketide and alkaloid scaffolds. Results: QNS synthesizes quinolone as the single product, whereas ACS produces acridone as the major product. Conclusion: QNS and ACS are novel quinolone-and acridone-producing type III PKSs, respectively. Significance: Structure-function analyses of QNS and ACS provide insights into molecular bases for alkaloid biosyntheses.

Exploiting the Biosynthetic Potential of Type III Polyketide Synthases

“…The V98A substitution, hypothesized to be important for m-substituted phenyl substrate selectivity [24], was only found in R. graveolens ACS [58] but not in the other ACS and QNSs identified so far [59]. C. microcarpa ACS was found to be a highly promiscuous enzyme with a large active site cavity, being able to accept different starter CoAs to generate chalcone, benzophenone, and phloroglucinol scaffolds in addition to the production of 4-hydroxy-N-methylquinolone (29), 1,3-dihydroxy-N-methylacridone (30), and N-methyl-anthraniloyltriacetic acid lactone (31, Scheme 9).…”

“…Recently, the crystal structures of ACS (PDB ID: 3WD7) and QNS (PDB ID: 3WD8) from Citrus microcarpa were solved and wide active site entrances were found in both PKSs, which enabled the access of the bulky N-methylanthraniloyl-CoA [59]. The V98A substitution, hypothesized to be important for m-substituted phenyl substrate selectivity [24], was only found in R. graveolens ACS [58] but not in the other ACS and QNSs identified so far [59].…”

Structural Basis for the Formation of Acylalkylpyrones from Two β-Ketoacyl Units by the Fungal Type III Polyketide Synthase CsyB

“…Notably, triketide or tetraketide pyrone byproducts are not formed by the condensations of fatty acyl-CoA with two or three molecules of malonyl-CoA. Interestingly, the in vitro H 2 18 O incorporation experiment indicated that one of the two 18 O atoms is enzymatically incorporated into the product most likely by the nucleophilic cleavage of the thioester bond of the Cys-bound intermediate to generate the ␤-keto acid as the intermediate (whereas the other 18 O atom is exchangeable and spontaneously incorporated into the molecule). These results thus excluded the possibility of the ␤-ketoacyl-CoA intermediate but suggested that CsyB catalyzes the coupling of the ␤-keto acid intermediate and the enzyme-bound ␤-ketoacyl unit (Fig.…”

“…As a result, the obtained product 3b afforded parent ion peaks [M ϩ H] ϩ at m/z 253 and m/z 255 in addition to m/z 251, indicating the incorporation of two 18 O atoms into the product (Fig. 8A).…”

Tropane and Granatane Alkaloid Biosynthesis: A Systematic Analysis

“…Type III PKSs are promiscuous enzymes that have a broad tolerance for diverse substrates and are able to catalyze multiple reactions [84,85]. Type III PKSs that use cyclic nitrogen-containing substrates have been previously characterized for their roles in alkaloid production [86][87][88]. However, unlike these previous studies the predicted substrate in TA metabolism, N-methyl-∆ 1 -pyrrolinium cation (19) [81].…”