Sima Aurelia, Parisotto Maxime, Mader Sylvie, Bhat Pangala V
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.
Biochim Biophys Acta. 2009 Dec;1790(12):1660-4. doi: 10.1016/j.bbagen.2009.09.004. Epub 2009 Sep 18.
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.
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.
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.
These data extend the kinetic characterization of RALDH3 and 4, providing their specificities for retinal isomer substrates.
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.
视网膜脱氢酶（RALDHs）催化视网膜脱氢生成视黄酸（RAs），而视黄酸是胚胎发育和组织分化所必需的。本研究旨在确定两种小鼠RALDHs，即RALDH3和RALDH4对视网膜异构体底物的详细动力学特性，以更好地界定它们在视黄酸异构体合成中的特异性。
RALDH3和RALDH4在大肠杆菌中作为带His标签的蛋白表达并亲和纯化。用视网膜异构体底物进行酶动力学研究。通过高压液相色谱分析酶促产物。
RALDH3以高催化效率氧化全反式视网膜（Vmax/Km = 77.9），但对9-顺式或13-顺式视网膜底物均无活性。另一方面，RALDH4对全反式视网膜底物无活性，对9-顺式视网膜氧化表现出高活性（Vmax/Km = 27.4），并以较低的催化效率氧化13-顺式视网膜（Vmax/Km = 8.24）。β-紫罗兰酮是RALDH4活性的有效抑制剂，分别以0.60和0.32的抑制常数竞争性抑制9-顺式和13-顺式视网膜氧化，但对RALDH3活性无影响。二价阳离子MgCl2使RALDH4对13-顺式视网膜的氧化活性提高了3倍，对9-顺式视网膜氧化无显著影响，并轻微激活RALDH3活性。
这些数据扩展了RALDH3和RALDH4的动力学特征，提供了它们对视网膜异构体底物的特异性。
RALDHs的动力学特征应为确定参与视网膜异构体特异性的氨基酸残基以及这些酶在特定组织中视黄酸合成中的作用提供有用信息。
