Kiyofumi Wanibuchi

An acridone-producing novel multifunctional type III polyketide synthase from Huperzia serrata.

A cDNA encoding a novel plant type III polyketide synthase was cloned and sequenced from the Chinese club moss Huperzia serrata (Huperziaceae). The deduced amino acid sequence of Hu. serrata polyketide synthase 1 showed 44–66% identity to those of other chalcone synthase superfamily enzymes of plant origin. Further, phylogenetic tree analysis revealed that Hu. serrata polyketide synthase 1 groups with other nonchalcone‐producing type III polyketide synthases. Indeed, a recombinant enzyme expressed in Escherichia coli showed unusually versatile catalytic potential to produce various aromatic tetraketides, including chalcones, benzophenones, phloroglucinols, and acridones. In particular, it is remarkable that the enzyme accepted bulky starter substrates such as 4‐methoxycinnamoyl‐CoA and N‐methylanthraniloyl‐CoA, and carried out three condensations with malonyl‐CoA to produce 4‐methoxy‐2′,4′,6′‐trihydroxychalcone and 1,3‐dihydroxy‐N‐methylacridone, respectively. In contrast, regular chalcone synthase does not accept these bulky substrates, suggesting that the enzyme has a larger starter substrate‐binding pocket at the active site. Although acridone alkaloids have not been isolated from Hu. serrata, this is the first demonstration of the enzymatic production of acridone by a type III polyketide synthase from a non‐Rutaceae plant. Interestingly, Hu. serrata polyketide synthase 1 lacks most of the consensus active site sequences with acridone synthase from Ruta graveolens (Rutaceae).

Novel type III polyketide synthases from Aloe arborescens.

Aloe arborescens is a medicinal plant rich in aromatic polyketides, such as pharmaceutically important aloenin (hexaketide), aloesin (heptaketide) and barbaloin (octaketide). Three novel type III polyketide synthases (PKS3, PKS4 and PKS5) were cloned and sequenced from the aloe plant by cDNA library screening. The enzymes share 85–96% amino acid sequence identity with the previously reported pentaketide chromone synthase and octaketide synthase. Recombinant PKS4 and PKS5 expressed in Escherichia coli were functionally identical to octaketide synthase, catalyzing the sequential condensations of eight molecules of malonyl‐CoA to produce octaketides SEK4/SEK4b. As in the case of octaketide synthase, the enzymes are possibly involved in the biosynthesis of the octaketide barbaloin. On the other hand, PKS3 is a multifunctional enzyme that produces a heptaketide aloesone (i.e. the aglycone of aloesin) as a major product from seven molecules of malonyl‐CoA. In addition, PKS3 also afforded a hexaketide pyrone (i.e. the precursor of aloenin), a heptaketide 6‐(2‐acetyl‐3,5‐dihydroxybenzyl)‐4‐hydroxy‐2‐pyrone, a novel heptaketide 6‐(2‐(2,4‐dihydroxy‐6‐methylphenyl)‐2‐oxoethyl)‐4‐hydroxy‐2‐pyrone and octaketides SEK4/SEK4b. This is the first demonstration of the enzymatic formation of the precursors of the pharmaceutically important aloesin and aloenin by a wild‐type PKS obtained from A. arborescens. Interestingly, the aloesone‐forming activity was maximum at 50 °C, and the novel heptaketide pyrone was non‐enzymatically converted to aloesone. In PKS3, the active‐site residue 207, which is crucial for controlling the polyketide chain length depending on the steric bulk of the side chain, is uniquely substituted with Ala. Site‐directed mutagenesis demonstrated that the A207G mutant dominantly produced the octaketides SEK4/SEK4b, whereas the A207M mutant yielded a pentaketide 5,7‐dihydroxy‐2‐methylchromone.

Structural basis for the one-pot formation of the diarylheptanoid scaffold by curcuminoid synthase from Oryza sativa

Curcuminoid synthase (CUS) from Oryza sativa is a plant-specific type III polyketide synthase (PKS) that catalyzes the remarkable one-pot formation of the C6-C7-C6 diarylheptanoid scaffold of bisdemethoxycurcumin, by the condensation of two molecules of 4-coumaroyl-CoA and one molecule of malonyl-CoA. The crystal structure of O. sativa CUS was solved at 2.5-Å resolution, which revealed a unique, downward expanding active-site architecture, previously unidentified in the known type III PKSs. The large active-site cavity is long enough to accommodate the two C6-C3 coumaroyl units and one malonyl unit. Furthermore, the crystal structure indicated the presence of a putative nucleophilic water molecule, which forms hydrogen bond networks with Ser351-Asn142-H2O-Tyr207-Glu202, neighboring the catalytic Cys174 at the active-site center. These observations suggest that CUS employs unique catalytic machinery for the one-pot formation of the C6-C7-C6 scaffold. Thus, CUS utilizes the nucleophilic water to terminate the initial polyketide chain elongation at the diketide stage. Thioester bond cleavage of the enzyme-bound intermediate generates 4-coumaroyldiketide acid, which is then kept within the downward expanding pocket for subsequent decarboxylative condensation with the second 4-coumaroyl-CoA starter, to produce bisdemethoxycurcumin. The structure-based site-directed mutants, M265L and G274F, altered the substrate and product specificities to accept 4-hydroxyphenylpropionyl-CoA as the starter to produce tetrahydrobisdemethoxycurcumin. These findings not only provide a structural basis for the catalytic machinery of CUS but also suggest further strategies toward expanding the biosynthetic repertoire of the type III PKS enzymes.

Enzymatic Formation of Unnatural Novel Chalcone, Stilbene, and Benzophenone Scaffolds by Plant Type III Polyketide Synthase

A C(19) hexaketide stilbene and a C(21) heptaketide chalcone were synthesized by Aloe arborescens octaketide synthase (OKS), a plant-specific type III polyketide synthase (PKS). Remarkably, the C(21) chalcone-forming activity was dramatically increased in a structure-guided OKS N222G mutant that produces a C(20) decaketide SEK15 from 10 molecules of malonyl-CoA. The findings suggested further strategies for production of unnatural polyketides by combination of the precursor-directed biosynthesis and the structure-guided engineering of type III PKS.

Crystallization and preliminary crystallographic analysis of an acridone-producing novel multifunctional type III polyketide synthase from Huperzia serrata

Polyketide synthase 1 (PKS1) from Huperzia serrata is a plant-specific type III polyketide synthase that shows an unusually versatile catalytic potential, producing various aromatic tetraketides, including chalcones, benzophenones, phlorogulucinols and acridones. Recombinant H. serrata PKS1 expressed in Escherichia coli was crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to space group I222 or I2(1)2(1)2(1), with unit-cell parameters a = 73.3, b = 85.0, c = 137.7 A, alpha = beta = gamma = 90.0 degrees. Diffraction data were collected to 2.0 A resolution using synchrotron radiation at BL24XU of SPring-8.

Crystallization and preliminary crystallographic analysis of an octaketide-producing plant type III polyketide synthase

Octaketide synthase (OKS) from Aloe arborescens is a plant-specific type III polyketide synthase that produces SEK4 and SEK4b from eight molecules of malonyl-CoA. Recombinant OKS expressed in Escherichia coli was crystallized by the hanging-drop vapour-diffusion method. The crystals belonged to space group I422, with unit-cell parameters a = b = 110.2, c = 281.4 A, alpha = beta = gamma = 90.0 degrees . Diffraction data were collected to 2.6 A resolution using synchrotron radiation at BL24XU of SPring-8.

Enzymatic formation of an aromatic dodecaketide by engineered plant polyketide synthase.

Octaketide synthase (OKS) from Aloe arborescens is a plant-specific type III polyketide synthase (PKS) that catalyzes iterative condensations of eight molecules of malonyl-CoA to produce the C(16) aromatic octaketides SEK4 and SEK4b. On the basis of the crystal structures of OKS, the F66L/N222G double mutant was constructed and shown to produce an unnatural dodecaketide TW95a by sequential condensations of 12 molecules of malonyl-CoA. The C(24) naphthophenone TW95a is a product of the minimal type II PKS (whiE from Streptomyces coelicolor), and is structurally related to the C(20) decaketide benzophenone SEK15, the product of the OKS N222G point mutant. The C(24) dodecaketide naphthophenone TW95a is the first and the longest polyketide scaffold generated by a structurally simple type III PKS. A homology model predicted that the active-site cavity volume of the F66L/N222G mutant is increased to 748Å(3), from 652Å(3) of the wild-type OKS. The structure-based engineering thus greatly expanded the catalytic repertoire of the simple type III PKS to further produce larger and more complex polyketide molecules.

Crystallization and preliminary X-ray analysis of 4-coumarate:CoA ligase from Arabidopsis thaliana.

4-Coumarate:CoA ligase 2 (4CL2) from Arabidopsis thaliana catalyzes the ATP-dependent formation of the 4-coumaroyl-CoA thioester through the formation of 4-coumarate-AMP. Recombinant 4CL2 protein was expressed in Escherichia coli and crystallized by the sitting-drop vapour-diffusion method. The crystals belonged to space group P2(1), with unit-cell parameters a=91.6, b=55.5, c=124.4 Å, α=γ=90.0, β=111.1°.

Crystallization and preliminary crystallographic analysis of a plant type III polyketide synthase that produces benzalacetone

Benzalacetone synthase (BAS) from Rheum palmatum is a plant-specific type III polyketide synthase that catalyzes the one-step decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce the diketide 4-(4-hydroxyphenyl)-but-3-en-2-one. Recombinant BAS expressed in Escherichia coli was crystallized by the sitting-drop vapour-diffusion method. The crystals belong to space group P2(1), with unit-cell parameters a = 54.6, b = 89.6, c = 81.1 A, alpha = gamma = 90.0, beta = 100.5 degrees . Diffraction data were collected to 1.8 A resolution using synchrotron radiation at BL24XU of SPring-8.

Expression, purification and crystallization of a plant type III polyketide synthase that produces diarylheptanoids.

Curcuminoid synthase (CUS) from Oryza sativa is a plant-specific type III polyketide synthase that catalyzes the one-pot formation of bisdemethoxycurcumin by the condensation of two molecules of 4-coumaroyl-CoA and one molecule of malonyl-CoA. Recombinant CUS was expressed in Escherichia coli and crystallized by the sitting-drop vapour-diffusion method. The crystals belonged to space group P2(1), with unit-cell parameters a=72.7, b=97.2, c=126.2 A, alpha=gamma=90.0, beta=103.7 degrees. A diffraction data set was collected in-house to 2.5 A resolution.