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

CORREGIDO, MARÍA CECILIA; IGLESIAS, ALBERTO ÁLVARO; PIATTONI, CLAUDIA VANESA

BUENOS AIRES

Congreso; 53 th Annual Meeting Argentine Society for Biochemistry and Molecular Biology; 2019

Nitrosomonas europaea is a chemolithoautotroph that obtains energy through the oxidation of ammonia to nitrite in the presence of oxygen and fixes CO2 through the Benson-Calvin cycle (BCc). In the BCc (typical of photosynthetic organisms), ATP and NADPH are utilized to reduce 3P-glycerate (3PGA) to glyceraldehyde-3-phosphate (Ga3P); this being opposite to what occurs in glycolysis, where Ga3P is oxidized to 3PGA to produce energetic or reductive power. In this metabolic enclave, Ga3P dehydrogenases (Ga3PDHases) play a critical role. In plants, four Ga3PDHases were identified: GAPA/B (EC 1.2.1.13, chloroplastidic), GAPC (EC 1.2.1.12; both, plastidic or cytosolic) and GAPN (EC 1.2.1.9, cytosolic). Based on sequence homology we identified in the N. europaea genome the genes Neu0327 and Neu2000 as coding for putative GAPC and GAPN, respectively. To further explore the probable role of these two genes in triose-P metabolism in the bacterium we cloned and expressed them in Escherichia coli; then we purified the proteins and characterized them kinetically. Neu0327 expression produced a typical GAPC like Ga3PDHase, catalyzing the reversible oxidation of Ga3P to 1,3bisP-glycerate, using specifically NAD+ as cofactor and with ~6-fold higher activity in the glycolytic direction of reaction. On the other hand, the protein product of Neu2000 irreversibly oxidized Ga3P to 3P-glycerate using NAD+ or (preferentially, with 33-fold higher catalytic efficiency) NADP+, thus resembling a GAPN enzyme. However, the activity of the latter with Ga3P was negligible when compared to that assayed using succinate semialdehyde (SSA) as a substrate; which suggests that Neu2000 codes for an SSA dehydrogenase (EC 1.2.1.16, SSADHase). Altogether, our results support a scenario for N. europaea where only one NAD+/NADH dependent Ga3PDHase is involved in the metabolism of triose-P (via the BCc as well glycolysis/gluconeogenesis); while the protein product of Neu2000 would preferentially act as an SSADHase with low potential to have a GAPN function.