[1S03a] 06. Enzyme Engineering/Enzymology - Part 2

2:55 PM - 3:10 PM JST(5:55 AM - 6:10 AM UTC)

[1ENZ-09] Discovery of a novel ADP-dependent phosphite dehydrogenase catalyzing unusual substrate-level phosphorylation

○Gamal Nasser Abdel-Hady^1,2, Takafumi Yamanaka^1,3, Asmaa Ali Ahmed¹, Takahisa Tajima¹, Akio Kuroda¹, Ryuichi Hirota¹ (1. Unit of Biotechnology, Division of Biological and Life Sciences, Graduate School of Integrated Sciences for Life, Hiroshima University, Hiroshima (Japan), 2. Department of Genetics, Faculty of Agriculture, Minia University, Minia (Egypt), 3. Department of Agriculture Microbiology, Faculty of Agriculture, Minia University, Minia (Egypt))

Keywords:

dissimilatory phosphite-oxidizing microorganisms (DPOMs),substrate-level phosphorylation,metagenome,ATP formation

Dissimilatory phosphite oxidation (DPO), the microbial oxidation of phosphite (Pt, HPO₃²⁻) to phosphate (Pi, HPO₄²⁻), is one of the most energetically favorable chemotrophic electron-donating processes owing to the extremely low redox potential of Pt (E^o′= −690 mV). Currently, only two dissimilatory phosphite-oxidizing microorganisms (DPOMs) have been isolated that rely on Pt oxidation for their energy metabolism: the Gram-positive Phosphitispora fastidiosa and the Gram-negative Desulfotignum phosphitoxidans. In both organisms, the key enzyme responsible for this metabolism is an AMP-dependent phosphite dehydrogenase (ApdA), which phosphorylates AMP to ADP while reducing NAD⁺ to NADH. Then, the resulting two ADP molecules are converted to ATP and AMP by an auxiliary myokinase enzyme. In this study, we identified a novel ApdA homolog enzyme (designated Sh-ApdA) through metagenomic analysis of DPOMs enriched from a river sediment culture utilizing Pt as a sole electron donor. This enzyme has been heterologously expressed in Escherichia coli and purified to homogeneity. Unlike canonical ApdA, Sh-ApdA directly catalyzes the substrate-level phosphorylation of ADP, rather than AMP, producing both ATP and NADH, using Pt as both the phosphoryl and electron donors. Quantitative detection of ATP production from ADP using luciferase-coupled assays confirmed its direct ATP-generating activity. The recombinant Sh-ApdA protein exhibited maximum activity at pH 7.0. Structural modelling analysis suggested that Sh-ApdA possesses an expanded ADP-binding pocket compared with canonical ApdA, enabling relaxed ADP accommodation and facilitating critical interactions between the ADP hydroxyl group and key catalytic residues. These findings demonstrate that Sh-ApdA is a novel enzyme capable of producing both NADH and ATP from the reducing power of Pt oxidation, expanding our understanding of microbial energy metabolism and providing a novel strategy for biotechnological applications