Poster: Photosynthesis

Abs # 332: C4-form phsophoenolpyruvate carboxylase (PEPC) of Zea mays: Desensitization to feedback inhibitors by site-directed mutagenesis.

Presenter:	Mihara, Yuko , miharayu@kais.kyoto-u.ac.jp
Authors	Mihara, Yuko  (A)   Terada, Akiko  (A)   Furumoto, Tsuyoshi  (A)   Matsumura, Hiroyoshi  (B)   Kai, Yasushi  (B)   Izui, Katsura  (A)
Affiliations:	(A): Labolatory of Plant Physiology, Graduate School of Biostudies, Kyoto University (B): Department of Materials Chemistry, Graduate School of Engineering, Osaka University

PEPC catalyzes the primary CO₂-fixation reaction in C4 photosynthesis. The maize PEPC is activated by glucose 6-phosphate (G6P) and glycine, and feedback inhibited by L-malate (MA) or aspartate (Asp) in an allosteric manner. The enzyme is regulated also by reversible phosphorylation. Recent studies intending to confer C4-like metabolism to C3 plants to improve photosynthetic productivity revealed the necessity of genetically modified PEPC which exerts high activity in a "C3 environment" (Hausler et al., J. Exp. Bot. 53, 591-607 (2002)). Here we report successful desensitization to feedback inhibitors by site-directed mutagenesis. Previously we reported the 3-dimensional structures of PEPC of E. coli complexed with Asp ( Kai et al., Proc. Natl. Acad. Sci. USA, 96, 823-828 (1999)) and PEPC of Zea mays comlexed with sulfate anion at G6P binding site (Matsumura et al., Structure, 10, 1721-1730 (2002)). The former structural data indicated the involvement of 4 conserved residues for the binding of inhibitor. These residues are Arg647, Lys835, Arg894 and Asn968 in numbering of Zea mays PEPC. Recombinant mutant enzymes of K835G, R894G and K835G/K894G were prepared and characterized. Each of K835G and R894G showed a marked desensitization to MA and Asp retaining almost the same catalytic activity and sensitivities to activators as wild-type enzyme.