title: Nucleoside-diphosphate kinase from Streptomyces coelicolor.
abstract: Nucleoside-diphosphate (NDP) kinase was purified from crude extracts of Streptomyces coelicolor to over 90% homogeneity in a single step using an adenosine 3',5'-cyclic monophosphate (cAMP) binding column. The specific activity of protein in the fraction eluted with cAMP (400 U/mg) was about 3600-fold higher than that in the crude extract. This enzyme was autophosphorylated in the presence of [y-32P]ATP. The high-energy phosphoenzyme intermediate was stable in alkali and highly labile in acid; this suggests the presence of an N-phosphate amino acid (most probably a histidine residue). A tetrameric form of the 15-kDa protein was suggested by its apparent molecular mass (66 kDa) on a gel filtration column. The measured Michaelis constant (Km) for ATP was 85 microM. The IC50 for cAMP of 6 mM suggested weak competitive inhibition. However, no evidence that cAMP acts as an allosteric effector was obtained. The ndk gene from S. coelicolor was isolated and sequenced. The deduced amino acid sequence was very similar to other NDP kinases. However, unique characteristics were also noted, including a truncated C-terminus that makes it one of the smallest NDP kinases reported in the literature.
authors.affiliation: Department of Microbiology, Biozentrum, University of Basel, Switzerland.
authors.forename: M
authors.initials: M
authors.lastname: Brodbeck
authors.forename: A
authors.initials: A
authors.lastname: Rohling
authors.forename: W
authors.initials: W
authors.lastname: Wohlleben
authors.forename: C J
authors.initials: CJ
authors.lastname: Thompson
authors.forename: U
authors.initials: U
authors.lastname: Süsstrunk
mesh_terms: D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009701:Nucleoside-Diphosphate Kinase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology*
chemical_list: D000242:Cyclic AMP; D009701:Nucleoside-Diphosphate Kinase