Sandbox Reserved 1475

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This Sandbox is Reserved from November 5 2018 through January 1, 2019 for use in the course "CHEM 4923: Senior Project taught by Christina R. Bourne at the University of Oklahoma, Norman, USA. This reservation includes Sandbox Reserved 1471 through Sandbox Reserved 1478.

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Retinal Dehydrogenase Type Two StructureRetinal Dehydrogenase Type Two Structure

spinqualitylabelsall models Retinal Dehydrogenase Type Two with NAD bound (PDB entry 1bi9) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

This molecular structure is Retinal Dehydrogenase Type Two (RalDH2) which was extracted from Rattus norvegicus. This enzyme is part of the super family Aldehyde Dehydrogenase. The sample was a crystallization of a clone of RalDH2 that was given as a gift from J. L. Napoli. It has been shown that RalDH2, the cellular binding protein type I, and the retinol binding protein receptor all co-localize in tissues that require Vitamin A (retinol) for normal development in rat embryos post-gastrulation, as discussed by "personal communication" to the authors of the "The Structure of Retinal Dehydrogenase Type II at 2.7 Angstrom Resolution: Implications for Retinal Specificity". RalDH2 is the enzyme that produces retinoic acid in mouse embryos during gastrulation, and is found to be surrounding the primitive node, which is where retinal is converted into retinoic acid. The function of this enzyme is to catalyze the oxidation of retinal to retinoic acid. Retinoic acid produces a putative morphogen that initiates pattern formation in the early embryo. This is the last step in the formation of the hormone from Vitamin A (retinol). Vitamin A that has been metabolized can produce retinoid derivatives which function in either vision or growth and development. RalDH2 is expressed in Escherichia coli strain BL21(DE3)

FunctionFunction

The reaction of this enzyme is [(retinal) + (NAD+) + (H2O) ↔ (retinoic acid) + (NADH) + (H+)]. The main function of this enzyme is to produce Retinoic acid. RalDH2 requires (NAD+) as a cofactor. In the oxidoreductase reaction, NAD+ acts as an electron acceptor. Once the NAD+ is bound, hydrogen bonds form with non-polar residues and one basic Lysine residue. Chloride ions participate in hydrophobic interactions with Arginine residues. These interactions cause a structural change to occur in the RalDH2 enzyme which causes it to form a more favorable folded confirmation. In the enzyme a large binding cavity is formed. Structural changes occur to stabilize the tertiary structure of RalDH2

Structural highlightsStructural highlights

spinqualitylabelsall models Figure 2 Nucleotide-Binding domain - orange, Catalytic domain - green, Tetramerization domain - blue (PDB entry 1bi9) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

Figure 1 Nucleotide-binding domain - orange, Catalytic domain - green, Tetramerization domain - blue. Imagine modified in Chimera from (PDB entry 1bi9). Chain D shown with NAD substrate shown in pink

Figure 3 Section (a) is the dimer of RalDH2 with the green spheres representing the amino and carboy termini of the substrate access channel loop. Section (b) shows the same orientation as section (a) with both dimers present and then the two dimers spun 90 degrees on the X-axis.

Figure4 Chain D of RalDH2 with Cys-302 in yellow, Glu-268 in green, and Asn-187 in red. Image modified in and taken from Chimera from (PDB entry 1bi9)

The structure was based on the mitochondrial aldehyde dehydrogenase type two. RalDH2 in a monomer made up of 3 domains: a nucleotide-binding domain (1-136, 161-270), a catalytic domain (271-484), and a tetramerization domain (137-160, 485-484) as shown in Figure 1. The tetramer can be envisioned as an "X", with nucleotide-binding sites at the tips of the "X", and the tetramerization domains as the equatorial portion of the "X" as seen in Figure 2. The is presented by the alpha1 helix and beta11 strand of one nucleotide-binding domain, with the same alpha1 helix and beta11 strand of it's dimer (Figure 2, the purple highlighted region). Although the beta strands are far apart, ordered water molecules are present to create beta-sheet contacts. The is presented by the beta18 of the catalytic domain beta-sheet of one monomer and the beta19 of the tetramerization domain of it's dimer(Figure 2, the pink highlighted region). This dimerization interaction creates an "embrace" between the beta-sheet's contact, and creates a channel for the substrate to access the active site.

Cofactor NAD and Cl ionsCofactor NAD and Cl ions

The crystal structure was cocrystallized with , and was determined at a 2.7 Angstrom resolution (Figure 2 Chain D present with NAD represented in pink). NAD+ acts as a cofactor and is the electron acceptor in RalDH2 oxidoructase reaction as seen in the reaction presented above. RalDH2 must be folded in a proper manner for its enzymatic function to occur. The folding of the enzyme is partially due to the interactions of NAD+ and Chloride ions. When NAD+ is present hydrogen bonds with Glu and Ser form, van der Waals interactions with non-polar residues and one polar residue (Lys) forms. The interaction with Lys-192 provides the transition state stability, making for a favorable confirmation. This conformational change is important since in the absence of NAD+, no crystal structures grew in any of the screened conditions. The Chloride ions participate in hydrophobic interactions with Arg which also help maintain the folded structure. With the cofactor NAD+ present the catalytic domain of RalDH2 is highly mobile and needs the selective substrate present to immobilize the catalytic domain. The binding of hydrophobic substrate and the NAD+ cofactor are needed to stabilize the catalytic domain. Short chain aldehydes do not have a large enough hydrophobic surface to bury inside the channel, therefor cannot work as suitable substrates. The long tails of the long chained aldehydes are needed to interact with the catalytic Cys-302 through hydrogen bonds. Short chained aldehydes can have hydrogen bond interactions with Cys-302 however are not large enough to fully bury the whole access channel, which is needed for the catalytic domain to be immobilized.

Cys-302Cys-302

In Figure 3 it is possible to see the active site, which is where the substrate interacts with Cys-302. The Cys-302 residue, highlighted in yellow in Figure 4, acts as a nucleophilic active site on each domain as a hydrogen-bond turn that is enclosed deep inside the substrate access channel. This is where the large substrate molecules can gain access to the catalytic Cys-302. The side chain of Cys-302 is the nucleophile that attaches to the substrate retinol (at its carbonyl) when it is deprotonated. To get Cys-302 deprotonated, the amino acid Glu-268, highlighted in green in Figure 4, is needed as the proton acceptor. The amine backbone of Glu-268 helps stabilize the negatively charged transition state, which helps the enzyme result in an energetically favorable conformation. Asn-187, highlighted in red in Figure 4, is also used as a transition site stabilizer and is on all four domains. It works in a similar fashion as Glu-268, in that Asn-187 amine backbone is used to stabilize the negatively charged transition state.

EnergeticsEnergetics

Figure 5 Relative efficiencies of RalDH2 for aldehyde substrates. Image reference.

In Figure 5 it is visible to see that acetaldehyde and benzaldehyde both have really high Km values, 645uM and 305uM respectfully, and relatively low Vmax values, 139nmol/min/mg and 200nmol/min/mg respectfully. Octantal and decanal both have really low Km values, 5uM and 3uM respectfully, and relatively high Vmax values, 152nmol/min/mg and 214nmol/min/mg respectfully. Acetaldehyde and benzaldehyde are both short chained aldehydes compared to octantal and decanal aldehydes. It is easier to compare the ratio of Vmax/Km. An energetically favorable substrate would display a ratio of Vmax/Km that has a large magnitude. As seen in Figure 5, both the long chained octantal and decanal aldehydes had large Vmax/Km values, 152 AND 214 respectfully. The substrate that RalDH2 uses to actually convert Vitamin A (Retinol) to retinoic acid is retinal in its "all-trans" form. As seen in Figure 5 the Km value for the this substrate is the smallest out all that were tested, and the Vmax values was comparatively high. The Vmax/Km was also pretty large at a value of 49± 6.

Relevance - DiseaseRelevance - Disease

Figure 6 "Morphological abnormalities of RalDH2 knock out embryos".

Multiple complications can occur if there is a deficiency of RalDH2 in mammals. If there were to be a RalDH2 deficiency during the embryonic development, possible congenital malformations can occur. The complications include defects such as lack of axial rotation, incomplete neural tube closure, and lack of heart looping and chamber morphogenesis. With the study of mouse with their RalDH2 emzyne knocked out, hearts consisted of single, medial, dilated cavities. The mice displayed their frontonasal region to be truncated, and their otocysts to be reduced. In Figure 6, the comparison of wild-type embryos compared to ones that are RalDH2 negative. It is visible to see that there is lack of embryonic turning, associated with a truncation of the posterior region. The WT 8.5 doc embryo is the wild-type embryo before turning has occurred. Section b displays a wild-type embryo compared to defective embryos, section c-d. The e and f show the embryos of a wild-type embryo compared to one that was low in the RalDH2 enzyme, respectfully.