New pieces to the carbon metabolism puzzle of Nitrosomonas europaea: Kinetic characterization of glyceraldehyde-3 phosphate and succinate semialdehyde dehydrogenases

Abstract

Nitrosomonas europaea is a chemolithotroph that obtains energy through the oxidation of ammonia to hydroxylamine while assimilates atmospheric CO 2 to cover the cell carbon demands for growth. This microorganism plays a relevant role in the nitrogen biogeochemical cycle on Earth but its carbon metabolism remains poorly characterized. Based on sequence homology, we identified two genes (cbbG and gabD) coding for redox enzymes in N. europaea. Cloning and expression of the genes in Escherichia coli, allowed the production of recombinant enzymes purified to determine their biochemical properties. The protein CbbG is a glyceraldehyde-3-phosphate (Ga3P) dehydrogenase (Ga3PDHase) catalyzing the reversible oxidation of Ga3P to 1,3-bis-phospho-glycerate (1,3bisPGA), using specifically NAD + /NADH as cofactor. CbbG showed ∼6-fold higher K m value for 1,3bisPGA but ∼5-fold higher k cat for the oxidation of Ga3P. The protein GabD irreversibly oxidizes Ga3P to 3Pglycerate using NAD + or NADP + , thus resembling a non-phosphorylating Ga3PDHase. However, the enzyme showed ∼6-fold higher K m value and three orders of magnitude higher catalytic efficiency with succinate semialdehyde (SSA) and NADP + . Indeed, the GabD protein identity corresponds to an SSA dehydrogenase (SSADHase). CbbG seems to be the only Ga3PDHase present in N. europaea; which would be involved in reducing triose-P during autotrophic carbon fixation. Otherwise, in cells grown under conditions deprived of ammonia and oxygen, the enzyme could catalyze the glycolytic step of Ga3P oxidation producing NADH. As an SSADHase, GabD would physiologically act producing succinate and preferentially NADPH over NADH; thus being part of an alternative pathway of the tricarboxylic acid cycle converting α-ketoglutarate to succinate. The properties determined for these enzymes contribute to better identify metabolic steps in CO 2 assimilation, glycolysis and the tricarboxylic acid cycle in N. europaea. Results are discussed in the framework of metabolic pathways that launch biosynthetic intermediates relevant in the microorganism to develop and fulfill its role in nature.