Purification and comparison of phosphoglycerate kinases from nitrifying bacteria

Abstract

Phosphoglycerate kinases [PGK, EC 2.7.2.3] were purified as electrophoretically homogeneous proteins from four nitrifying bacteria: Nitrosomonas europaea ATCC 25978T (Ns), Nitrosomonas sp. TNO632 (TNO), Nitrosomonas sp. K1 (K1), Nitrobacter winogradskyi IFO 14297 (Nb) and nonsulfur bacterium Rhodopseudomonas palustris JCM2524 (Rp). The enzymes were all monomers with molecular masses of 44.6, 44.3, 43.7, 46.1, and 43.4 kDa, respectively. Ns-PGK and Nb-PGK had the same pH-activity curves with an optimum pH range of 8.0–8.5. The enzymes were stable in the pH range 7.0–9.0 when kept at 4°C for 48 h. The temperature optima of Ns-PGK and Nb-PGK were 50 and 40°C, respectively. Both enzymes were strongly inhibited by pCMB and SDS at 1 mM, ATP was effective, while other nucleotides did not serve as a phosphate donor. The N-terminal amino acid sequences of Ns-PGK and Nb-PGK were maximally homologous (90–95%) with TNO-PGK and Rp-PGK, respectively. However, the degree of PGK homology was as low as 45–59% between the two nitrifying bacteria genera. As previously observed, all Nitrosomonas and Nitrobacter strains constitute a monophyletic assemblage within the beta and alpha subdivisions of the proteobacteria, respectively. The differences in the N-terminal amino acid sequences of the PGKs coincided with the taxonomic differences of the bacterial genera according to the molecular phylogenetic tree.