Activities of 6-phosphofructokinases and inorganic pyrophosphatase in aerobic methylotrophic bacteria

Abstract

The classical radioisotopic and enzymological studies of J.R. Quayle led to the discovery of the ribulosemonophosphate (RuMP) cycle of formaldehyde assimilation in methanotrophs and methylobacteria [1, 2]. The key reactions of the RuMP cycle catalyzed, by 3-hexulosephosphate synthase (HPS) and phospho-3hexuloisomerase, form fructose-6-phosphate from formaldehyde and ribulose-5-phosphate. It was believed that further phosphorylation of fructose-6phosphate to fructose-1,6-bisphosphate is carried out by ATP-dependent 6-phosphofructokinase (ATP-PFK), although its low activity (2‐8 mU/mg protein) in methanotrophs questioned the significance of the glycolytic pathway of phosphosugar transformation in the RuMP cycle. The discovery of the activity of pyrophosphatedependent 6-phosphofructokinase (PP i -PFK) and a high intracellular concentration of inorganic pyrophosphate in three species of methanotrophs employing different pathways of e 1 compound assimilation stimulated the study of the role of PP i and reappraisal of the concepts of the organization and regulation of the RuMP cycle in aerobic methylotrophic bacteria of various taxonomic positions [3]. Along these lines, in this work, the activities of PP i - and ATP-dependent 6-phosphofructokinases and pyrophosphatase (PP i -ase) were determined in a broad spectrum of aerobic methanotrophs and methylobacteria employing various pathways of C 1 assimilation. The work used strains of obligate methanotrophs and methylobacteria from the collection of the Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences. Cells were harvested in the early stationary phase, as described earlier [3, 4]. A 12-h Escherichia coli K12 culture grown in liquid medium with glucose was also used. Cells were separated from the medium by centrifugation, washed with 50 mM phosphate buffer (pH 7), and destroyed with an IBPM-press in a frozen state. To obtain cell-free extracts, cell homogenates were centrifuged (12000 g , 20 min) and dialyzed against the same buffer. The activities of PP i -PFK (EC 2.7.1.90), ATP-PFK (EC 2.7.1.11), and inorganic pyrophosphatase (EC 3.6.1.1) were determined as described earlier [4]. The data presented are the means of three measurements. The results of determination of the activities of PP i PFK, ATP-PFK, and PP i -ase in obligate and facultative methylobacteria are shown in the table. Since the detected activity of ATP-PFK could in fact be the activity of PP i -PFK at the expense of PP i admixtures, a preparation of yeast inorganic pyrophosphatase (0.2 U/ml) was introduced into the incubation mixture for ATPPFK determination in further experiments. Under these conditions, the ATP-PFK activity was not revealed. The highest PP i -PFK activity was revealed in type I methanotrophs, which assimilate formaldehyde via the RuMP cycle. In type II methanotrophs with the serine pathway, the activity of PP i -PFK was lower than in type I methanotrophs. The reversibility of this reaction and the necessity of the synthesis of hexose phosphates from triose phosphates in type II methanotrophs suggests the involvement of PP i -PFK in the gluconeogenesis of these bacteria. The lowest values of the PP i -PFK activity were recorded in type X methanotrophs of the genera Methylococcus and Methylocaldum , which simultaneously employ three pathways of C 1 assimilation. The activity of PP i -ase in cell-free extracts of methanotrophs was considerably lower than in cell-free extracts of most of the methylobacteria studied and E. coli K12.