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. 2019 Feb 14;7(2):52.
doi: 10.3390/microorganisms7020052.

ATP- and Polyphosphate-Dependent Glucokinases from Aerobic Methanotrophs

Alexander S Reshetnikov  1 , Natalia P Solntseva  2 , Olga N Rozova  3 , Ildar I Mustakhimov  4   5 , Yuri A Trotsenko  6   7 , Valentina N Khmelenina  8
Affiliations

ATP- and Polyphosphate-Dependent Glucokinases from Aerobic Methanotrophs

Alexander S Reshetnikov et al. Microorganisms. .

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.

Keywords: ATP-glucokinase; Methylomonas sp. 12; methanotrophs; polyphosphate-glucokinase.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis of 100 μg recombinant ATP-dependent glucokinase and 20 μg polyP-dependent glucokinase from Methylomonas sp. 12. M-markers of molecular masses, kDa.
Figure 2
Figure 2
Effect of the pH (A) and the temperature (B) on the activity of ATP-dependent glucokinase from Methylomonas sp. 12.
Figure 3
Figure 3
Effect of the temperature (A) and the pH (B) on the activity of polyphosphate-dependent glucokinase from Methylomonas sp. 12.
Figure 4
Figure 4
Phylogenetic tree constructed from the amino acid sequences of various putative and characterized bacterial ATP- and polyP-dependent glucokinases. The characterized enzymes are in bold and the amino acid accession numbers are in brackets. ATP-dependent glucokinases in the Group B are marked by asterisks. The scale bar corresponds to the number of substitutions per site.
See this image and copyright information in PMC

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