Hiroyuki Morita

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.

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.

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.