The [Ni-Fe] hydrogenase from the thermophilic bacterium Acetomicrobium flavidum.
Biochemical analysis of the soluble hydrogenase from the thermophilic organism Acetomicrobium flavidum revealed that the enzyme is an alpha 2 beta 2 tetramer, with the alpha and beta subunits having a molecular mass of 50 kDa and 25 kDa, respectively. The most important biochemical properties of the enzyme are a specific activity of 77 mumol min-1 (mg protein)-1, a Km for methylviologen of 0.2 mM, a pH optimum of 7.5 and a T50 of about 70 degrees C. In addition, the enzyme is remarkably stable to oxygen inactivation, retaining full activity after 24 h exposure to air. By using oligodeoxynucleotides designed on the basis of the N-terminal sequences of the two subunits, the corresponding genes have been isolated and sequenced. When compared to the other hydrogenases so far characterized, the A. flavidum hydrogenase appears to be a typical [Ni-Fe] enzyme. The hydrogenase was expressed in Escherichia coli at high levels in a soluble form but it was not active. The analysis of the recombinant large subunit showed that it was not post-translationally processed at its C-terminus.
Mura
G M
GM
ENIRICERCHE S.p.A. - Laboratories of Genetic Engineering and Microbiology, Via Maritano, 26 San Donato Milanese 20097 Milan, Italy.
Pedroni
P
P
ENIRICERCHE S.p.A. - Laboratories of Genetic Engineering and Microbiology, Via Maritano, 26 San Donato Milanese 20097 Milan, Italy.
Pratesi
C
C
ENIRICERCHE S.p.A. - Laboratories of Genetic Engineering and Microbiology, Via Maritano, 26 San Donato Milanese 20097 Milan, Italy.
Galli
G
G
ENIRICERCHE S.p.A. - Laboratories of Genetic Engineering and Microbiology, Via Maritano, 26 San Donato Milanese 20097 Milan, Italy.
Serbolisca
L
L
ENIRICERCHE S.p.A. - Laboratories of Genetic Engineering and Microbiology, Via Maritano, 26 San Donato Milanese 20097 Milan, Italy.
Grandi
G
G
ENIRICERCHE S.p.A. - Laboratories of Genetic Engineering and Microbiology, Via Maritano, 26 San Donato Milanese 20097 Milan, Italy.
D000595:Amino Acid Sequence; D001421:Bacteria, Anaerobic / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D006864:Hydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping
D017931:DNA Primers; D004269:DNA, Bacterial; D011994:Recombinant Proteins; C043296:nickel-iron hydrogenase; D006864:Hydrogenase