Kinetic characterization of recombinant mouse retinal dehydrogenase types 3 and 4 for retinal substrates.
BACKGROUND
Retinal dehydrogenases (RALDHs) catalyze the dehydrogenation of retinal into retinoic acids (RAs), which are required for embryogenesis and tissue differentiation. This study sought to determine the detailed kinetic properties of 2 mouse RALDHs, namely RALDH3 and 4, for retinal isomer substrates, to better define their specificities in RA isomer synthesis.


METHODS
RALDH3 and 4 were expressed in Escherichia coli as His-tagged proteins and affinity-purified. Enzyme kinetics were performed with retinal isomer substrates. The enzymatic products were analyzed by high pressure liquid chromatography.


RESULTS
RALDH3 oxidized all-trans retinal with high catalytic efficiency (Vmax/Km=77.9) but did not show activity for either 9-cis or 13-cis retinal substrates. On the other hand, RALDH4 was inactive for all-trans retinal substrate, exhibited high activity for 9-cis retinal oxidation (Vmax/Km=27.4), and oxidized 13-cis retinal with lower catalytic efficiency (Vmax/Km=8.24). beta-ionone, a potent inhibitor of RALDH4 activity, suppressed 9-cis and 13-cis retinal oxidation competitively with inhibition constants of 0.60 and 0.32, respectively, but had no effect on RALDH3 activity. The divalent cation MgCl2 activated 13-cis retinal oxidation by RALDH4 by 3-fold, did not significantly influence 9-cis retinal oxidation, and slightly activated RALDH3 activity.


CONCLUSIONS
These data extend the kinetic characterization of RALDH3 and 4, providing their specificities for retinal isomer substrates.


CONCLUSIONS
The kinetic characterization of RALDHs should give useful information in determining amino acid residues that are involved in the specificity for retinal isomers and on the role of these enzymes in the synthesis of RAs in specific tissues.
Sima
Aurelia
A
Laboratory of Nutrition and Cancer, Centre de recherché du Centre hospitalier de l'Université de Montréal (CRCHUM), Hôtel-Dieu, and the Department of Medicine Université de Montréal, Montreal, Quebec, Canada.
Parisotto
Maxime
M
Mader
Sylvie
S
Bhat
Pangala V
PV
D000818:Animals; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D066298:In Vitro Techniques; D007536:Isomerism; D007700:Kinetics; D051379:Mice; D011994:Recombinant Proteins / Q000378:metabolism; D050697:Retinal Dehydrogenase / Q000378:metabolism*; D012172:Retinaldehyde / Q000031:analogs & derivatives* / Q000493:pharmacokinetics; D012176:Retinoids / Q000493:pharmacokinetics*; D013379:Substrate Specificity; D014212:Tretinoin / Q000031:analogs & derivatives / Q000493:pharmacokinetics
D011994:Recombinant Proteins; D012176:Retinoids; D014212:Tretinoin; C547414:RALDH4 protein, mouse; D050697:Retinal Dehydrogenase; C508905:retinaldehyde dehydrogenase 3, mouse; D012172:Retinaldehyde