On the metabolism of glyceraldehyde-3-phosphate in Nitrosomonas europaea

PIATTONI CV; CORREGIDO MC; IGLESIAS AA; ASENCION DIEZ MD

Buenos Aires

Congreso; Joint Meeting of Bioscience Societies - LIII Argentine Society for Biochemical and Molecular Biology Research (SAIB) Annual meeting; 2017

ON THE METABOLISM OF GLYCERALDEHYDE-3-PHOSPHATE IN NITROSOMONAS EUROPAEAMaria Cecilia Corregido, Claudia Vanesa Piattoni, MatíasDamián Asencion Diez, Alberto Álvaro IglesiasInstituto de Agrobiotecnología del Litoral (CONICET-UNL)Nitrosomonas europaea is a chemolithoautotroph that obtainsenergy through the oxidation of ammonia to nitrite in the presenceof oxygen and fixes CO2 through the Benson-Calvin cycle (BCc).In the BCc, ATP and NADPH are utilized to reduce 3P-glycerate(3PGA) to glyceraldehyde-3-phosphate (Ga3P); this being oppositeto what occurs in glycolysis, where Ga3P is oxidized to 3PGA to produceenergetic or reductive power. In this metabolic enclave, Ga3Pdehydrogenases (Ga3PDHases) play a critical role. In plants, fourGa3PDHases were identified: GAPA/B (EC 1.2.1.13, chloroplastidic),GAPC (EC 1.2.1.12; both, plastidic or cytosolic) and GAPN (EC1.2.1.9, cytosolic). Based on sequence homology we identified inthe N. europaea genome the genes Neu0327 and Neu2000 as codingfor putative GAPC and GAPN, respectively. To further explorethe probable role of these two genes in triose-P metabolism in thebacterium we cloned and expressed them in E. coli; then we purifiedthe proteins and characterized them kinetically. Neu0327 expressionproduced a typical GAPC like Ga3PDHase, catalyzing the reversibleoxidation of Ga3P to 1,3bisP-glycerate, using specifically NAD+ ascofactor and with ~6-fold higher activity in the glycolytic directionof reaction. On the other hand, the protein product of Neu2000 irreversiblyoxidized Ga3P to 3P-glycerate using NAD+ or (preferentially,with 33-fold higher catalytic efficiency) NADP+, thus resemblinga GAPN enzyme. However, the activity of the latter with Ga3Pwas negligible when compared to that assayed using succinatesemialdehyde (SSA) as a substrate; which suggests that Neu2000codes for an SSA dehydrogenase (EC 1.2.1.16, SSADHase). Altogether,our results support a scenario for N. europaea where onlyone NAD+/NADH dependent Ga3PDHase is involved in the metabolismof triose-P (via the BCc as well glycolysis/gluconeogenesis);while the protein product of Neu2000 would preferentially act as anSSADHase with low potential to have a GAPN function.