title: The properties and potential metabolic role of glucokinase in halotolerant obligate methanotroph Methylomicrobium alcaliphilum 20Z.
abstract: Aerobic bacteria utilizing methane as the carbon and energy source do not use sugars as growth substrates but possess the gene coding for glucokinase (Glk), an enzyme converting glucose into glucose 6-phosphate. Here we demonstrate the functionality and properties of Glk from an obligate methanotroph Methylomicrobium alcaliphilum 20Z. The recombinant Glk obtained by heterologous expression in Escherichia coli was found to be close in biochemical properties to other prokaryotic Glks. The homodimeric enzyme (2 × 35 kDa) catalyzed ATP-dependent phosphorylation of glucose and glucosamine with nearly equal activity, being inhibited by ADP (Ki= 2.34 mM) but not affected by glucose 6-phosphate. Chromosomal deletion of the glk gene resulted in a loss of Glk activity and retardation of growth as well as in a decrease of intracellular glycogen content. Inactivation of the genes encoding sucrose phosphate synthase or amylosucrase, the enzymes involved in glycogen biosynthesis via sucrose as intermediate, did not prevent glycogen accumulation. In silico analysis revealed glk orthologs predominantly in methanotrophs harboring glycogen synthase genes. The data obtained suggested that Glk is implicated in the regulation of glycogen biosynthesis/degradation in an obligate methanotroph.
authors.affiliation: Laboratory of Methylotrophy, Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Prospect Nauki 5, Pushchino, Russia, 142290.|Pushchino State Institute of Natural Sciences, Prospect Nauki 3, Pushchino, Russia, 142290.
authors.forename: Ildar I
authors.initials: II
authors.lastname: Mustakhimov
authors.affiliation: Laboratory of Methylotrophy, Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Prospect Nauki 5, Pushchino, Russia, 142290.
authors.forename: Olga N
authors.initials: ON
authors.lastname: Rozova
authors.affiliation: Pushchino State Institute of Natural Sciences, Prospect Nauki 3, Pushchino, Russia, 142290.
authors.forename: Natalia P
authors.initials: NP
authors.lastname: Solntseva
authors.affiliation: Laboratory of Methylotrophy, Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Prospect Nauki 5, Pushchino, Russia, 142290. khmelenina@rambler.ru.
authors.forename: Valentina N
authors.initials: VN
authors.lastname: Khmelenina
authors.affiliation: Laboratory of Methylotrophy, Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Prospect Nauki 5, Pushchino, Russia, 142290.
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Reshetnikov
authors.affiliation: Laboratory of Methylotrophy, Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Prospect Nauki 5, Pushchino, Russia, 142290.|Pushchino State Institute of Natural Sciences, Prospect Nauki 3, Pushchino, Russia, 142290.
authors.forename: Yuri A
authors.initials: YA
authors.lastname: Trotsenko
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D050260:Carbohydrate Metabolism; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D005941:Glucokinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005964:Glucosyltransferases / Q000235:genetics; D006003:Glycogen / Q000096:biosynthesis; D053858:Metabolic Networks and Pathways; D008772:Methylococcaceae / Q000737:chemistry / Q000145:classification / Q000201:enzymology*; D009154:Mutation; D010766:Phosphorylation; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013395:Sucrose / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D013395:Sucrose; D006003:Glycogen; D005964:Glucosyltransferases; C022698:sucrose-phosphate synthase; D005941:Glucokinase
keywords: Glucokinase; Glycogen; Methanotrophic bacteria; Methylomicrobium alcaliphilum 20Z; Sucrose