Choline monooxygenase, an unusual iron-sulfur enzyme catalyzing the first step of glycine betaine synthesis in plants: prosthetic group characterization and cDNA cloning.
Plants synthesize the osmoprotectant glycine betaine via the route choline --> betaine aldehyde --> glycine betaine. In spinach, the first step is catalyzed by choline monooxygenase (CMO), a ferredoxin-dependent stromal enzyme that has been hypothesized to be an oligomer of identical subunits and to be an Fe-S protein. Analysis by HPLC and matrix-assisted laser desorption ionization MS confirmed that native CMO contains only one type of subunit (Mr 42,864). Determination of acid-labile sulfur and nonheme iron demonstrated that there is one [2Fe-2S] cluster per subunit, and EPR spectral data indicated that this cluster is of the Rieske type--i.e., coordinated by two Cys and two His ligands. A full-length CMO cDNA (1,622 bp) was cloned from spinach using a probe generated by PCR amplification for which the primers were based on internal peptide sequences. The ORF encoded a 440-amino acid polypeptide that included a 60-residue transit peptide. The deduced amino acid sequence included two Cys-His pairs spaced 16 residues apart, a motif characteristic of Rieske-type Fe-S proteins. Larger regions that included this motif also showed some sequence similarity (approximately 40%) to Rieske-type proteins, particularly bacterial oxygenases. Otherwise there was very little similarity between CMO and proteins from plants or other organisms. RNA and immunoblot analyses showed that the expression of CMO in leaves increased several-fold during salinization. We conclude that CMO is a stress-inducible representative of a new class of plant oxygenases.
Rathinasabapathi
B
B
Horticultural Sciences Department, University of Florida, Gainesville 32611, USA.
Burnet
M
M
Russell
B L
BL
Gage
D A
DA
Liao
P C
PC
Nye
G J
GJ
Scott
P
P
Golbeck
J H
JH
Hanson
A D
AD
D000595:Amino Acid Sequence; D001483:Base Sequence; D001622:Betaine / Q000378:metabolism*; D002384:Catalysis; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004578:Electron Spin Resonance Spectroscopy; D007506:Iron-Sulfur Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism*; D010944:Plants / Q000378:metabolism*
D018076:DNA, Complementary; D007506:Iron-Sulfur Proteins; D001622:Betaine; C105448:choline monooxygenase; D010105:Oxygenases