Mitochondrial Aldehyde Dehydrogenase
Created by Andy Kirst
Mitochondrial aldehyde dehydrogenase (PDB ID: 1o01), commonly referred to as ALDH2, is the second enzyme of the major oxidative pathway of ethanol metabolism (1) in Homo sapiens. ALDH2 is a member of the aldehyde dehydrogenase family, all of which are enzymes whose primary responsibility is to catalyze the oxidation of aldehydes to carboxylic acids (2). Specifically, the function of ALDH2 is to break down acetaldehyde produced by ethanol, as acetaldehyde is considered to be a toxic metabolite (1). Because this protein family’s function relates to oxidation, they are classified as oxidoreductases, which are enzymes that catalyze the transfer of electrons from one molecule (an electron donor) to another molecule (an electron acceptor).
ALDH2’s involvement in the ethanol metabolism pathway is necessary in order to safely break down any ethanol consumed. This pathway involves three steps: ethanol is first converted into acetaldehyde, which is then converted with the help of mitochondrial aldehyde dehydrogenase to acetic acid, which is finally converted into acetyl CoA. Once acetyl CoA is formed, it is free to enter the Krebs cycle (3).
ALDH2 has many interesting aspects of its structure that are critical for its primary function of catalyzing the conversion of acetaldehyde to acetic acid via the second step in the ethanol metabolism pathway. The protein is composed of eight identical chains, each consisting of 500 amino acids, for a total of 4000 amino acids comprising the enzyme (1). Furthermore, the active version of ALDH2 can be described as a dimer of dimers, or alternatively as a homotetramer with several important cofactors attached to it (4). Of these cofactors, magnesium and nicotinamide-adenine-dinucleotide (NAD+) both serve key functional roles for ALDH2 in enabling it to catalyze the acetaldehyde breakdown. One important note about ALDH2 is that while an active enzyme only consists of four subunits, the biological unit, or asymmetric unit, of this enzyme exists as two identical tetramers in total, thus accounting for the eight chains.
In addition to its own unique structure, ALDH2 is found to be complexed with several ligands and cofactors that are essential for ALDH2’s function. When ALDH2 binds an aldehyde, such as acetaldehyde, as a ligand, it utilizes nicotinamide adenine dinucleuotide (NAD+) as a cofactor in order to fulfill its role as an oxidoreductase. In this situation, the aldehyde acts as an electron donor while NAD+ acts as an electron acceptor, with the final reaction yielding a carboxylic acid and NADH. The detailed mechanism of the reaction involves the aldehyde entering the active site of ALDH2 and interacting with the cofactor, NAD+. ALDH2 is also capable of recruiting additional cofactors in order to alter the rate of the oxidation-reduction reaction. One such cofactor is magnesium ions, which, when complexed with ALDH2, aldehyde, and NAD+/NADH can help speed up the deacylation rate of the aldehyde to increase the overall reaction rate (5).
The secondary structure of each individual subunit of ALDH2 consists of 14 alpha helices and 20 beta strands, with specific alpha helices and beta strands having roles in the functional domains of the enzyme (2). ALDH2 has been identified to possess three major functional domains, consisting of a catalytic, coenzyme binding, and oligomerization domain (2). The secondary structures of an ALDH2 subunit can be labeled with the beta sheets being numbered and alpha helices being lettered to more clearly see which specific secondary structures are involved with which domain. The coenzyme-binding domain consists of strands 1-4, 7-11, and helices A-G and N. The oligomerization domain is comprised of strands 5, 6, and 19, while the catalytic domain is made up of strands 12-18 and helices H-M.
As previously stated, a necessary step in the ability of ALDH2 to carry out its function is the binding of the cofactor, NAD+. This occurs at the coenzyme-binding domain, which consists of residues 8-135 and 159-270 (2). One NAD+ binds to each of the four subunits before any acetaldehyde can bind, showing that this enzyme follows a single displacement mechanism. Each portion of the NAD+ molecule binds so as to be stabilized by specific non-covalent interactions from the enzyme. The adenine ring is stabilized by hydrophobic interactions created by alpha helices D-E and the ribose oxygen atoms form hydrogen bonds with side chains of Lys-192, Glu-195, and Ile-166, while the phosphate oxygen forms a hydrogen bond with Trp-168’s indole nitrogen (2).
The catalytic domain, which contains the active site, is located from residues 271-470 with the actual substrate-binding site located on the opposite face of where the NAD+ binds. One key characteristic of the active site is that it contains a Rossman fold, which is a structure composed of five to six parallel beta strands linked to two pairs of alpha helices whose function, combined with the cofactor, helps with isomerization of ALDH2 during the reaction while still keeping the active site in a functional state (2). The two most important residues in providing the catalytic function for the reaction are Cys-302 and Glu-268. Cys-302 functions as the active site nucleophile and Glu-268 functions as the general base that activates a water molecule for hydrolysis of the acyl-enzyme intermediate (2). Lastly, the oligomerization domain is involved in subunit interactions to complete the enzyme’s functional domains.
Mitochondrial aldehyde dehydrogenase, being a part of a large family of aldehyde dehydrogenases and an even larger class of oxidoreductases, has considerable sequence homology with other proteins of its class and family. Mitochondrial aldehyde dehydrogenase even exists in an isoform with cytosolic aldehyde dehydrogenase, which is identical in structure and function but operates in a different region of the cell. Additionally, human ALDH2 can be seen to have considerable homology across various species, including sheep, mice, and bovine. When alpha subunits of bovine and human ALDH2 are superimposed, one can easily note the incredible similarity between two. This homology helps represent the fact that large parts of the protein sequence for this enzyme are conserved evolutionarily and represent a potential common ancestry (1). For example, looking deeper into the bovine and human ALDH2 alpha subunits, both of the critical reaction residues (Cys-302 and Glu-268) are found in the exact same location in the sequence, helping to show just how necessary these two residues are in their specific position in order to carry out the enzyme’s function.
Recent research indicates that a point mutation that results in a Glu-268 to Lys substitution can have profound impacts on ALDH2’s ability to carry out catalysis. This point mutation increases the Km of ALDH2 significantly, thereby lowering its affinity for acetaldehyde and increasing the buildup of acetaldehyde during ethanol consumption (1). Approximately 50% of the Asian population has one inactive variant of ALDH2, which might explain the significantly lower incidences of alcoholism in Asians (2). The reasoning behind this is that acetaldehyde buildup causes headaches and vomiting, which might decrease one’s desire to consume more ethanol.
The structure of ALDH2 is an example of how form follows function in several ways. The three functional domains each contain specific amino acid residues that allow them to interact with specific cofactors, substrates, and other subunits in order to carry out the catalysis of acetaldehyde to acetic acid conversion. Additionally, the cofactors that ALDH2 binds to are specific so as to provide certain roles in the mechanism of the reaction. NAD+ acts as an electron acceptor, while magnesium ions help to both stabilize the phosphate from NAD+ and also to open up additional active sites on ALDH2 to increase the overall reaction rate. These cofactors, in addition to the internal primary and secondary structure of ALDH2 show explicitly why the enzyme is suited to carry out catalysis of acetaldehyde to acetic acid in the mitochondria. This reaction has an essential role in helping to metabolize toxic metabolites, which is especially important for humans with respect to the popular social activity of consuming alcohol and its side effects on the human body. Perhaps with more research into ALDH2 and its role in ethanol metabolism, more connections can be made with studies focusing on some of the deleterious effects of alcoholism that many humans deal with every day.