Poster: Enzymology

Abs # 241: Characterization of Arabidopsis proline oxidase expressed in E. coli

Presenter:	Yoshida, Takayuki , takay@nihonkai.kanazawa-u.ac.jp
Authors	Yoshida, Takayuki  (A)   Kato, Youichi  (A)   Sakamoto, Toshio  (A)   Wada, Keishiro  (A)
Affiliations:	(A): Division of Life Science, Graduate School of Natural Science and Technology, Kanazawa University

Proline is one of the most common compatible osmolytes in water-stressed plants. A number of plants accumulate proline under stressed conditions, and proline is rapidly metabolized after relief from the stress. But the enzymes involved in the proline metabolic pathway are poorly characterized. Proline oxidase (POX) is the first enzyme in the proline metabolism, catalyzing the conversion of proline to pyrroline-5-carboxylate (P5C). The cDNA clone for POX has been isolated from Arabidopsis thaliana as ERD5 (early-responsive to dehydration), but POX protein remains to be characterized enzymatically because of difficulty to solubilize POX from plant mitochondria. In this study, the Arabidopsis POX has been expressed as an active form in E. coli cells. The POX activity was recovered in 100,000 g supernatant of the E.coli lysate expressing Arabidopsis POX cDNA . The enzyme activity was measured by the rate of production of P5C using an amino acid analyzer, and by the rate of oxygen consumption using Clark-type oxygen electrode. Oxygen consumption was accompanied with the P5C formation. The Km value for proline was 20mM, and its optimal pH was 9.0. Divalent cations such as Mg²⁺ at a concentration of around 10mM were required for the enzyme activity. Potassium cyanide inhibited the enzyme activity, but not sodium azide. These results suggest that oxygen is the electon acceptor for the oxidation of proline by POX.