Characterization of a bifunctional HPr kinase/phosphorylase from Leuconostoc mesenteroides SY1.
The hprK gene encoding bifunctional HPrK/P (kinase/ phosphorylase) was cloned from L. mesenteroides SY1, a strain isolated from kimchi. hprK was transcribed as a monocistronic gene. His-tagged HPrH16A and HPrK/P were produced in E. coli BL21(DE3) using pET26b(+) and purified. HPrK/P phosphorylation assay with purified proteins showed that the kinase activity of HPrK/P increased at slightly acidic pHs. Divalent cations such as Mg2+ and Mn2+ and glycolytic intermediates such as fructose-1, 6-bisphosphate (FBP) and phosphoenolpyruvate (PEP) increased the kinase activity of HPrK/P, but inorganic phosphate strongly inhibited it. Kinetic studies for the kinase activity of HPrK/P showed that the apparent Km values were 0.18 and 14.57 microM for ATP and HPr, respectively. The Km value for the phosphorylase activity of HPrK/P was 14.16 microM for P-Ser-HPr (HPr phosphorylated at the serine residue).
Park
Jae-Yong
JY
Institute of Agriculture & Life Science, Gyeongsang National University,Jinju 660-701, Korea.
Lee
Kang Wook
KW
Lee
Ae Ran
AR
Jeong
Woo Ju
WJ
Chun
Jiyeon
J
Lee
Jong Hoon
JH
Kim
Jeong Hwan
JH
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D007934:Leuconostoc / Q000201:enzymology*; D008969:Molecular Sequence Data; D006005:Phosphorylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic; D014675:Vegetables / Q000382:microbiology
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D006005:Phosphorylases; C110847:HPr kinase; D017346:Protein Serine-Threonine Kinases