Cloning and expression of the enolase gene from Desulfovibrio vulgaris (Miyazaki F).
The gene encoding an enolase from Desulfovibrio vulgaris (Miyazaki F) was cloned and overexpressed in Escherichia coli. A 2.1-kb DNA fragment, isolated from D. vulgaris (Miyazaki F) by double digestion with PstI and BamHI, contained an enolase gene (eno) and part of the methylenetetrahydrofolate dehydrogenase gene (folD). The nucleotide sequence of eno indicates that the protein monomer is composed of 434 amino acids. An expression system for eno under control of the T7 promoter was constructed in E. coli. The purified His-tagged enolase formed a homooctamer and was active in the formation of phosphoenolpyruvate (PEP) as well as in the reverse reaction, the formation of D-(+)-2-phosphoglyceric acid (2-PGA). The pH dependence and kinetic properties of the recombinant enolase from the sulfate-reducing bacterium were also studied. The amounts of eno mRNA when the bacterium was grown on glycerol or glucose were compared to that when D. vulgaris was grown on lactate.
Kitamura
Masaya
M
Department of Applied and Bioapplied Chemistry, Graduate School of Engineering, Osaka City University, Sumiyoshi-ku, Osaka 558-8585, Japan. kitamura@bioa.eng.osaka-cu.ac.jp
Takayama
Yuki
Y
Kojima
Shuichi
S
Kohno
Kyoko
K
Ogata
Hideaki
H
Higuchi
Yoshiki
Y
Inoue
Hideo
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D016969:Desulfovibrio vulgaris / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D004926:Escherichia coli / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D019344:Lactic Acid / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010751:Phosphopyruvate Hydratase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction
D012333:RNA, Messenger; D011994:Recombinant Proteins; D019344:Lactic Acid; D010751:Phosphopyruvate Hydratase; D005947:Glucose; D005990:Glycerol