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Microorganisms 2019, 7(2), 52; https://doi.org/10.3390/microorganisms7020052

ATP- and Polyphosphate-Dependent Glucokinases from Aerobic Methanotrophs

1
Laboratory of Methylotrophy, G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Federal Research Center “Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences”, Pushchino 142290, Russia
2
Department of Microbiology and Biotechnology, Pushchino State Institute of Natural Sciences, Prospect Nauki 3, Pushchino 142290, Russia
*
Author to whom correspondence should be addressed.
Received: 26 December 2018 / Revised: 1 February 2019 / Accepted: 12 February 2019 / Published: 14 February 2019
(This article belongs to the Special Issue Recombinant Protein Expression in Microorganisms)
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Abstract

The genes encoding adenosine triphosphate (ATP)- and polyphosphate (polyP)-dependent glucokinases (Glk) were identified in the aerobic obligate methanotroph Methylomonas sp. 12. The recombinant proteins were obtained by the heterologous expression of the glk genes in Esherichia coli. ATP-Glk behaved as a multimeric protein consisting of di-, tri-, tetra-, penta- and hexamers with a subunit molecular mass of 35.5 kDa. ATP-Glk phosphorylated glucose and glucosamine using ATP (100% activity), uridine triphosphate (UTP) (85%) or guanosine triphosphate (GTP) (71%) as a phosphoryl donor and exhibited the highest activity in the presence of 5 mM Mg2+ at pH 7.5 and 65 °C but was fully inactivated after a short-term incubation at this temperature. According to a gel filtration in the presence of polyP, the polyP-dependent Glk was a dimeric protein (2 × 28 kDa). PolyP-Glk phosphorylated glucose, mannose, 2-deoxy-D-glucose, glucosamine and N-acetylglucosamine using polyP as the phosphoryl donor but not using nucleoside triphosphates. The Km values of ATP-Glk for glucose and ATP were about 78 μM, and the Km values of polyP-Glk for glucose and polyP(n=45) were 450 and 21 μM, respectively. The genomic analysis of methanotrophs showed that ATP-dependent glucokinase is present in all sequenced methanotrophs, with the exception of the genera Methylosinus and Methylocystis, whereas polyP-Glks were found in all species of the genus Methylomonas and in Methylomarinum vadi only. This work presents the first characterization of polyphosphate specific glucokinase in a methanotrophic bacterium. View Full-Text
Keywords: ATP-glucokinase; polyphosphate-glucokinase; methanotrophs; Methylomonas sp. 12 ATP-glucokinase; polyphosphate-glucokinase; methanotrophs; Methylomonas sp. 12
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Reshetnikov, A.S.; Solntseva, N.P.; Rozova, O.N.; Mustakhimov, I.I.; Trotsenko, Y.A.; Khmelenina, V.N. ATP- and Polyphosphate-Dependent Glucokinases from Aerobic Methanotrophs. Microorganisms 2019, 7, 52.

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