Identification of the single amino acid involved in quenching the ent-kauranyl cation by a water molecule in ent-kaurene synthase of Physcomitrella patens.
ent-Kaurene is a tetracyclic diterpene hydrocarbon and a biosynthetic intermediate of the plant hormone gibberellins. In flowering plants, ent-kaurene is biosynthesized from geranylgeranyl diphosphate (GGDP) by two distinct cyclases, ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS). Recently, the moss Physcomitrella patens ent-kaurene biosynthetic gene was cloned and functionally characterized. The bifunctional ent-kaurene synthase [P. patens CPS/KS (PpCPS/KS)] produces both ent-kaurene and 16α-hydroxy-ent-kaurane from GGDP via ent-copalyl diphosphate. Here, we cloned and analyzed the function of a cDNA encoding bifunctional ent-kaurene synthase from the liverwort Jungermannia subulata [J. subulata CPS/KS (JsCPS/KS)]. JsCPS/KS catalyzes the cyclization reaction of GGDP to produce ent-kaurene but not 16α-hydroxy-ent-kaurane, even though the PpCPS/KS (881 amino acids) and JsCPS/KS (886 amino acids) sequences share 60% identity. To determine the regions and amino acids involved in 16α-hydroxy-ent-kaurane formation, we analyzed the enzymic functions of JsCPS/KS and PpCPS/KS chimeric proteins. When the C-terminal region of PpCPS/KS was exchanged with the JsCPS/KS C-terminal region, the chimeric cyclases produced only ent-kaurene. The replacement of PpCPS/KS Ala710 with Met or Phe produced a JsCPS/KS-type cyclase that converted GGDP to ent-kaurene as the sole product. In contrast, replacing Ala710 with Gly, Cys or Ser did not affect the PpCPS/KS product profile as much as replacement of Cys of JsCPS/KS by Ala. Thus, the hydrophobicity and size of the side chain residue at the PpCPS/KS amino acid 710 is responsible for quenching the ent-kauranyl cation by the addition of a water molecule.
Institute of Agriculture, Tokyo University of Agriculture and Technology, Japan. hkawaide@cc.tuat.ac.jp
Hiroshi
H
Kawaide
Ken-ichiro
K
Hayashi
Ryo
R
Kawanabe
Yuka
Y
Sakigi
Akihiko
A
Matsuo
Masahiro
M
Natsume
Hiroshi
H
Nozaki
D019883:Alkyl and Aryl Transferases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019068:Bryopsida / Q000201:enzymology* / Q000235:genetics; D002412:Cations / Q000737:chemistry*; D008401:Gas Chromatography-Mass Spectrometry; D035926:Hepatophyta / Q000201:enzymology; D008954:Models, Biological; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D014867:Water / Q000737:chemistry*
D002412:Cations; D010940:Plant Proteins; D014867:Water; D019883:Alkyl and Aryl Transferases; C090963:ent-kaurene synthetase A