Characterization and Molecular Cloning of a Novel Enzyme, Inorganic Polyphosphate/ATP-Glucomannokinase, of Arthrobacter sp. Strain KM

Abstract

A bacterium exhibiting activities of several inorganic polyphosphate [poly(P)]-and ATP-dependent kinases, including glucokinase, NAD kinase, mannokinase, and fructokinase, was isolated, determined to belong to the genus Arthrobacter, and designated Arthrobacter sp. strain KM. Among the kinases, a novel enzyme responsible for the poly(P)-and ATP-dependent mannokinase activities was purified 2,200-fold to homogeneity from a cell extract of the bacterium. The purified enzyme was a monomer with a molecular mass of 30 kDa. This enzyme phosphorylated glucose and mannose with a high affinity for glucose, utilizing poly(P) as well as ATP, and was designated poly(P)/ATP-glucomannokinase. The K m values of the enzyme for glucose, mannose, ATP, and hexametaphosphate were determined to be 0.50, 15, 0.20, and 0.02 mM, respectively. The catalytic sites for poly(P)-dependent phosphorylation and ATP-dependent phosphorylation of the enzyme were found to be shared, and the poly(P)-utilizing mechanism of the enzyme was shown to be nonprocessive. The gene encoding the poly(P)/ATP-glucomannokinase was cloned from Arthrobacter sp. strain KM, and its nucleotide sequence was determined. This gene contained an open reading frame consisting of 804 bp coding for a putative polypeptide with a calculated molecular mass of 29,480 Da. The deduced amino acid sequence of the polypeptide exhibited homology to the amino acid sequences of the poly(P)/ATP-glucokinase of Mycobacterium tuberculosis H37Rv (level of homology, 45%), ATP-dependent glucokinases of Corynebacterium glutamicum (45%), Renibacterium salmoninarum (45%), and Bacillus subtilis (35%), and proteins of bacteria belonging to the order Actinomyces whose functions are not known. Alignment of these homologous proteins revealed seven conserved regions. The mannose and poly(P) binding sites of poly(P)/ATP-glucomannokinase are discussed.Inorganic polyphosphate [poly(P)] is an energy-and phosphorus-rich biopolymer that is present in a variety of organisms. The energy contained in phosphodiester bonds in poly(P) is thermodynamically equivalent to the energy of ATP and can be utilized indirectly and directly for the phosphorylation of cellular molecules (13). The presence of various poly(P)-dependent enzymes responsible for the phosphorylation reaction in microbes has been reported previously. These enzymes include poly(P) kinase, glucokinase, NAD kinase, AMP phosphotransferase (3), and 1,3-diphosphoglycerate phosphotransferase (14).Among these enzymes, poly(P) kinase, which is found only in prokaryotic cells and catalyzes the formation of poly(P) through addition of the terminal phosphate of ATP to a growing poly(P) chain, has been studied extensively and has been well characterized (13). In addition to poly(P) kinase, poly(P)/ ATP-glucokinase and poly(P)/ATP-NAD kinase have also been well studied and have been shown to catalyze the phosphorylation of glucose and NAD, respectively, by use of poly(P) and ATP. Poly(P)/ATP-glucokinase was first found by Szymona and Ostrowski in Mycobacterium ph...

Crystal Structures ofEscherichia coliATP-Dependent Glucokinase and Its Complex with Glucose

“…3). A similar motif [X 2 -D-(I/L/V)-G-G-(S/T-X 3 ); conserved Asp and Gly residues are in boldface] is conserved as well in group III (ROK) glucokinases (30,45) and is equivalent to a portion of the phosphate 1 motif identified originally by Bork et al (13). The two conserved Gly residues contribute to formation of the loop and could form main chain hydrogen bonds with the ATP phosphates.…”

“…The ATP/ polyphosphate glucokinase from Mycobacterium tuberculosis (30) and glucomannokinase from Anthrobacter sp. strain KM (45) as well as the strictly polyphosphate-dependent GlK from Microlunatus phosphovorus (66) are also members of this group.…”

Crystal Structure of Bacterial Inorganic Polyphosphate/ATP-glucomannokinase

“…Note that poly(P)-GK is poly(P)-specific, although poly(P)/ATPtype enzymes use poly(P) and ATP. Although the primary structures of these poly(P)-dependent kinases were determined (1)(2)(3)(4), the lack of a crystal structure has prevented us from clarifying the poly(P)-utilizing mechanism of these unique kinases. Poly(P)-dependent kinases have ATP-specific partners, and a knowledge of the crystal structure of poly(P)-dependent kinase may aid in understanding the structural relationship of poly(P)-dependent kinase to the ubiquitous ATP-specific kinase, i.e.…”

“…strain KM, is a monomer with 30 kDa that phosphorylates glucose and mannose with preference for glucose through the use of poly(P) and ATP (3). The primary structure of GMK shows high homology with those of poly(P)-and poly(P)/ATP-GKs and other Gram-positive bacterial GKs but little with those of Gram-negative bacterial GKs and eucaryotic hexokinases (HKs), except for a few conserved motifs (3,6). HK shows a broad specificity for hexose, whereas GK has high specificity for glucose (7,8).…”

Structure and Function of NAD Kinase and NADP Phosphatase: Key Enzymes That Regulate the Intracellular Balance of NAD(H) and NADP(H)

“…strain KM. 51) An enzyme that phosphorylates glucose and mannose using poly(P) and ATP was purified from this bacterium. It was designated poly(P)/ATP-glucomannokinase (GMK), and on the basis of its primary structure, it was found to belong to the gram-positive bacterial glucokinase family.…”