Les Rétinal déshydrogénases (RALDHs) catalysent irréversiblement la déshydrogénation du Rétinal en Acide Rétinoïque (AR) qui est impliqué dans l’embryogenèse et la différenciation tissulaire. Pour comprendre le rôle dans la biosynthèse de l’AR des RALDHs type 3 et 4 de souris, nous avons déterminé leurs propriétés cinétiques ainsi que leur comportement en présence de différents inhibiteurs. Les tests enzymatiques sont effectués avec une préparation d’enzyme recombinante, tagguée avec 6 histidines, purifiée sur colonne Ni-NTA (Qiagen). L’activité enzymatique est évaluée en quantifiant la production d’AR par chromatographie liquide à haute performance (HPLC) en phase inversée. Les constantes cinétiques ont été déterminées pour les isomères du rétinal tout-trans, 9-cis et 13-cis. La RALDH4 catalyse les isomères 9-cis et 13-cis de rétinal, elle présente un faible KM (3μM) pour les deux isomères et a une efficacité catalytique élevée pour le 9-cis rétinal 3.4 fois supérieure au 13-cis rétinal. La RALDH3 est spécifique au tout-trans rétinal avec un KM de 4 μM et une efficacité élevée. β-Ionone, inhibiteur possible pour la RALDH4, inhibe l’activité avec le rétinal 9-cis et 13-cis, mais n’influence pas l’activité de la RALDH3. Le para-hydroxymercuribenzoïque (p-HMB) inhibe l’activité de deux isoenzymes. Le cation MgCl2 augmente par 3 fois l’oxydation du rétinal 13-cis par la RALDH4, diminue l’oxydation du 9-cis rétinal et influence faiblement la RALDH3. Ces données enrichissent les connaissances sur les caractéristiques cinétiques des RALDHs recombinantes de souris de types 3 et 4 et fournissent des éclaircissements sur la biogenèse de l’acide rétinoïque in vivo. / SUMMARY Retinal dehydrogenases (RALDHs) catalyze the dehydrogenation of retinal into retinoic acids (RA) that 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 as His-tagged proteins and affinity-purified. RALDH3 oxidized all-trans retinal with high catalytic efficiency but did not show activity for either 9-cis or 13-cis retinal substrates. RALDH4 was inactive for all-trans retinal substrate, exhibited high activity for 9-cis retinal oxidation, and oxidized 13-cis retinal with lower catalytic efficiency. β-ionone, a potent inhibitor of RALDH4 activity, suppressed 9-cis and 13-cis retinal oxidation competitively, but had no effect on RALDH3 activity. The p-HMB inhibited the activity for both RALDH3 and RALDH4. The divalent cation MgCl2 activated 13-cis retinal oxidation by RALDH4 by 3-fold, slightly decreased 9-cis retinal oxidation, and did not significantly influence RALDH3 activity. These data extend the kinetic characterization of RALDH3 and 4, providing their specificities for retinal isomer substrates, which should help in determining their functions in the synthesis of RAs in specific tissues.

Les Rétinal déshydrogénases (RALDHs) catalysent irréversiblement la déshydrogénation du Rétinal en Acide Rétinoïque (AR) qui est impliqué dans l’embryogenèse et la différenciation tissulaire. Pour comprendre le rôle dans la biosynthèse de l’AR des RALDHs type 3 et 4 de souris, nous avons déterminé leurs propriétés cinétiques ainsi que leur comportement en présence de différents inhibiteurs. Les tests enzymatiques sont effectués avec une préparation d’enzyme recombinante, tagguée avec 6 histidines, purifiée sur colonne Ni-NTA (Qiagen). L’activité enzymatique est évaluée en quantifiant la production d’AR par chromatographie liquide à haute performance (HPLC) en phase inversée. Les constantes cinétiques ont été déterminées pour les isomères du rétinal tout-trans, 9-cis et 13-cis. La RALDH4 catalyse les isomères 9-cis et 13-cis de rétinal, elle présente un faible KM (3μM) pour les deux isomères et a une efficacité catalytique élevée pour le 9-cis rétinal 3.4 fois supérieure au 13-cis rétinal. La RALDH3 est spécifique au tout-trans rétinal avec un KM de 4 μM et une efficacité élevée. β-Ionone, inhibiteur possible pour la RALDH4, inhibe l’activité avec le rétinal 9-cis et 13-cis, mais n’influence pas l’activité de la RALDH3. Le para-hydroxymercuribenzoïque (p-HMB) inhibe l’activité de deux isoenzymes. Le cation MgCl2 augmente par 3 fois l’oxydation du rétinal 13-cis par la RALDH4, diminue l’oxydation du 9-cis rétinal et influence faiblement la RALDH3. Ces données enrichissent les connaissances sur les caractéristiques cinétiques des RALDHs recombinantes de souris de types 3 et 4 et fournissent des éclaircissements sur la biogenèse de l’acide rétinoïque in vivo. / SUMMARY Retinal dehydrogenases (RALDHs) catalyze the dehydrogenation of retinal into retinoic acids (RA) that 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 as His-tagged proteins and affinity-purified. RALDH3 oxidized all-trans retinal with high catalytic efficiency but did not show activity for either 9-cis or 13-cis retinal substrates. RALDH4 was inactive for all-trans retinal substrate, exhibited high activity for 9-cis retinal oxidation, and oxidized 13-cis retinal with lower catalytic efficiency. β-ionone, a potent inhibitor of RALDH4 activity, suppressed 9-cis and 13-cis retinal oxidation competitively, but had no effect on RALDH3 activity. The p-HMB inhibited the activity for both RALDH3 and RALDH4. The divalent cation MgCl2 activated 13-cis retinal oxidation by RALDH4 by 3-fold, slightly decreased 9-cis retinal oxidation, and did not significantly influence RALDH3 activity. These data extend the kinetic characterization of RALDH3 and 4, providing their specificities for retinal isomer substrates, which should help in determining their functions in the synthesis of RAs in specific tissues.

<p>Several plant defense systems consist of enzymes that act on glucosides and produce a toxic compound. In the intact plant tissue the substrate and enzyme are kept apart. The system studied here consists of the substrate 2-O-β-D-glucopyranosyl-4-dihydroxy-1,4-benzoxazin-3-one and the enzyme glucan 1,3-β-glucosidase in rye. The aim was to determine the properties of a cell wall β-glucosidase. Two different systems for expression and purification of β-glucosidase fused to a tag were used: a 6xHistidine tag system and a thioredoxin tag system. The sequence of the β-glucosidase had previously been determined so now the gene was subcloned into E.coli. A direct PCR on colonies, a test expression, a restriction digestion of plasmids and sequencing was made to analyze the transformation, which all turned out successful. Then the β-glucosidase solubility was determined. Finally a purification of the β-glucosidase from E.coli under native conditions and a pNPG assay was carried out. For the (His)6-tagged protein, the recombinant β-glucosidase tended to end up in the insoluble pelleted fraction which indicated formation of inclusion bodies. The cell wall 1,3-β-glucosidase was soluble with the thioredoxin system, but the percentage of soluble protein fraction was around 5% only of the total protein. In eluates from a nickel-nitrilotriacetic acid column the presence of recombinant protein was confirmed with Western blot, but contaminating bands were also present. Purified elauted fractions did not exhibit detectable β-glucosidase activity. It was not possible to purify active enzyme. From a BLAST search it was clear that the most similar enzymes all had putative glycosylation sites and lack of glycosylation could be a reason for the protein not to fold properly.</p>

Several plant defense systems consist of enzymes that act on glucosides and produce a toxic compound. In the intact plant tissue the substrate and enzyme are kept apart. The system studied here consists of the substrate 2-O-β-D-glucopyranosyl-4-dihydroxy-1,4-benzoxazin-3-one and the enzyme glucan 1,3-β-glucosidase in rye. The aim was to determine the properties of a cell wall β-glucosidase. Two different systems for expression and purification of β-glucosidase fused to a tag were used: a 6xHistidine tag system and a thioredoxin tag system. The sequence of the β-glucosidase had previously been determined so now the gene was subcloned into E.coli. A direct PCR on colonies, a test expression, a restriction digestion of plasmids and sequencing was made to analyze the transformation, which all turned out successful. Then the β-glucosidase solubility was determined. Finally a purification of the β-glucosidase from E.coli under native conditions and a pNPG assay was carried out. For the (His)6-tagged protein, the recombinant β-glucosidase tended to end up in the insoluble pelleted fraction which indicated formation of inclusion bodies. The cell wall 1,3-β-glucosidase was soluble with the thioredoxin system, but the percentage of soluble protein fraction was around 5% only of the total protein. In eluates from a nickel-nitrilotriacetic acid column the presence of recombinant protein was confirmed with Western blot, but contaminating bands were also present. Purified elauted fractions did not exhibit detectable β-glucosidase activity. It was not possible to purify active enzyme. From a BLAST search it was clear that the most similar enzymes all had putative glycosylation sites and lack of glycosylation could be a reason for the protein not to fold properly.