Purification and comparison of phosphoglycerate kinases from nitrifying bacteria

Phosphoglycerate kinases [PGK, EC 2.7.2.3] were purified as electrophoretic ally homogeneous proteins from four nitrifying bacteria: Nitrosomonas europ aea ATCC 25978T (Ns), Nitrosomonas sp. TNO632 (TNO), Nitrosomonas sp. K1 (K 1), Nitrobacter winogradskyi IFO 14297 (Nb) and nonsulfur bacterium Rhodops eudomonas palustris JCM2524 (Rp). The enzymes were all monomers with molecu lar masses of 44.6, 44.3, 43.7, 46.1, and 43.4 kDa, respectively. Ns-PGK an d Nb-PGK had the same pa-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 degrees C for 48 h. The temperature optima of Ns-PGK and Nb-PGK were 50 and 40 degre es 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 phosph ate donor. The N-terminal amino acid sequences of Ns-PGK and Nb-PGK were ma ximally 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 subd ivisions of the proteobacteria, respectively. The differences in the N-term inal amino acid sequences of the PGKs coincided with the taxonomic differen ces of the bacterial genera according to the molecular phylogenetic tree.