Open AccessNucleoside‐Diphosphate Kinase from Streptomyces coelicolor
Author(s) -
Brodbeck Michel,
Rohling Annette,
Wohlleben Wolfgang,
Thompson Charles J.,
Süsstrunk Urs
Publication year - 1996
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1996.0208u.x
Subject(s) - streptomyces coelicolor , nucleoside diphosphate kinase , biochemistry , nucleoside , kinase , allosteric regulation , chemistry , nucleotide , amino acid , enzyme , protein kinase a , stereochemistry , biology , gene , mutant
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 [γ‐ 32 P]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 ( K m ) for ATP was 85 μM. The IC 50 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 truncaled C‐terminus that makes it one of the smallest NDP kinases reported in the literature.