Identification and characterization of a second butyrate kinase from Clostridium acetobutylicum ATCC 824.
A gene encoding a new butyrate kinase isozyme (BKII) was identified from the C. acetobutylicum ATCC 824 DNA database. The enzyme was expressed in Escherichia coli, purified, and characterized. The purified enzyme exhibited a subunit molecular mass of 43 kDa by SDS-PAGE, and a native molecular mass of 80 kDa by gel filtration suggesting it functions as a dimer. In the butyryl phosphate-forming direction the optimal pH of BKII was 8.5. The enzyme had a Km of 0.62 M and a turn over rate of 2.2 x 10(5)/sec (Vmax of 165 units/mg). The presence of a mRNA encoding the BKII was demonstrated using a reverse transcription PCR reaction. The expression of the BKII in Clostridium acetobutylicum ATCC 824 was further examined by Western blot analysis using a polyclonal antibody prepared against recombinant BKII.
Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77005-1892, USA.
K X
KX
Huang
S
S
Huang
F B
FB
Rudolph
G N
GN
Bennett
D000595:Amino Acid Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid
D012333:RNA, Messenger; D017851:Phosphotransferases (Carboxyl Group Acceptor); C019285:butyrate kinase