Properties of recombinant ATP-dependent fructokinase from the halotolerant methanotroph Methylomicrobium alcaliphilum 20Z.
In the cluster of genes for sucrose biosynthesis and cleavage in Methylomicrobium alcaliphilum 20Z, a gene whose encoded sequence showed high similarity to sugar kinases of the ribokinase family was found. By heterologous expression of this gene in Escherichia coli cells and following metal chelate affinity chromatography, the electrophoretically homogenous recombinant enzyme with six histidine residues on the C-end was obtained. The enzyme catalyzes ATP-dependent phosphorylation of fructose into fructose-6-phosphate but is not active with other sugars as phosphoryl acceptors. The fructokinase of M. alcaliphilum 20Z is most active in the presence of Mn(2+) at pH 9.0 and 60°C, being inhibited by ADP (K(i) = 2.50 ± 0.03 mM). The apparent K(m) values for fructose and ATP are 0.26 and 1.3 mM, respectively; the maximal activity is 141 U/mg protein. The enzyme shows the highest similarity of translated amino acid sequence with putative fructokinases of methylotrophic and autotrophic proteobacteria whose fruK gene is located in the gene cluster of sucrose biosynthesis. The involvement of fructokinase in sucrose metabolism in M. alcaliphilum 20Z and other methanotrophs and autotrophs is discussed.
Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, pr. Nauki 5, 142290 Pushchino, Moscow Region, Russia.
S Y
SY
But
O N
ON
Rozova
V N
VN
Khmelenina
A S
AS
Reshetnikov
Y A
YA
Trotsenko
D000255:Adenosine Triphosphate / Q000378:metabolism*; D052818:Autotrophic Processes; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004795:Enzyme Stability; D005631:Fructokinases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005636:Fructosephosphates / Q000378:metabolism; D007700:Kinetics; D008697:Methane / Q000378:metabolism; D008772:Methylococcaceae / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013395:Sucrose / Q000378:metabolism
D001426:Bacterial Proteins; D005636:Fructosephosphates; D011994:Recombinant Proteins; D013395:Sucrose; C027618:fructose-6-phosphate; D000255:Adenosine Triphosphate; D005631:Fructokinases; C046173:fructokinase; D008697:Methane