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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.

SUBMITTER: Mori T

PROVIDER: S-EPMC3789980 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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

Mori Takahiro T Shimokawa Yoshihiko Y Matsui Takashi T Kinjo Keishi K Kato Ryohei R Noguchi Hiroshi H Sugio Shigetoshi S Morita Hiroyuki H Abe Ikuro I

The Journal of biological chemistry 20130820 40

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 prod ...[more]

PMID: 23963450

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Project description:In the native pathway to therapeutic cannabinoid biosynthesis in Cannabis sativa, the three-step production of a key intermediate, olivetolic acid, is catalysed by the enzymes tetraketide synthase (TKS; linear tetraketide intermediate production in two stages) and olivetolic acid cyclase (OAC; final C2 → C7 aldol condensation). In the absence of OAC, a nonenzymatic C2 → C7 decarboxylative aldol condensation of the tetraketide intermediate occurs forming olivetol. TKS is a type III polyketide synthase, and the question arises why it is unable to form olivetolic acid directly, but instead forms this unwanted side product. We determined the TKS, CoA complex structure, and performed structurally guided mutagenesis studies to identify potential residues responsible for cyclization pathway discrimination in type III polyketide synthases. Prior studies suggested an 'aldol switch' is necessary to allow linear tetraketide intermediate release prior to cyclization, thereby enabling subsequent olivetolic acid production by OAC. However, our studies do not support the presence of a universal or predictable 'aldol switch' consensus sequence. Instead, we propose the mode of ordered active site water activation between type III polyketide synthases catalysing different cyclization mechanisms is subtle and homologue-specific. Our work indicates that subtle structural variations between homologous enzymes can have a major mechanistic impact on the catalytic outcome. This highlights the importance of embedding high-resolution structural analysis of multiple enzyme homologues with classical site-directed mutagenesis studies when investigating highly similar enzymes with different mechanistic pathway outcomes. ENZYMES: TKS, EC 2.3.1.206; OAC, EC 4.4.1.26; chalcone synthase, EC 2.3.1.74; stilbene synthase, EC 2.3.1.95; 2-PS, EC 2.3.1.-. ACCESSION NUMBERS: The atomic coordinates and structure factors for the crystal structure of TKS have been deposited in the Protein Data Bank with accession number 6GW3.

Dataset Information

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

Publications

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

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