AMP-dependent phosphite dehydrogenase, a phosphorylating enzyme in dissimilatory phosphite oxidation

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Abstract

Oxidation redox potential of phosphite (E0′= −690 (HPO mV) 3 2− which ) to phosphate renders phosphite (HPO4 2−) an releases excellent electrons electron at a donor very low for microbial energy metabolism. To date, two pure cultures of strictly anaerobic bacteria have been isolated that run their energy metabolism on the basis of phosphite oxidation, the Gram-negative Desulfotignum phosphitoxidans (DSM 13687) and the Gram-positive Phosphitispora fastidiosa (DSM 112739). Here, we describe the key enzyme for dissimilatory phosphite oxidation in these bacteria. The enzyme catalyzed phosphite oxidation in the presence of adenosine monophosphate (AMP) to form adenosine diphosphate (ADP), with concomitant reduction of oxidized nicotinamide adenine dinucleotide (NAD+) to reduced nicotinamide adenine dinucleotide (NADH). The enzyme of P. fastidiosa was heterologously expressed in Escherichia coli. It has a molecular mass of 35.2 kDa and a high affinity for phosphite and NAD+. Its activity was enhanced more than 100-fold by addition of ADP-consuming adenylate kinase (myokinase) to a maximal activity between 30 and 80 mU x mg protein−1. A similar NAD-dependent enzyme oxidizing phosphite to phosphate with concomitant phosphorylation of AMP to ADP is found in D. phosphitoxidans, but this enzyme could not be heterologously expressed. Based on sequence analysis, these phosphite-oxidizing enzymes are related to nucleotide-diphosphate-sugar epimerases and indeed represent AMP-dependent phosphite dehydrogenases (ApdA). A reaction mechanism is proposed for this unusual type of substrate-level phosphorylation reaction.

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Mao, Z., Fleming, J. R., Mayans, O., Frey, J., Schleheck, D., Schink, B., & Müller, N. (2023). AMP-dependent phosphite dehydrogenase, a phosphorylating enzyme in dissimilatory phosphite oxidation. Proceedings of the National Academy of Sciences of the United States of America, 120(45). https://doi.org/10.1073/pnas.2309743120

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