Carbohydrate Metabolism in Nitrosomonas europaea: Characterization of ADP-glucose pyrophosphorylase

M. MACHTEY; M.L. KUHN; M. ALEANZI; M.A. BALLICORA; A.A. IGLESIAS

Puerto Madryn

Congreso; XLVI Reunión Anual de SAIB; 2010

SAIB

CARBOHYDRATE METABOLISM IN Nitrosomas europaea: C H A R A C T E R I Z AT I O N O F A D P - G L U C O S E PYROPHOSPHORYLASE Machtey, M; Kuhn M , Aleanzi MC , Ballicora M , Iglesias AA 1Inst. Agrobiotecnología Litoral (UNL-CONICET), Santa Fe; 2Dept. Chemistry, Loyola Univ. Chicago, USA. E-mail: mmachtey@fbcb.unl.edu.ar Glycogen and starch serve as important energy and carbon storage compounds for nearly all living organisms. In bacteria and plants, glgCgene) aswell useUTPas rangewas displaying S values of 0.5 mM, 1 mM, and 29 mM, with similar p y g pp y y (activator). The distinctive kinetic and regulatory properties of NeuADPGlc PPase are relevant to understand metabolism of chemolitotrophes. ADP-glucose pyrophosphorylase (ADPGlc PPase, the product of the glgC gene) catalyzes the rate-limiting step of the polysaccharide biosynthetic pathway. Transcripts of the glgC and glgA (glycogen synthase) genes in N. europaea were evidenced. The recombinant ADPGlc PPase (NeuADPGlc PPase) enzyme was previously expressed in our laboratory in an active form. In the present work, the enzyme was characterized regards the influence of physicochemical variables (temperature, pH), stability as well as its ability to use different divalent metals as cofactors. Also, we determined that the enzyme can use UTP as an alternative substrate. NeuADPGlc PPase remained stable after 2 hours incubation between 0-45oC, and more than 24 hours at room temperature. Optimum pH range was between 9.5-10.0, showing high stability at alkaline pH (~9). The enzyme utilized different divalent cations 2+ 2+ 2+ with a preference order of Co > Mn > Mg ; respectively V . The apparent affinity of the enzyme for the metal increased max significantly at alkaline pH or in the presence of pyruvate.