Cloning and structure-function analyses of quinolone- and acridone-producing novel type III polyketide synthases from Citrus microcarpa

Abstract

Two novel type III polyketide synthases, quinolone synthase (QNS) and acridone synthase (ACS), were cloned from Citrus microcarpa (Rutaceae). The deduced amino acid sequence of C. microcarpa QNS is unique, and it shared only 56-60% identities with C. microcarpa ACS, Medicago sativa chalcone synthase (CHS), and the previously reported Aegle marmelos QNS. In contrast to the quinolone- and acridone-producing A. marmelos QNS, C. microcarpa QNS produces 4-hydroxy-N-methylquinolone as the "single product" by the one-step condensation of N-methylanthraniloyl-CoA and malonyl-CoA. However, C. microcarpa ACS shows broad substrate specificities and produces not only acridone and quinolone but also chalcone, benzophenone, and phloroglucinol from 4-coumaroyl-CoA, benzoyl-CoA, and hexanoyl-CoA, respectively. Furthermore, the x-ray crystal structures of C. microcarpa QNS and ACS, solved at 2.47- and 2.35-Å resolutions, respectively, revealed wide active site entrances in both enzymes. The wide active site entrances thus provide sufficient space to facilitate the binding of the bulky N-methylanthraniloyl-CoA within the catalytic centers. However, the active site cavity volume of C. microcarpa ACS (760 Å(3)) is almost as large as that of M. sativa CHS (750 Å(3)), and ACS produces acridone by employing an active site cavity and catalytic machinery similar to those of CHS. In contrast, the cavity of C. microcarpa QNS (290 Å(3)) is significantly smaller, which makes this enzyme produce the diketide quinolone. These results as well as mutagenesis analyses provided the first structural bases for the anthranilate-derived production of the quinolone and acridone alkaloid by type III polyketide synthases.

Keywords: Biosynthesis; Cloning; Enzymes; Polyketides; Structural Biology.

FIGURE 1.

Proposed mechanism for the formation of alkaloids and polyketides by type III PKSs. Enzyme reaction products from malonyl-CoA and the following: N-methylanthraniloyl-CoA (A), 4-coumaroyl-CoA (B), benzoyl-CoA (C), and hexanoyl-CoA (D). C. microcarpa QNS produces 4-hydroxy-N-methylquinolone as the sole product from N-methylanthraniloyl-CoA and triketide lactones from benzoyl-CoA and hexanoyl-CoA, whereas C. microcarpa ACS produces all of the products.

FIGURE 2.

Sequence alignment of C. microcarpa QNS/ACS with other plant type III PKSs. CmQNS, C. microcarpa QNS; CmACS, C. microcarpa ACS; AmQNS, A. marmelos QNS; RgACS, R. graveolens ACS; MsCHS, M. sativa chalcone synthase; HsPKS1, H. serrata PKS1; RpBAS, R. palmatum BAS. The catalytic triad (Cys-164, His-303, and Asn-336) is colored red, and the active site residues, 132, 133, 137, 194, 197, 211, 215, 256, 265, 338, 375, are highlighted in blue (numbering in M. sativa CHS).

FIGURE 3.

Phylogenetic tree analysis of plant and bacterial type III PKSs. Multiple sequence alignment, performed with ClustalW (1.8). The scale represents 0.1 amino acid substitutions per site. Abbreviations used are as follows: ALS, aloesone synthase; BBS, bibenzyl synthase; BIS, biphenyl synthase; BPS, benzophenone synthase; CTAS, 4-coumaroyltriacetic acid synthase; CUS, curcuminoid synthase; CURS, curcumin synthase; DCS, diketide-CoA synthase; HKS, hexaketide synthase; OKS, octaketide synthase; ORAS, 3′-oxoresorcinolic acid synthase; PCS, pentaketide chromone synthase; 2PS, 2-pyrone synthase; STS, stilbene synthase; THNS, tetrahydroxynaphthalene synthase; VPS, valerophenone synthase. The β-ketoacyl carrier protein synthase III (KAS III and FABH) enzymes of E. coli were employed as out groups.

FIGURE 4.

HPLC elution profiles of the enzyme reaction products of C. microcarpa QNS and ACS. A–D, enzyme reaction products of C. microcarpa (Cm) ACS and QNS from N-methylanthraniloyl-CoA (A), 4-coumaroyl-CoA (B), benzoyl-CoA (C), and hexanoyl-CoA, and malonyl-CoA (D). Note that by acid treatment naringenin chalcone is converted to racemic naringenin (5,7,4′-trihydroxyflavanone) through a nonstereospecific ring-C closure.

FIGURE 5.

Overall structures of C. microcarpa QNS and ACS. A, C. microcarpa QNS; B, C. microcarpa ACS. The structures are represented as schematics. The catalytic Cys-164 and the substrate entrance are represented by a CPK model and an arrow, respectively. The CoASH molecules in C. microcarpa ACS are depicted as blue stick models.

FIGURE 6.

Comparison of the active site entrances of C. microcarpa QNS/ACS and other type III PKSs. A, C. microcarpa QNS; Bl C. microcarpa ACS, and C, M. sativa CHS. Arrows indicate the substrate entrance in each structure.

FIGURE 7.

Comparison of the active site structures of C. microcarpa QNS and ACS and other type III PKSs, and their schematic representations. A, C. microcarpa QNS; B, C. microcarpa ACS; C, M. sativa CHS, and D, R. palmatum BAS. The naringenin in M. sativa CHS and the bound monoketide intermediate in R. palmatum BAS are shown as magenta and pink stick models, respectively. Arrows indicate the substrate entrance in each structure.

FIGURE 8.

HPLC elution profiles of the enzyme reaction products of C. microcarpa QNS and ACS mutants. A, enzyme reaction products of C. microcarpa QNS, wild-type, and Y197A mutant, from 4-coumaroyl-CoA. B and C, enzyme reaction products of C. microcarpa ACS, wild-type, and mutants (S132M, T194M, and T197Y), from N-methylanthraniloyl-CoA (B), and 4-coumaroyl-CoA (C). Note that by acid treatment naringenin chalcone is converted to racemic naringenin (5,7,4′-trihydroxyflavanone) through a nonstereospecific ring-C closure.