Ent-kaurene synthase from the fungus Phaeosphaeria sp. L487. cDNA isolation, characterization, and bacterial expression of a bifunctional diterpene cyclase in fungal gibberellin biosynthesis.
ent-Kaurene is the first cyclic diterpene intermediate of gibberellin biosynthesis in both plants and fungi. In plants, ent-kaurene is synthesized from geranylgeranyl diphosphate via copalyl diphosphate in a two-step cyclization catalyzed by copalyl diphosphate synthase and ent-kaurene synthase. A cell-free system of the fungus Phaeosphaeria sp. L487 converted labeled geranylgeranyl diphosphate to ent-kaurene. A cDNA fragment, which possibly encodes copalyl diphosphate synthase, was isolated by reverse transcription-polymerase chain reaction using degenerate primers based on the consensus motifs of plant enzymes. Translation of a full-length cDNA sequence isolated from the fungal cDNA library revealed an open reading frame for a 106-kDa polypeptide. The deduced amino acid sequence shared 24 and 21% identity with maize copalyl diphosphate synthase and pumpkin ent-kaurene synthase, respectively. A fusion protein produced by expression of the cDNA in Escherichia coli catalyzed the two-step cyclization of geranylgeranyl diphosphate to ent-kaurene. Amo-1618 completely inhibited the copalyl diphosphate synthase activity of the enzyme at 10(-6) M, whereas it did not inhibit the ent-kaurene synthase activity even at 10(-4) M. These results indicate that the fungus has a bifunctional diterpene cyclase that can convert geranylgeranyl diphosphate into ent-kaurene. They may be separate catalytic sites for the two cyclization reactions.
Kawaide
H
H
Laboratory for Plant Hormone Function, Frontier Research Program, The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-01, Japan.hkwaide@postman.riken.go.jp
Imai
R
R
Sassa
T
T
Kamiya
Y
Y
D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D001203:Ascomycota / Q000201:enzymology*; D001483:Base Sequence; D002474:Cell-Free System; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification; D004224:Diterpenes / Q000378:metabolism; D045786:Diterpenes, Kaurane; D008401:Gas Chromatography-Mass Spectrometry; D005875:Gibberellins / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D014166:Transferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003313:Zea mays / Q000201:enzymology
D018076:DNA, Complementary; D004224:Diterpenes; D045786:Diterpenes, Kaurane; D005875:Gibberellins; D010940:Plant Proteins; C002627:kaurene; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C090963:ent-kaurene synthetase A; C105712:ent-kaurene synthetase B