Alcohol Dehydrogenase
Created by Max Friedfeld
Alcohol dehydrogenase (ADH) is a protein found in nearly all organisms, including animals, plants, fungi, and archaea. ADH is used to convert alcohols to aldehydes or ketones or to perform the reverse reaction. One well-characterized form of the protein is found in the thermophilic archaea Sulfolobus solfataricus. The protein, SsADH (PDB ID = 1JVB), exists as a homotetramer (isoelectric point = 7.32, molecular weight = 150220 Da), similar to other ADH proteins found in archaea.1 In other organisms, alcohol dehydrogenase exists in different forms (horse liver ADH is a homodimer), but the function of ADH is similar in many organisms: to catalyze the reversible reaction of alcohol oxidation to aldehydes or ketones.2 ADH proteins from Archaea organisms have recently become a topic of study for two reasons: these organisms and their proteins are highly stable and thrive in high temperatures, and these ADH proteins, while often having dissimilar sequences, exhibit similar folding patterns with each other and with other eukaryotic ADH proteins.2
The widespread nature of ADHs indicates a significant biological role of the protein. Since alcohols are toxic to most plants and animals, the ADH conversion may act as a toxin-removal mechanism. Yeast cells use ADH to perform the reverse reaction of reducing ketones and aldehydes to alcohols, possibly as a way to store chemical energy through forming the byproduct NAD+.
In performing these transformations, ADH employs NAD+ as a stoichiometric oxidant (NAD+ is reduced to NADH) to achieve the oxidation of the alcohol substrate. SsADH, like other ADH proteins, contains a zinc cation that assists with alcohol oxidation through coordination to the hydroxyl group.2 The active site of SsADH consists of a hydrophobic channel that the alcohol moves into, with the zinc cation at the bottom of the channel.1
The primary structure of SsADH is similar to that of other homotetrameric forms of ADH found in archaea organisms, with one major difference being the incorporation of a second zinc cation into each monomer of SsADH.1 To determine the primary structural similarities of SsADH with that of other ADH proteins, a BLAST (Basic Local Alignment Search Tool) search was performed. SsADH is structurally similar to an ADH protein synthesized from another thermophile, Aeropyrum pernix.3 This tetrameric alcohol dehydrogenase protein (PBI ID = 1H2B) has an E value of 2 x 10-60 when compared with SsADH, indicating the two exhibit primary structure similarities. A search in the Dali server revealed that the tertiary structures of ApADH and SsADH also are similar. When compared with SsADH, ApADH was found to have a Z score of 43.2 and a Root Mean Squared Deviation of 2.4 Å, indicating these two proteins have a similar tertiary structure.4 Additionally, their quaternary structures are similar; in fact, both are tetramers.3 This indicates that the proteins perform very similar biological functions for their host organisms. The similarity in structure is a result of evolutionary changes in these two organisms’ ancestors and indicates that these two thermophiles are evolutionary related, since the genes that expresses the ADH proteins have a similar code.
One crystal structure1 of a tetrameric ADH comes from the hyperthermophile Solfolobus solfataricus and catalyzes the dehydrogenation of 2-ethoxyethanol to yield 2-acetaldehyde (Eq 1).
This protein (SsADH) is useful for study because three forms of the protein have been crystallized: both the apo (apo-SsADH, PDB ID = 1JVB)2 and holo forms (holo-SsADH, PDB ID = 1R37)5 in addition to a single-point mutant (PDB ID = 1NVG).6 Holo-SsADH and the single point mutant are very structurally similar to apo-SsADH, with a root mean square deviation of 1.8 Å and 0.4 Å respectively. An ADH from another archaeon, Aeropyrum pernix, that has recently been crystallized5 (PDB ID = 1H2B) is also very structurally similar to SsADH, with a root mean square deviation of 2.4 Å.4
Each subunit of the homo-tetrameric SsADH contains two domains, a coenzyme-binding domain (responsible for binding NAD, the oxidizing coenzyme used by the protein) and a catalytic domain (where alcohol oxidation takes place). The coenzyme-binding domain contains a parallel beta-sheet and six helices, while the catalytic domain contains antiparallel beta-strands surrounded by five alpha-helices. Residues in the sequence not assigned to helices or sheets account for 36 percent of residues in each subunit (126 residues out of 347 are random coil). The calcuated Ramachandran plot for SsADH supports this; nearly all the residues fall in the beta sheet or alpha helix domains of the Ramachandran plot. Additional residues are in the left handed helix domain; residues in no common domain are most likely random coil. The Ramachandran plot can be viewed in the third tab ("Ramachandran_plot").
The quaternary structure of SsADH involves the association of four subunits into a dimer of dimers. Strong ionic and hydrogen bonds hold the subunits together. For example, the crystal structure reveals that His-110 of subunit A is ionically bound to Glu-288 of subunit B (bond distance is 3.1 Å). Networks of hydrogen bonds and ionic interactions like this help contribute to the great theromstability of this protein. In fact, Sulfolobus solfataricus grows at an incredible 87 ºC and pH = 3.5; the proteins of hyperthermophiles are exciting areas of study due to their stability and potential applications in industrial synthesis.1
One important feature of each subunit is the environment of the two zinc centers. The catalytic zinc center is bound to four amino acid residues in a tetrahedral arrangement: Cys-38, His-68, Cys-154, and Glu-69. The zinc-bound glutamate residue is a recurring motif in ADH proteins and plays an important role in catalysis. In holo-SsADH, 2-ethoxyethanol displaces Glu-69 as the fourth coordination position for the zinc ion; weak binding of this residue to the zinc cation is essential for catalyst activity. The inner coordination sphere of the catalytic zinc ion in SsADH is similar to that of other thermophiles; in bacterial and eukaryotic ADH proteins however, a water molecule is complexed with the catalytic zinc ion instead of Glu.7 Coordination of the alcohol group aids in the hydride transfer from the alcohol to NAD+, forming the ketone/aldehyde and NADH. The second zinc center is bound by Cys-101, Cys-103, Cys-112, and Glu-98. Removal of this zinc ion from the subunit has little effect on catalysis but reduces the thermal integrity of the protein; thus this structural zinc cation is essential in allowing the organism to survive in its uniquely hot and acidic environment.
The catalytic domain, in which both the catalytic zinc ion and the structural zinc ion are located, is responsible for coordinating the substrate alcohol. As mentioned previously, dissociation of Glu-69 from the zinc cation allows for coordination of the substrate alcohol; in the case of holo-SsADH, the substrate is 2-ethoxyethanol. The substrate is positioned such that the methylene carbon donating a hydride is 3.3 Å from the accepting carbon of NAD+. Ser-40 and His-43 help transfer the hydroxyl proton to the solvent to complete the oxidation of the alcohol. Hydrophobic residues in the catalytic domain shield the active site from solvent; Ser-40 and Trp-95 compose the inner region, Phe-49, Ile-120, Leu-295, Trp-117 and Leu-272 constitute the central region of the substrate pocket, and the outer region is composed of Asn-51 and Leu-52.
In the apo-holo transition, the catalytic domain and the coenzyme domain move closer together. Residues 270-275 interact primarily with the coenzyme and undergo a significant change upon coordination, allowing the loop of residues 46-62 (which primarily interacts with substrate) to move closer to the coenzyme domain.
The coenzyme binding site contains polar residues that are able to bind NAD+, which is converted to NADH during catalysis. Important in determining NAD+ specificity are Asp-203 and the glycine-rich segment 178GAGGGLG184, which directly hydrogen-bonds with the pyrophosphate group of NAD+. Additionally, three residues, Val-270, Thr-153, and Leu-183, hydrophobically interact with the nicotinamide moiety.
Analysis of the structure of a single-point mutation of SsADH revealed that strong coenzyme binding slows the rate of catalysis.5 Upon changing Asn249 to Trp249 (N249Y, PDB ID = 1NVG), the coenzyme affinity decreased, resulting in an increase in the rate of catalysis. This study suggests that coenzyme dissociation is rate-limiting. The mutation causes distortions in the coenzyme binding site; specifically, Val-270 is now oriented the carbonyl oxygen cannot hydrogen bond with the substrate, thus reducing coenzyme affinity. This, coupled with changes in other substrate interactions, decreases substrate affinity for the protein.