Enzymatic characterization of recombinant mouse retinal dehydrogenase type 1.
Retinal dehydrogenases (RALDHs) convert retinal into retinoic acids (RAs), which are important signaling molecules in embryogenesis and tissue differentiation. We expressed mouse RALDH type 1 (mRALDH1) in Escherichia coli and studied the kinetic properties of the recombinant enzyme for retinal substrates. Purified recombinant mRALDH1 catalyzed the oxidation of all-trans and 9-cis retinal but not 13-cis retinal, and exhibited two pH optimums, 7.8 and 9.4, for all-trans and 9-cis retinal substrates, respectively. The K(m) for all-trans retinal (11.6 micro M) was 3-fold higher than for 9-cis retinal (3.59 micro M). However, the conversion efficiencies of either all-trans or 9-cis retinal to the respective RAs were similar. MgCl(2) inhibited the oxidation of both all-trans and 9-cis retinal. Chloral hydrate and acetaldehyde competitively suppressed all-trans retinal oxidation with inhibition constants (K(i)) of 4.99 and 49.4 micro M, respectively. Retinol, on the other hand, blocked the reaction uncompetitively. These data extend the kinetic characterization of mRALDH1, provide insight into the possible role of this enzyme in the biogenesis of RAs, and should give useful information on the determination of amino acid residues that play crucial roles in the catalysis of all-trans and 9-cis retinal.
Gagnon
Isabelle
I
Laboratory of Nutrition and Cancer, Research Centre, Centre hospitalier de l'Universite de Montreal-Hotel-Dieu, 3850 St. Urbain Street, Montreal, Quebec, Canada H2W 1T7.
Duester
Gregg
G
Bhat
Pangala V
PV
D000445:Aldehyde Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D004224:Diterpenes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D051379:Mice; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D050697:Retinal Dehydrogenase; D012172:Retinaldehyde / Q000378:metabolism*; D013045:Species Specificity
D004224:Diterpenes; D012333:RNA, Messenger; D011994:Recombinant Proteins; C031338:13-cis-retinal; C031390:9-cis-retinal; D000445:Aldehyde Oxidoreductases; D050697:Retinal Dehydrogenase; D012172:Retinaldehyde