D-3-Hydroxybutyrate Dehydrogenase
Created by R.J. Santucci
D-3-hydroxybutyrate dehydrogenase (pdb ID = 2ZTL) from pseudomonas fragi is a cytosolic catalytic enzyme that facilitates the reversible reaction between D-3-hydroxybutyrate and acetoacetate (1). As a catalyst, D-3-hydroxybutyrate dehydrogenase (HBDH) works to increase both the forward and reverse rates of this reaction (1). Acetyl-CoA is formed in the body by the oxidation of fatty acids and can be metabolized to form acetoacetate and D-3-hydroxybutyrate under low concentrations of carbohydrates (1, 2). Acetoacetate, D-3-hydroxybutyrate, and acetone are together called ketone bodies, which are high-energy compounds that can be used for energy when there is a deficiency of glucose in the body (2). Brain tissue cannot use fatty acids for energy when glucose levels deviate from normal conditions, and so ketone bodies are used as a source of energy accounting for almost two-thirds of the brain’s energy source in times of low sugar concentrations (2). Excess concentrations of these ketone bodies can have a negative effect in the body, causing ketoacidosis (2). Ketoacidosis is a common ailment for diabetic patients, especially children (3). HBDH is of great interest because it provides a way to readily access the amount of ketone bodies present in a patient, which is useful for monitoring a patient’s risk for Diabetic Ketoacidosis (DKA) (2). The molecular weight of HBDH is 26,684.48 Da and it has an isoelectric point of 6.59, calculated from ExPASy (4).
HBDH is classified in the enzymatic family of short-chained dehydrogenase/reductase, SDR, due to its function and architecture. There is often a lot of similarities in the three dimensional structures of the enzymes of the SDR family, despite the low similarity in primary sequence (about 15-30%) (1). HBDH is a homotetramer of 4 subunits each with a length of 260 residues (1). Each subgroup contains two critical regions that are important for substrate binding. One region is a Rossmann fold, while the other is the substrate-binding loop (1). The substrate-binding region is comprised of residues 190-216 (1). This region is of interest because it is what performs the conformation change in the protein from an open conformation in the absence of a co-factor to the closed conformation in the presence of a co-factor (namely, NAD+) (1). Interestingly, the substrate-binding loop is disordered when coordinated to the cofactor but is ordered when it is not present (1). This was not expected because for most SDR enzymes the substrate-binding loop is ordered in the presence of a cofactor and disordered in its absence (1).
HBDH’s function is to oxidize the D-enantiomer of 3-hydroxybutyrate into acetoacetate and to reduce acetoacetate into D-3-hydroxybutyrate (D-3-HB) (2). This is performed through a hydride transfer aided by the presence of NAD+/NADH (2). The overall function of the HBDH protein is to bring together the NAD cofactor and the substrate (D-3-HB/acetoacetate) in a way that facilitates the reduction-oxidation reaction and make it more favorable than it would be otherwise. Substrate binding occurs through interactions with the protein and the carboxyl and 3-methyl groups of the substrate, thus securing the substrate in place (1). The hydrophobic pocket that coordinates with the 3-methyl group is comprised of Ala-143, His-144, Gly-186, Trp-187, and Trp-257 (1). The same is true for the carboxyl group and the residues Gln-94, His-144, and Lys-152 (1). It is believed that substrate must be the D-enantiomer because the proper alignment of the hydroxyl group (3R-configuration) is well accommodated by the catalytic tetrad of Asn-114, Ser-142, Tyr-155, and Lys-159 and properly primed for a hydride transfer (1). Once in place, the substrate is secured by a substrate-binding loop composed of the residues Thr-190 to Leu-216 (1). The substrate-binding loop is disordered when the protein is complexed with NAD cofactor. The disorder in this segment makes it more malleable than it would be otherwise and is likely a key factor in how it binds to the substrate (5). The process in which the substrate-binding loop moves into position is through rotation about two “hinges” within the protein (1). There are actually two significant conformational changes that occur about these two hinges. The first is the aforementioned change from open to closed conformation in the presence of the NAD cofactor (1). This change prepares the protein or substrate binding by aligning the substrate-binding loop in the proper orientation and by disordering the loop (1). The second change occurs upon substrate binding, in which the loop moves to further secure the substrate to the protein. The two hinge regions for both of these conformational changes are comprised of residues Arg-189 through Leu-192 and Leu-215 through Ser-217 (1). The substrate binding loop is able to move into place through the rotation about these hinges. Within each hinge there is a critical residue that acts as the rotation axis for the entire hinge. One hinge is more heavily dependent on the Thr-190 than it is on the other residues of the hinge (1). The other hinge has two different residues that act as the main point of rotation, depending on the type of rotation. It appears that for the conformational change upon cofactor binding Leu-216 is the most dependent residue through which the most torsion occurs, while for the conformational change upon substrate binding Leu-215 is the important residue (1). The importance of these residues was determined through selective mutagenesis; a process in which a residue of interest is replaced with another residue and then changes in structure, chemistry, etc are accessed and residue importance is determined.
When fully coordinated with its ligands and substrates, the enzyme contains four NAD+ ligands, four L-3-hydroxybutyrate substrates, two Mg2+ ions, and 836 water units (1). The NAD+ ligand functions to bring the protein into its closed conformation (1).
The two main structural features of this protein are the Rossmann fold and the substrate-binding region (1). The Rossmann fold is known to be an important region for binding nucleotides like NAD+ (6). The substrate-binding loop contains two helixes and a connecting loop between the two of them (1).
Alcaligenes faecalis D-3-dehydroxybutyrate dehydrogenase (pdb ID = 2YZ7) is the same protein as pseudomonas fragi D-3-dehydroxybutyrate dehydrogenase with respect to amino acid primary sequence, but is from a different organism and thus has a slightly different architecture. Park et al. suggest that proteins can have different structures when assembled in different organisms, and thus can have different substrate binding properties (7). The Dali Server is a program that compares a protein of interest (POI) to another protein on the basis of tertiary architecture (8). The comparison of these two proteins on Dali Server gave a Z-score of 42.6. This is a high Z-score, which indicates that the two proteins are very similar in structure. BLAST is another program that compares a POI to a different protein on the basis of the primary amino acid sequence (9). The e-value for this comparison was 2*10-154. This extremely low e-value indicates that the two have the same amino acid sequence, as should be expected. Nakashima et al. addresses the similarity between the subunits of these two proteins and affirms that they are indeed very similar (1).