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Phosphofructokinases from extremely thermophilic microorganisms

The distribution of phosphofructokinase (PFK) phosphoryl donor subtypes [ATP-, ADP- and pyrophosphate (PPᵢ)] in the deeply rooted phylogenetic lineages of thermophiles is of interest with respect to the evolution of PFK activity and of the Embden-Meyerhof (EM) pathway. To gain additional insight into the understanding of this key enzyme in the central metabolism within the three domains of life, PFKs with different phosphoryl donor specificities were studied from some extremely thermophilic bacteria, archaea and non-thermophilic bacteria. Results from a survey of species from the order Spirochaetales showed that all of the tested species of Spirochaeta, both thermophilic and mesophilic species/strains, possessed a PPᵢ-dependent PFK (PPᵢ-PFK) activity. However, ATP dependent-PFK activities were found to be predominant in some strains of Leptospira and Treponema. Overall, the results suggest that the presence of a PPᵢ-PFK might be a reliable phenotypic marker for the genus Spirochaeta and that there are some potentially interesting differences in how the catabolism of saccharides is controlled among members of genera within the Spirochaetales. The PPᵢ-PFK from an extremely thermophilic bacterium Dictyoglomus thermophilum Rt46 B.1 has been purified and characterised. Biochemical studies with the Dictyoglomus native enzyme showed that this enzyme possesses some properties that are similar to other bacterial PPᵢ-PFKs. The enzyme is homodimeric, non-allosteric and possesses an acidic pH optimum for the forward reaction and a neutral to slightly alkaline optimum for the reverse reaction. The enzyme requires Mg²⁺ for optimal activity and has significant activity with tripolyphosphate (PPPᵢ) and polyphosphate (polyP, n=15±3). The Dictyoglomus enzyme is extremely sensitive to Cu²⁺. The Dictyoglomus PPᵢ-PFK-encoding gene (pfp) was also sequenced, cloned and the enzyme expressed in Escherichia coli. The full-length sequence of the pfp gene was obtained using degenerate PCR and inverse-PCR. Sequence analysis and a phylogenetic comparison suggest that the Dictyoglomus PPᵢ-PFK represents an ancient lineage and is closely related to those PPᵢPFKѕ from the archaeal Thermoproteus tenax, bacterial Mycobacterium tuberculosis and Amycolatopsis methanolica, and the ATP-PFK from Streptomyces coelicolor, all of which belong to group III PFKѕ. A biochemical comparison between the Dictyoglomus native and recombinant enzymes demonstrated that they possessed a high degree of similarity in most of properties examined, e.g. the optimal pH values and Mg²⁺ concentration for activity, lack of allosteric response to metabolites, thermostability, most kinetic parameters and extreme sensitivity to Cu²⁺. An ATP-PFK from the hyperthermophilic crenarchaeon Desulfurococcus amylolyticus was also purified to homogeneity and characterised. The enzyme was confirmed as an ATP-dependent enzyme possessing no activity with either ADP or PPᵢ. The Desulfarococcus enzyme was not significantly affected by traditional allosteric modulators, e.g. phosphoenolpyruvate (PEP), citrate, succinate or fructose- 2,6-bisphosphate (F-2,6-P₂). This enzyme similar to what was found for the Dictyoglomus PFK is also extremely sensitive to Cu²⁺. Two genes from the hyperthermophilic bacterium Thermotoga maritima, one encoding a PPᵢ- PFK and the other an ATP-PFK, were cloned, expressed and characterised. Both enzymes were shown to be extremely thermostable, activated by KC1 and strongly inhibited by Cu²⁺ and Zn²⁺. The PPᵢ-PFK enzyme is indicated to be a non-allosteric homodimer which catalyses a near-reversible reaction. Significantly, the apparent Kₘ values for the phosphoryl donors PPᵢ, PPPᵢ and polyP (n=15±3) for the forward reaction were 67 μM, 10 μM and 3.8 μM respectively, and thus the enzyme might operate in vivo as a polyP-dependent PFK. The ATP-PFK exhibited significant activity with other nucleotide triphosphates, GTP (42% of control activity with ATP), UTP (14%), CTP (13%) and TTP (10%), but its activity was not significantly affected by common allosteric effectors, i.e. PEP, although it was partially inhibited by citrate. Surprisingly, the ATP-PFK was strongly inhibited by PPᵢ, PPPᵢ and polyP. The inhibition by PPᵢ could be partially relieved by nucleotide diphosphates including ADP, GDP, TDP and UDP. The T. maritima ATP-PFK is thus likely to be modulated by PPᵢ and/or polyP which would represent a novel mechanism for controlling glycolytic flux. Taken together, the data suggests that the distribution, subtypes and regulatory mechanisms of PFK in the control of the EM pathway in extremely thermophilic organisms are much more complex than we expected. As originally intended, the results from this work has provided additional insights into the understanding of the evolution of this key enzyme in glycolysis.
Type of thesis
The University of Waikato
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