Catalytic and molecular properties of the quinohemoprotein tetrahydrofurfuryl alcohol dehydrogenase from Ralstonia eutropha strain Bo.
The quinohemoprotein tetrahydrofurfuryl alcohol dehydrogenase (THFA-DH) from Ralstonia eutropha strain Bo was investigated for its catalytic properties. The apparent k(cat)/K(m) and K(i) values for several substrates were determined using ferricyanide as an artificial electron acceptor. The highest catalytic efficiency was obtained with n-pentanol exhibiting a k(cat)/K(m) value of 788 x 10(4) M(-1) s(-1). The enzyme showed substrate inhibition kinetics for most of the alcohols and aldehydes investigated. A stereoselective oxidation of chiral alcohols with a varying enantiomeric preference was observed. Initial rate studies using ethanol and acetaldehyde as substrates revealed that a ping-pong mechanism can be assumed for in vitro catalysis of THFA-DH. The gene encoding THFA-DH from R. eutropha strain Bo (tfaA) has been cloned and sequenced. The derived amino acid sequence showed an identity of up to 67% to the sequence of various quinoprotein and quinohemoprotein dehydrogenases. A comparison of the deduced sequence with the N-terminal amino acid sequence previously determined by Edman degradation analysis suggested the presence of a signal sequence of 27 residues. The primary structure of TfaA indicated that the protein has a tertiary structure quite similar to those of other quinoprotein dehydrogenases.
Zarnt
G
G
Institut für Mikrobiologie, Martin-Luther-Universität Halle-Wittenberg, Halle, Germany.
Schräder
T
T
Andreesen
J R
JR
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D020118:Cupriavidus necator / Q000201:enzymology* / Q000235:genetics; D005663:Furans / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D013237:Stereoisomerism
D005663:Furans; D000429:Alcohol Oxidoreductases; C019641:alcohol dehydrogenase (acceptor); C018675:tetrahydrofurfuryl alcohol