Characterization of retinaldehyde dehydrogenase 3

Abstract

RALDH3 (retinal dehydrogenase 3) was characterized by kinetic and binding studies, protein engineering, homology modelling, ligand docking and electrostatic-potential calculations. The major recognition determinant of an RALDH3 substrate was shown to be an eight-carbon chain bonded to the aldehyde group whose kinetic influence (kcat/Km at pH 8.5) decreases when shortened or lengthened. Surprisingly, the b-ionone ring of all-trans-retinal is not a major recognition site. The dissociation constants (Kd) of the complexes of RALDH3 with octanal, NAD+ and NADH were determined by intrinsic tryptophan fluorescence. The similarity of the Kd values for the complexes with NAD+ and with octanal suggests a random kinetic mechanism for RALDH3, in contrast with the ordered sequential mechanism often associated with aldehyde dehydrogenase enzymes. Inhibition of RALDH3 by tri-iodothyronine binding in competition with NAD+, predicted by the modelling, was established kinetically and by immunoprecipitation. Mechanistic implications of the kinetically influential ionizations with macroscopic pKa values of 5.0 and 7.5 revealed by the pH-dependence of kcat are discussed. Analogies with data for non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans, together with the present modelled structure of the thioacyl RALDH3, suggest (a) that kcat characterizes deacylation of this intermediate for specific substrates and (b) the assignment of the pKa of the major ionization (approximating to 7.5) to the perturbed carboxy group of Glu280 whose conjugate base is envisaged as supplying general base catalysis to attack of a water molecule. The macroscopic pKa of the minor ionization (5.0) is considered to approximate to that of the carboxy group of Glu488.

Figure 1. Graphic representations of the model of mouse RALDH3

(A) Overview of the subunit structure of RALDH3 looking down into the substrate-binding tunnel with Cys³¹⁴ (nucleophile) and NAD⁺ shown at the bottom of the tunnel. (B) A model of retinal binding to RALDH3 showing NAD⁺ and Cys³¹⁴. In this model the NAD⁺, shown in ball-and-stick representation, is in the out position and Glu²⁸⁰ in the in position. Also shown are hydrophobic amino acids that line the tunnel, namely Phe¹⁸², Trp¹⁸⁹, Phe³⁰⁸, Phe⁴⁷¹, Tyr⁴⁷², Phe⁴⁷⁷ and Val¹³¹. The β-ionone ring sits above the binding tunnel and may make few or no interactions with the enzyme. (C) Modelled tetrahedral intermediate state showing the proposed tetrahedral species with the oxyanion stabilized by hydrogen bonding to Asn¹⁸¹ and the amide of Cys³¹⁴. The water molecule is activated by Glu²⁸⁰ in the in position in this model. Glu²⁸⁰ is adjacent to Glu⁴⁸⁸.

Figure 2. Examples of (A) kinetic saturation curves at 37 °C and pH 8.5 and (B) binding curves

(A) Saturation of RALDH3 (10 nM) by NAD⁺ with an octanal concentration of 100 μM. (B) Specific binding of octanal by the RALDH3 F471L mutant.

Figure 3. Demonstration of competitive inhibition by T₃ of the NAD⁺ oxidation of octanal catalysed by RALDH3

The concentration of octanal was maintained at 100 μM; the plotted v–[NAD⁺] data pairs are means of replicates with S.D. values less than 10% of the v values.

Figure 4. pH-dependence of k_cat for the NAD⁺ oxidation of octanal at 37 °C catalysed by RALDH3: demonstration of a major and a minor kinetically influential ionization

The data points are means for three replicates with S.D. values less than 10% of the k_cat values. The plotted data points are means for three replicates with S.D. values less than 10% of the k_cat values, and appear to conform to a single ionization curve over the pH range ≈6–9. These data fit well to the continuous line theoretical for a pH-dependent rate equation for two reactive protonic states (eqn 3 of the text) with pK_I=5.0, _cat(1)=0.16 s⁻¹ (the minor ionization) and pK_II=7.5, _cat(2)=2.14 s⁻¹.

Scheme 1. Protonic dissociation from a two-site acid

The relationships between the microscopic (group) acid dissociation constants (K_A, K_B, K′_A, K′_B) and the macroscopic (molecular) constants (K_I and K_II) characteristic of the two stages of ionization of the dicarboxy couple (Glu²⁸⁰ and Glu⁴⁸⁸) XH₂ to XH⁻ to X²⁻ are given by eqns (4) and (5) in the text.