Characterization of NADH-insensitive phosphoribulokinase from a marine, obligately chemolithoautotrophic hydrogen- and sulfur-oxidizing bacterium Hydrogenovibrio marinus MH-110.

Phosphoribulokinase (PRK) catalyzes the ATP-dependent phosphorylation of ribulose 5-phosphate (Ru5P) to produce ribulose 1,5-bisphosphate in the Calvin-Benson-Bassham cycle. Of PRK's four classifications, class III consists of PRKs from pseudomonadota and α-cyanobacteria. They are known as octameric enzymes regulated allosterically by NADH. In this study, we characterized the PRK from a hydrogen- and sulfur-oxidizing chemolithoautotroph Hydrogenovibrio marinus MH-110 (HmPRK). Phylogenetic analysis revealed that PRKs from organisms of Piscirickettsiaceae, including H. marinus, formed a clearly separate clade within class III. HmPRK lacks most of the conserved arginine residues which are involved in regulation by NADH. Analysis of the PRK activity in the cell-free extract of H. marinus and the purified recombinant HmPRK (rHmPRK) expressed in Escherichia coli revealed the activity as unaffected by NADH. The molecular mass of rHmPRK estimated from size-exclusion chromatography was ca. 75.4 kDa, suggesting it might form a dimer. The optimum temperature and pH of rHmPRK were 30 °C and 7.4, respectively. The enzyme had apparent Km (Ru5P) of 495 ± 108 μM and apparent Km (ATP) of 526 ± 253 μM; apparent Vmax of 298 ± 22 μmol ATP consumed min-1 mg protein-1. Regarding metabolite regulation, the enzyme activity was inhibited by phosphoenolpyruvate and slightly by AMP. These findings indicate that HmPRK has unique properties distinct from those of the canonical class III PRKs.