Expression, purification and crystallization of a plant polyketide cyclase fromCannabis sativa

Abstract

Plant polyketides are a structurally diverse family of natural products. In the biosynthesis of plant polyketides, the construction of the carbocyclic scaffold is a key step in diversifying the polyketide structure. Olivetolic acid cyclase (OAC) from Cannabis sativa L. is the only known plant polyketide cyclase that catalyzes the C2-C7 intramolecular aldol cyclization of linear pentyl tetra--ketide-CoA to generate olivetolic acid in the biosynthesis of cannabinoids. The enzyme is also thought to belong to the dimeric + barrel (DABB) protein family. However, because of a lack of functional analysis of other plant DABB proteins and low sequence identity with the functionally distinct bacterial DABB proteins, the catalytic mechanism of OAC has remained unclear. To clarify the intimate catalytic mechanism of OAC, the enzyme was overexpressed in Escherichia coli and crystallized using the vapour-diffusion method. The crystals diffracted X-rays to 1.40 Å resolution and belonged to space group P3 1 21 or P3 2 21, with unit-cell parameters a = b = 47.3, c = 176.0 Å . Further crystallographic analysis will provide valuable insights into the structurefunction relationship and catalytic mechanism of OAC.

Structural basis for olivetolic acid formation by a polyketide cyclase from Cannabis sativa

“…For the expression of SeMet labeled OAC, 25 mg·L −1 selenomethionine, 100 mg·L −1 each of l ‐Lys, l ‐Thr, l ‐Ile, l ‐Leu, l ‐Val and l ‐Phe and 1.0 m m IPTG was then added, and the cells were cultured further for 24 h at 17 °C. The SeMet labeled OAC was purified using the same procedure as that for the non‐labeled recombinant OAC .…”

“…All crystallization experiments were performed using the sitting‐drop vapor diffusion technique, with incubation at 5 °C. Diffraction quality SeMet labeled and native wild‐type OAC apo crystals were obtained as previously reported . Crystals of the OAC–OA complex were obtained under conditions with 100 m m Tris/HCl (pH 8.8) and 25% (w/v) PEG6000 by co‐incubating 25 mg·mL −1 OAC with 50 m m OA.…”

“…D) . The enzyme is a homodimeric protein, with each subunit consisting of 101 amino acid residues , and shares 30% – 50% identity with the other plant DABB superfamily proteins, such as the structurally characterized and functionally uncharacterized heat stable protein (AtHS1) from Arabidopsis thaliana , boiling stable protein (SP1) from Populus tremula and At5g22580 from A. thaliana . In contrast, the enzyme exhibits low identity (< 20%) with the bacterial DABB proteins (Fig.…”

A Novel Class of Plant Type III Polyketide Synthase Involved in Orsellinic Acid Biosynthesis from Rhododendron dauricum

“…The recombinant C. sativa OAC was bacterially expressed and purified as a homogeneous protein with a molecular mass of ∼12 kDa, as described previously ( Yang et al, 2015 ).…”

How structural subtleties lead to molecular diversity for the type III polyketide synthases

“…The OAC crystal struc-tures revealed a unique active-site architecture with a 9-Å-long pentyl-binding pocket, which is a narrow hydrophobic pocket in the interior of each monomer (Fig. 10, B and C) that binds the pentyl moiety of the olivetolic acid (77,78). Furthermore, His 78 , which forms a hydrogen network with Tyr 72 , protrudes toward the C1 and C6 positions of the olivetolic acid molecule, thus facilitating the C2-C7 aldol-type cyclization of the substrate.…”