Aldehyde Dehydrogenase
Created by Lauren Vrablik
The protein aldehyde dehydrogenase, also referred to as Mop, is an aldehyde oxidoreductase of the xanthine oxidase family (5). It is a biological enzyme physiologically found as a homodimer (5, 6), but was crystallized as a single chain protein from Desulfovibrio gigas as documented in the protein data bank (pdb ID: 3L4P) (8). The molecular weight and isoelectric point (pI) of aldehyde dehydrogenase are 97,034.61Da and 5.69, respectively, as calculated using ExPasy.
As previously mentioned, aldehyde dehydrogenase (aldehyde oxidoreductase, Mop) is a member of the xanthine oxidase family: a class of biological enzymes which oxidize aldehyde substrates into carboxylic acids. In general, this reaction is extremely important biologically for detoxification and other metabolic reactions. Specifically, aldehyde dehydrogenase was reported to react well with the following aldehyde substrates: acetaldehyde, propionaldehyde, benzaldehyde (7) and salicylaldehyde (1).
The xanthine oxidase family is distinguished by the incorporation of molybdenum, a metal ion that is central to the molybdopterin cofactor. These enzymes are also unique, since they employ water molecules, rather than O2 as the source of oxygen to be inserted into the substrate (8). Aldehyde dehydrogenase separates itself, functionally, from other oxidoreductases in several ways. It is uniquely NAD-independent, using its intrinsic molybdenum (Mo) active site in conjunction with other reducing ligands discussed later as the reducing center in order to oxidize the substrate. Also, unlike xanthine oxidase (pdb ID: 3ETR- chain N) aldehyde dehydrogenase does not have a flavin domain (6), which is used as an additional redox center in xanthine oxidase (5).
Aldehyde dehydrogenase contains several important ligands listed in its protein data bank (pdb) document and on Uniprot. The most critical of these is the molybdopterin-cytosine-dinucleotide-S-S cofactor. This is the site of aldehyde oxidation, and its function will be discussed in more detail later. Another highly important ligand is the Fe2S2 redox center, a characteristic ligand in enzymes of the xanthine oxidase family. Aldehyde dehydrogenase contains two of these in its physiologic form (5). Isopropanol is another ligand of critical importance to function. The isopropanol site is the eventual binding site for the aldehyde substrate (5, 6). This mechanism will also be explained in further detail later. The remaining reported ligands are various ions including calcium, chlorine, lithium and magnesium, as well as small molecules, namely urea and arsenite. Calcium ion, chlorine ion and urea have no documented functional significance and were likely added for crystallization. Lithium and Magnesium ions provide a structural role. Lithium coordinates Gly-693, Ala-649, and Glu-641, and Magnesium coordinates Glu-903 and Glu-899 (8). Arsenite is an inhibitor of the molybdenum active site, and directly coordinates to it (2, 8). It serves no functional purpose for the enzyme, and was included to better understand the coordination geometry of the active site during inhibition (8).
Aldehyde dehydrogenase is a homodimer in its physiologic form, and this is the structure that will be henceforth described, since this structure is extremely important to its functionality. The 907 amino acid residues making up the primary structure of the protein are folded into four distinct domains (5, 6). The two smaller domains are found at the N terminus and bind the two Fe2-S2 prosthetic groups. The larger C terminus domains coordinate the single molybdopterin cofactor which resides inside the protein at the end of a 15Ǻ “tunnel” through which the aldehyde substrate must pass (5, 6). Due to their respective prosthetic groups, the smaller domains are referred to as Fe-S I and II, and the larger domains are known as Mo I and II. Fe-S I contains residues 1-76, and is believed to be the site of substrate docking and an electron acceptor (6).
Judging from 3-D structural images of aldehyde dehydgrogenase, Fe-S I appears to contain mostly beta sheets and random coils. Fe-S II contains residues 84-156 and is made up of alpha helices and random coils. Fe-S I is located inside the protein near the molybdenum site. Fe-S II is located near the surface of the protein (8). The overall chemical reaction catalyzed by the aldehyde dehydrogenase enzyme is RH + H2O --> ROH + 2e- + 2H-, where RH is an aldehyde (8). The electrons given up come from molybdenum, which gets reduced as it oxidizes the aldehyde substrate. These electrons are then transferred to Fe-S I and Fe-S II, thus fulfilling their purpose as electron receptors (8).
Mo I and II include residues 196-581 and 582-907 respectively and involve both beta sheets and alpha helices (6). Overall, the protein is comprised of approximately 23% beta sheets, 32% alpha helices, and 45% random coils (8). Residues in Mo I and II coordinate the molybdenum cofactor through hydrogen bond interactions (6).
Other important residues have been documented. Probably the most critically important residue is Glu-869, which assists multiple times in the functionality of the enzyme. It accepts a proton from water which allows the hydroxyl group to be transferred onto the aldehyde substrate. It also transiently binds with molybdenum, allowing the release of the oxidized product (5). Leu-394, -497, -626, and Phe-425, -494 act as “doors” which mediate the access of substrates to the molybdenum active site. The “doors” separate the inner and outer compartments of the protein, opening to allow the substrate in, and then closing until the product is ready to be released (5). These “doors” exist halfway down the 15Ǻ “tunnel” mentioned previously, which is lined with hydrophobic residues (6).
The geometric structure of the molybdenum cofactor and surrounding protein is critical to enzyme function. Mylobdenum cofactor is pentacoordinated in a roughly square pyramidal geometry (5), and its molybdenum center has an initial oxidation number of 6. It is commonly referred to in the literature as Mo(VI). After transferring two electrons to the aldehyde substrate, the cofactor is reduced to Mo(IV) (5, 6, 8). In aldehyde dehydrogenase, the molybdenum cofactor is known as molybdopterin cysteine dinucleotide. In the desulfo, uninhibited form (pdb ID: 1VLB), the molybdenum atom is coordinated to three oxygen ligands: a water molecule, two oxo groups, and two dithiolene sulfur atoms (5, 6). In the resulfurated form of the protein, however, the apical oxo group is replaced by a sulfido group (5, 6, 8) (Figure 1).
In the arsenite-inhibited form of the protein (pdb ID: 3L4P), arsenite coordinates with an oxygen ligand, effectively eliminating the reducing center’s functionality. Thapper et. al. used electron paramagnetic resonance (EPR) experiments to analyze the effect of arsenite inhibition on non-reduced Mo(VI) centers versus reduced Mo(IV) centers. The results showed that arsenite coordinated differently depending on the center’s oxidative state. For Mo(VI), EPR studies showed that arsenite coordinated with the oxygen of the water ligand, known as the catalytic labile site, replacing the hydrogen ions. For a reduced center Mo(IV), however, the arsenite coordinated in the same way, but the non-apical oxo ligand was observed to have been replaced by a hydroxy ligand (8).
The mechanism for function is heavily based on the specific protein structure. The aldehyde substrate passes through the channel and the residue “doors” to access the active molybdenum site at the water ligand of molybdenum, which is aimed at the channel opening. It is suggested that the aldehyde docks at the isopropanol site through a Michaelis complex (5). The aldehyde changes its orientation in space to mimic an isopropyl molecule, with the carbonyl oxygen pointed downwards, allowing it to coordinate with molybdenum’s water through a hydrogen bond. The aldehyde’s terminal hydrogen atom is angled towards the apical group, and the R group of the aldehyde is faced away from molybdenum and towards the channel. Glu-869 accepts a proton from water, thus allowing water’s oxygen to attack the carbonyl carbon of the aldehyde. Molybdenum’s apical group accepts the aldehyde’s terminal hydrogen. At this intermediate stage, the aldehyde has been oxidized to a carboxylic acid, but its hydroxyl oxygen is still coordinated between molybdenum and the hydroxyl oxygen of Glu-869. A chain of water molecules exists within the center of aldehyde dehydrogenase. At this point, a transient hydrogen bond develops between the carbonyl oxygen of the carboxylic acid, thus changing the polarity of the system. Glu-869 releases from the carboxylic acid and binds transiently to molybdenum, thus forcing the severance of the carboxylic acid product. The hydrogen bond between the water molecule in the chain and the carboxylic acid product is also severed, allowing the product to be released from the enzyme. This chain water molecule then replaces the water ligand just lost by molybdenum, breaking the bond between molybdenum and Glu-869, restoring it to its original conformation, and permitting the process to be repeated (5, 6). Crystallized water molecules of importance to function are rarely seen. This marks another distinguishing factor about the unique functionality of aldehyde dehydrogenase.
According to the pdb document, atoms 936-2941 of aldehyde dehydrogenase represent water molecules. It is water 137 which is proposed to interact with the carboxylic acid product (6). Waters 137 and 138 are recorded to exhibit four hydrogen bonds, which supports the suggestion that these molecules provide the hydrogen bond interaction necessary to release the carboxylic acid product (5). The last water molecule in the internal chain is water molecule 105. This molecule is surrounded by very nonpolar residues, namely Phe-505, Phe-763, and Tyr-622 which limit its ability to hydrogen bond (5).
As aldehyde dehydrogenase is part of a larger class of enzymes (xanthine oxidases), its structure and function are similar to several other known proteins. The online programs Dali Server and PSI-BLAST were used to find comparison proteins.
The Dali Server returns proteins with similar tertiary structures to the query based on intermolecular distances which indicate folding patterns. The results are given a Z score, where a Z score above 2 means that the tertiary structure is significantly similar. Three proteins returned by the Dali Server were Xanthine Dehydrogenase in complex with bound inhibitopterin-6-aldehyde from Rhodobacter Capsulatus (Z-score: 42.5; pdb ID: 2W54), Quinoline 2-Oxidoreductase from Pseudomonas Putida (Z-score: 42.8; pdb ID: 1T3Q), and 4-Hydroxybenzoyl-CoA from Thauera Aromatica (Z-score: 44.4; pdb ID: 1SB3) (4).
PSI-BLAST finds proteins of similar amino acid sequence (primary structure) to the query protein by assigning an E value. This E value is a measure of the homology between the two proteins, with a larger E meaning they are more dissimilar. Three proteins of significant similarity returned by PSI-BLAST included chain A of Xanthine Dehydrogenase from Bos Taurus (E value: 5e-64; pdb ID: 1N5X), chain A of Rat Xanthine Dehydrogenase Triple Mutant from Rattus norvegicus (E value: 1e-60; pdb ID: 1WYG), and chain C of Xanthine Oxidase in complex with lumazine from Bos Taurus (E value: 7e-66, pdb ID: 3ETR).
Since aldehyde dehydrogenase is a member of the xanthine oxidase family, chain N of xanthine oxidase from Bos Taurus (pdb ID: 3ETR; Z-score: 42.1) was chosen as a comparison protein. According to its pdb document, xanthine oxidase contains 6 chains (7); however, only one chain was chosen to compare since aldehyde dehydrogenase only has one chain reported in its pdb document (8).
Xanthine oxidase contains only six ligands: calcium ion, flavin adenine dinucleotide, Fe2S2, pteridine-2,4(1H,3H)-dione, dioxothiomolybdenum(VI) ion, and phosphnic acidmono-(2-amino-5,6-dimercapto-4-oxo-3,7,8A,9,10,10A-hexahydro-4H-8-oxa-1,3,9,10-tetraaza-anthracen-7-ylmethyl) ester (7). A direct FASTA sequence comparison was done in PSI-BLAST which returned an E value of 8e-46. This value is slightly different than the E value initially returned in the general PSI-BLAST search, and this difference may be attributable to a different algorithm used in a direct FASTA comparison versus a general search for a query protein. The sequences share 44% similarity. The significant difference in structure comes from the fact that xanthine oxidase contains a flavin domain while aldehyde dehydrogenase does not. The flavin domain acts as an additional redox center, “extending the electron transfer pathway from the active site” (8) to the two Fe2S2 receptor groups seen in both proteins. It is suggested that this flavin domain in xanthine oxidase exists between the segment connecting the Mo I and Fe-S II domains seen in aldehyde dehydrogenase (6). Spectroscopic studies and electron paramagnetic resonance (EPR) experiments done to compare these two proteins show that the molybdenum centers in both are very similar. In fact, before xanthine oxidase was isolated, its structure and function was interpreted using data provided by aldehyde dehydrogenase (5) because it was the first molybdopterin-containing enzyme to have its structure determined (6).
Xanthine oxidase reacts similarly to aldehyde dehydrogenase when exposed to arsenite inhibitors. It is also inhibited at the catalytic labile site. However, while xanthine oxidase’s molybdenum center has the same geometry, it contains only one apical oxo ligand. The other oxo ligand seen in aldehyde dehydrogenase is a sulfa ligand in the case of xanthine oxidase. When inhibited, the arsenite coordinates between the catalytic labile site and the sulfa group. Both experience a reduction in their electron transfer pathways when inhibited (8).