Structure of the Cannabis sativa olivetol-producing enzyme reveals cyclization plasticity in Type III polyketide synthases.

FEBS J. 2019 Oct 12;:

Authors: Kearsey LJ, Prandi N, Karuppiah V, Yan C, Leys D, Toogood H, Takano E, Scrutton NS

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 (linear tetraketide intermediate production in two stages) and olivetolic acid cyclase (final C2→C7 aldol condensation). In the absence of olivetolic acid cyclase, a non-enzymatic C2→C7 decarboxylative aldol condensation of the tetraketide intermediate occurs forming olivetol. Tetraketide synthase 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 tetraketide synthase: coenzyme A 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 cyclisation, thereby enabling subsequent olivetolic acid production by olivetolic acid cyclase. 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.

PMID: 31605668 [PubMed – as supplied by publisher]

Source: ncbi 2

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