Isolation of a glucosamine-specific kinase, a unique enzyme of Vibrio cholerae.
We showed previously that chitin catabolism by the marine bacterium Vibrio furnissii involves at least three signal transduction systems and many genes, several of which were molecularly cloned, and the corresponding proteins were characterized. The predicted amino acid sequences of these proteins showed a high degree of identity to the corresponding proteins from Vibrio cholerae, whose complete genomic sequence has recently been determined. We have therefore initiated studies with V. cholerae. We report here a novel ATP-dependent glucosamine kinase of V. cholerae encoded by a gene designated gspK. The protein, GspK (31.6 kDa), was purified to apparent homogeneity from recombinant Escherichia coli. The product of the reaction was shown to be GlcN-6-P by matrix-assisted laser desorption/ionization-time of flight (MALDI mass spectrometry) and NMR. The K(m) values for GlcN, ATP, and MgCl(2) were 0.45, 2.4, and 2.2 mm, respectively, and the V(max) values were in the range 180-200 nmol/microg/min (approximately 6 nmol/pmol/min). Kinase activity was not observed with any other sugar, including: galactosamine, mannosamine, Glc, GlcNAc, GalNAc, mannose, 2-deoxyglucose, and oligosaccharides of chitosan. The enzyme is also ATP-specific. The kinase can be used to specifically determine micro quantities of GlcN in acid hydrolysates of glycoconjugates. The physiological function of this enzyme remains to be determined.
Park
Jae Kweon
JK
Department of Biology and the McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland 21218, USA.
Wang
Lai-Xi
LX
Roseman
Saul
S
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004926:Escherichia coli; D005944:Glucosamine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010770:Phosphotransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics; D014734:Vibrio cholerae / Q000201:enzymology*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D010770:Phosphotransferases; C458524:GspK protein, Vibrio cholerae; D017853:Phosphotransferases (Alcohol Group Acceptor); D005944:Glucosamine