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.
Highlights
Glucokinase (Glk, E.C. 2.7.1.63) catalyzes the phosphorylation of glucose into glucose 6-phosphate using nucleoside triphosphates and/or inorganic polyphosphate as a phosphoryl donor
In this paper we have demonstrated for the first time that the obligate methanotroph Methylomonas sp. 12 expresses two functional (ATP- and polyP-dependent) glucokinases
adenosine triphosphate (ATP)-dependent glucokinase is present in all sequenced methanotrophs, with the exception of the genera Methylosinus and Methylocystis, whereas polyP-Glks have been found only in methanotrophs of the genus Methylomonas, as well as in Methylomarinum vadi
Summary
Glucokinase (Glk, E.C. 2.7.1.63) catalyzes the phosphorylation of glucose into glucose 6-phosphate using nucleoside triphosphates and/or inorganic polyphosphate (polyP) as a phosphoryl donor. In Escherichia coli transporting glucose into cells via the phosphoenolpyruvate-dependent phosphotransferase system (PTS), Glk is not needed to support the growth on sugar as the deletion of the glk gene was not affected by the growth of the mutant strain [1,2]. All known methanotrophs are unable to grow on any sugars, most of them possess the Glk encoding genes [7]. ATP-dependent glucokinase (ATP-Glk) from the halotolerant methanotroph Methylomicrobium alcaliphilum 20Z has been biochemically characterized, and an essential role of the enzyme in the reutilization of free glucose derived from intracellular sucrose and glycogen has been revealed [7]. A preliminary genomic analysis showed that methanotrophs of the genus Methylomonas possess at least two glucokinase-like genes. The functioning of the glucokinase isozymes in methanotrophs has not been studied
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
