Poster: Salinity

Abs # 153: Functional characterization of choline monooxygenase, an enzyme for betaine synthesis in plants

Presenter:	Aoki, Kenji , c6020001@ccmailg.meijo-u.ac.jp
Authors	Aoki, Kenji  (A)   Hibino, Takashi  (A)   Ishikawa, Hiroshi  (A)   Waditee, Rungaroon  (A)   Takabe, Teruhiro  (A)
Affiliations:	(A): Meijo University

In plants, the first step in betaine synthesis was shown to be catalyzed by a novel Rieske-type iron-sulfur enzyme, choline monooxygenase (CMO). Although CMO so far has been found only in Chenopodiaceae and Amaranthaceae, the recent genome sequence suggests the presence of a CMO-like gene in Arabidopsis, a betaine non-accumulating plant. Purified CMO showed extremely low activity probably due to the labile property of the enzyme. For functional characterization of CMO, the expression in E. coli would be useful. We constructed the vectors in which the CDH gene of E. coli bet gene clusters was replaced with CMO or deleted, and used for the transformation of E. coli (DH5a) and fresh water cyanobacterium Synechococcus PCC7942, neither of which could synthesis betaine. For operation of CMO in these organisms, the Rieske-type iron-sulfur center must be assembled into the CMO polypeptide, and reduced ferredoxin is required. We found that E. coli cells in which choline dehydrogenase (CDH) was replaced with spinach CMO accumulate betaine and complement the salt-sensitive phenotype of the CDH-deleted E. coli mutant. Changes of Cys181 in spinach CMO to Ser, Thr, and Ala and His287 to Gly, Val, and Ala abolished the accumulation of betaine. The Arabidopsis CMO-like gene was transcribed in Arabidopsis, but its protein was not detected. When the Arabidopsis CMO-like gene was expressed in E. coli, the protein was detected, but was found not to promote betaine sysnthesis. Overexpression of spinach CMO in E. coli, Synechococcus, and in Arabidopsis conferred the resistance to abiotic stress. These facts clearly indicate that CMO, but not CMO-like protein, could oxidize choline and that Cys181 and His287 are involved in the binding of Fe-S cluster and Fe, respectively.a