Hiroshi Noguchi

The first plant type III polyketide synthase that catalyzes formation of aromatic heptaketide

A cDNA encoding a novel plant type III polyketide synthase (PKS) was cloned from rhubarb (Rheum palmatum). A recombinant enzyme expressed in Escherichia coli accepted acetyl‐CoA as a starter, carried out six successive condensations with malonyl‐CoA and subsequent cyclization to yield an aromatic heptaketide, aloesone. The enzyme shares 60% amino acid sequence identity with chalcone synthases (CHSs), and maintains almost identical CoA binding site and catalytic residues conserved in the CHS superfamily enzymes. Further, homology modeling predicted that the 43‐kDa protein has the same overall fold as CHS. This provides new insights into the catalytic functions of type III PKSs, and suggests further involvement in the biosynthesis of plant polyketides.

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.u2003serrata 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.u2003serrata 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.u2003serrata, this is the first demonstration of the enzymatic production of acridone by a type III polyketide synthase from a non‐Rutaceae plant. Interestingly, Hu.u2003serrata polyketide synthase 1 lacks most of the consensus active site sequences with acridone synthase from Ruta graveolens (Rutaceae).

Active site residues governing substrate selectivity and polyketide chain length in aloesone synthase

Aloesone synthase (ALS) and chalcone synthase (CHS) are plant‐specific type III poyketide synthases sharing 62% amino acid sequence identity. ALS selects acetyl‐CoA as a starter and carries out six successive condensations with malonyl‐CoA to produce a heptaketide aloesone, whereas CHS catalyses condensations of 4‐coumaroyl‐CoA with three malonyl‐CoAs to generate chalcone. In ALS, CHSs Thr197, Gly256, and Ser338, the active site residues lining the initiation/elongation cavity, are uniquely replaced with Ala, Leu, and Thr, respectively. A homology model predicted that the active site architecture of ALS combines a ‘horizontally restricting’ G256L substitution with a ‘downward expanding’ T197A replacement relative to CHS. Moreover, ALS has an additional buried pocket that extends into the ‘floor’ of the active site cavity. The steric modulation thus facilitates ALS to utilize the smaller acetyl‐CoA starter while providing adequate volume for the additional polyketide chain extensions. In fact, it was demonstrated that CHS‐like point mutations at these positions (A197T, L256G, and T338S) completely abolished the heptaketide producing activity. Instead, A197T mutant yielded a pentaketide, 2,7‐dihydroxy‐5‐methylchromone, while L256G and T338S just afforded a triketide, triacetic acid lactone. In contrast, L256G accepted 4‐coumaroyl‐CoA as starter to efficiently produce a tetraketide, 4‐coumaroyltriacetic acid lactone. These results suggested that Gly256 determines starter substrate selectivity, while Thr197 located at the entrance of the buried pocket controls polyketide chain length. Finally, Ser338 in proximity of the catalytic Cys164 guides the linear polyketide intermediate to extend into the pocket, thus leading to formation of the hepataketide in Rheum palmatum ALS.

Novel type III polyketide synthases from Aloe arborescens.

Aloeu2003arborescens 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 Escherichiau2003coli 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.u2003arborescens. Interestingly, the aloesone‐forming activity was maximum at 50u2003°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.

Enzymatic Reactions by Five Chalcone Synthase Homologs from Hop (Humulus lupulus L.)

The enzyme activities encoded in five cDNAs for chalcone synthase (CHS) homologs from hop were investigated. Only valerophenone synthase (VPS) and CHS_H1 showed both naringenin-chalcone and phlorisovalerophenone forming activity. Narigenin-chalcone production by VPS was much lower than by CHS_H1. Therefore, it is highly possible that flavonoid depends mainly on CHS_H1, while bitter acid biosynthesis depends mainly on VPS and CHS_H1.

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.

Molecular cloning, expression, and site-directed mutations of oxidosqualene cyclase from Cephalosporium caerulens

A cDNA for oxidosqualene:lanosterol cyclase (OSLC) was cloned and sequenced from the fungus Cephalosporium caerulens, that produces a steroidal antibiotic, helvolic acid. A 2280 bp open reading frame encoded an M(r) 87078 protein with 760 amino acids. The cDNA was functionally expressed in the OSLC-deficient mutant GIL77 strain of Saccharomyces cerevisiae. A truncated recombinant enzyme (Delta49N) starting from the second methionine (M50) residue was completely inactive, suggesting that ca. 30 additional hydrophilic amino acid residues at the N-terminal are essential for the folding of the enzyme. Furthermore, the active site residues, H234 and D456 (numbering in S. cerevisiae OSLC), were chosen for site-directed mutagenesis experiments; H234E, H234Y, H234F, D456E, D456N, and D456H mutants were inactive, while H234W and H234K mutants retained lanosterol-forming activity.

Crystallization and preliminary crystallographic analysis of a novel plant type III polyketide synthase that produces pentaketide chromone.

Pentaketide chromone synthase (PCS) from Aloe arborescens is a novel plant-specific type III polyketide synthase that catalyzes the formation of 5,7-dihydroxy-2-methylchromone from five molecules of malonyl-CoA. Recombinant PCS expressed in Escherichia coli was crystallized by the hanging-drop vapour-diffusion method. The crystals belonged to space group P2(1), with unit-cell parameters a = 73.2, b = 88.4, c = 70.0 A, alpha = gamma = 90.0, beta = 95.6 degrees . Diffraction data were collected to 1.6 A resolution using synchrotron radiation at BL24XU of SPring-8.