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Characterization of recombinant PPi-dependent 6-phosphofructokinases from Methylosinus trichosporium OB3b and Methylobacterium nodulans ORS 2060

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Abstract

The properties of the purified recombinant PPi-dependent 6-phosphofructokinases (PPi-PFKs) from the methanotroph Methylosinus trichosporium OB3b and rhizospheric phytosymbiont Methylobacterium nodulans ORS 2060 were determined. The dependence of activities of PPi-PFK-His6-tag from Ms. trichosporium OB3b (6 × 45 kDa) and PPi-PFK from Mb. nodulans ORS 2060 (4 × 43 kDa) on the concentrations of substrates of forward and reverse reactions conformed to Michaelis-Menten kinetics. Besides fructose-6-phosphate, the enzymes also phosphorylated sedoheptulose-7-phosphate. ADP or AMP (1 mM each) inhibited activity of the Ms. trichosporium PPi-PFK but did not affect the activity of the Mb. nodulans enzyme. Preference of PPi-PFKs to fructose-1,6-bisphosphate implied a predominant function of the enzymes in hexose phosphate synthesis in these bacteria. PPi-PFKs from the methylotrophs have low similarity of translated amino acid sequences (17% identity) and belong to different phylogenetic subgroups of type II 6-phosphofructokinases. The relationship of PPi-PFKs with microaerophilic character of Ms. trichosporium OB3b and adaptation of Mb. nodulans ORS 2060 to anaerobic phase of phytosymbiosis are discussed.

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Abbreviations

ATP-PFK:

ATP-dependent 6-phosphofructokinase

FBP:

fructose-1,6-bisphosphate

F6P:

fructose-6-phosphate

IPTG:

isopropyl β-D-thiogalactopyranoside

LB:

Luria-Bertani medium

PFK:

6-phosphofructokinase

PPi-PFK:

pyrophosphate-dependent 6-phosphofructokinase

PRK:

phosphoribulokinase

RuBisCO:

ribulose-1,5-bisphosphatecarboxylase/oxygenase

RuMP:

ribulose monophosphate

SBPase:

sedoheptulose-1,7-bisphosphatase

S7P:

sedoheptulose-7-phosphate

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Authors and Affiliations

  1. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, pr. Nauki 5, 142290, Pushchino, Moscow Region, Russia

    O. N. Rozova, V. N. Khmelenina & Y. A. Trotsenko

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  1. O. N. Rozova
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  2. V. N. Khmelenina
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  3. Y. A. Trotsenko
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Correspondence to V. N. Khmelenina.

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Published in Russian in Biokhimiya, 2012, Vol. 77, No. 3, pp. 363–371.

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Rozova, O.N., Khmelenina, V.N. & Trotsenko, Y.A. Characterization of recombinant PPi-dependent 6-phosphofructokinases from Methylosinus trichosporium OB3b and Methylobacterium nodulans ORS 2060. Biochemistry Moscow 77, 288–295 (2012). https://doi.org/10.1134/S0006297912030078

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  • DOI: https://doi.org/10.1134/S0006297912030078

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