Diol Dehydratase
Created by James Drake
Diol Dehydratase from Klebsiella Oxytoca is an enzyme that catalyzes the dehydration of 1,2-propanediol into propionaldehyde. More specifically, when substrate binding occurs via a potassium ion, heterolytic cleavage of a cobalamin-carbon bond in the coenzyme produces a radical that dehydrates the substrate. Diol dehydratase serves as an important step in the glycerol-lipid metabolism in bacteria. Metabolism of glycerol is one of the most important metabolic pathways for energy in bacteria. The enzyme is extracted and purified from Klebsiella Oxytoca, which is a rod-shaped bacteria that is Gram-negative and has a polysaccharide capsule. K. Oxytoca is primarily a pathogen found in nursing homes and hospitals, and can be easily spread by human-to-human contact (4). In healthy humans it is naturally present in the colon and oropharynx along with a very similar species, K. Pneumoniae, but becomes dangerous when spread to other parts of the body. Those at high risk to K. Oxytoca infection are those with diseases such as alcoholism, diabetes, and chronic bronchopulmonary disease. The molecular weight of diol dehydratase is 207,179.43 Daltons (Da) and has an isoelectric point (pI) of 5.22.
Subunit and Secondary Structure
The subunit structure of diol dehydratase is a dimer of a heterotrimer that is composed of three subunits, alpha (α), beta (β), and gamma (γ). The alpha subunit is the largest of all three with the 554 residues and is folded into a closed TIM beta/alpha-barrel (5). A TIM (triosephosphate isomerase) beta/alpha-barrel is composed of eight alpha helices and eight beta-strands. The parallel beta-strands compose the inner side of the barrel and the alpha helices compose the outer side (5). The entire alpha subunit is composed of 33 helices (281 residues) and 20 beta sheets (58 residues), making up 50% and 10% of the entire secondary structure, respectively. The interaction between alpha subunits is to exclusively in the dimerization of the heterotrimer (2). The beta subunit is medium sized with 224 residues and is folded into an anticodon binding domain (6). This binding domain has 3 layers of five mixed beta sheets strands, with an order of 21345 (5). Strand four is the only antiparallel sheet relative to the rest. The entire beta subunit is composed of 5 helices (59 residues) and 11 beta sheets (38 residues), making up 26% and 16% of the entire secondary structure, respectively. The gamma subunit is the smallest with only 173 residues and is an open three-helical up-and-down bundle (6). The core structure is three alpha helices. The entire gamma subunit is composed of 11 helices (86 residues) and 2 isolated beta bridges (2 residues), making up 49% and 1% of the entire secondary structure, respectively. The large percentage of alpha helices in the gamma subunit surround the outer part of the beta/alpha barrel by acting as a support to the overall alpha subunit structure. Since diol dehydratase is a dimer, there are two identical heterotrimers. Each heterotrimer is then composed of a single (α β γ)heterotrimer. Each individual heterotrimer can perform the enzyme dehydration independently and successfully of the other. if the enzyme were to disassociate, it would produce two dissimilar protein components designated by either F or S. The F and S components represent the monomeric beta subunit and the dimeric alpha-gamma complex, respectively.
The alpha subunit serves as the binding site for the substrate, which is bound inside the TIM barrel. The solvent channel is directly accessible from the outside of the enzyme and is ~15Å deep. The channel runs through the C-terminal ends of stands 2 and 3 of the beta barrel, which is a loop between Thr-172, Ser-202, Thr-207 and Ser-149, all of which are hydrophilic residues and serve as a substrate guide (7). The substrate coordinates with the K+ ion its two hydroxyl positions. The potassium ion keeps the substrate fixated so that the intermediates are in the proper position to ensure accurate hydrogen abstraction and recombination. The K+ ion lowers the OH group migration by 2.2kcal/mol, yet the transition state of the OH group migration is energetically higher that the hydrogen abstraction. The O2 atom of the substrate forms hydrogen bonds with the deprotonated carboxyl group of Asp-335 and His-143, while the O1 atom is hydrogen bound to the NH group of Gln-296 and the deprotonated carboxyl group on Glu-170 (8). The other carbon atoms and methyl group interact through some hydrophobic contact. The corrin ring of the coenzyme is located at the C-terminal side of the barrel and this area above the ring is the active site of the enzyme. Cobalamin is positioned at the beginning or cavity to protect the active site and reactive radical intermediates from any solvent molecules and is held in place by the beta subunit. The beta subunit surrounds the hydrophilic residues of the cobalamin, specifically making a hydrogen bonding with Pro-155. This barrel architecture is a “common molecular apparatus for radical reactions catalyzed by Ado-Cbl-dependant enzymes (2).”
Function
In coenzyme B12-dependent enzymes, substrate binding triggers the catalytic radical formation through the cobalt−carbon bond homolysis. The 1,2-propanediol substrate and essential potassium ion are located inside a beta/alpha barrel (β/α). The two hydroxyl groups of the substrate coordinate directly to the potassium ion. which is negatively bound to the inner part of the cavity (2). The high reactivity of free radicals, which originate from cofactors are utilized to catalyze the reaction. The enzyme must generate radicals in the active site, control these highly reactive radical intermediates, and decide when to undergo reactivation and inactivation. All of these responsibilities of the enzyme create problems for the adenosylcobalamin (Ado-Cbl)-dependent enzyme.
Dehydration of the substrate is triggered by the heterolytic cleavage of the coenzyme Co-C bond, which forms the adenosyl radical. The coenzyme complex has a unique and rare organometallic covalent bond between the cobalt and the C5’ of the adenosyl group (8). Ado-Cbl serves as a coenzyme for multiple enzymes, which are divided into three classes. Diol dehydratase is in the second class, which is characterized by catalyzing heteroatom elimination. Although the reaction may appear simple, the actual mechanism is complex. The initial migration of the hydroxyl group is stereospecific, in which the dehydration of the gem-diol is sterically control by the enzyme so that only one of the hydroxyl groups on the pro-chiral center is eliminated. Although the reaction is stereospecific, it can catalyze the conversion of both (R) and (S) 1,2-propanediols into (R) and (S) propionaldehyde, respectively. However, the binding affinity for diol dehydratase is much higher for the (S)-isomer. Each isomeric substrate is bound the enzyme differently with different catalytic efficiency and binding affinity (3).
The stereo-specificity heteroatom dehydration is unique and rare since most enzymes are extremely stereospecific for their respective substrates. The enzyme reactions is occurs when a hydrogen atom migrates from C1 atom of the substrate to an adjacent carbon atom in exchange for a hydroxyl group on the C2 to move in the opposite direction of the gem-diol. The hydrogen atom moves to the adjacent carbon atom without exchange with solvent protons. The migrating hydroxyl group is replaced by the hydrogen atom followed by inversion of the configuration at carbon two. Enantiomeric ethylene glycols are labeled with deuterium and tritium to produces a racemic mixture and indicate that rapid internal rotation of the intermediate occurs before the hydrogen recombination (2). The Ado-Cbl complex serves as an intermediate hydrogen carrier by first accepting a hydrogen atom from C1 of the substrate to C5 of the coenzyme, eventually re-donating the hydrogen back to C2. The enzyme-enzyme interaction leads to the activation of the cobalamin-carbon (Co-C) bond of the coenzyme, which is triggered by the binding of the substrate. The Co-C bond forms the adenosyl radical and cob(II)alamin. The newly created radical attacks the substrate and “abstracts” a hydrogen to form 5’-deoxyadenosine. The substrate radical rearranges to the product radical, which then abstracts a hydrogen from 5’-deoxyadenosine to give the final product of propionaldehyde and regenerates the coenzyme.
Regeneration of the coenzyme is extremely important, and can be inactivated by glycerol, other substrates and oxygen. When the enzyme is substrate free, two water molecules (W1 and W2) are found bound to potassium. Each water molecule is hydrogen bound by two residues; W1 is bound by Glu-170, and Gln-296, while W2 is bound by His-143, and Asp-335 (7). These are the same residues that bind to the substrate, except Ser-362. The amide oxygen on the serine changes position signaling the change between substrate-free and substrate-bound.
The heterolytic cleavage of the coenzyme Co-C bond creates a radical, and dehydrates the substrate, which carefully regulated by the enzyme structure. The tight interactions between the enzyme and the coenzyme at both the cobalamin moiety and the adenine ring of the adenosyl group produce angular strains, labializing the bond and inevitably breaking the Co-C bond (7). The Co-C bond cleavage has a bond disassociation energy of 30kcal/mol (8). The adenine-binding pocket is trapped by a hydrogen-bonding network, which consists of a water molecule and four amino acid residues, Ser-224, Ser-229, Ser-301, Gly-261. The ribose moiety is then rotated around the glycosidic linkage, giving the adenosyl radical access to the hydrogen atom on the substrate, which is then dehydrated. Before rotation, the substrate is 6.6Å apart from the C5’ radical center of the adenosyl group and way too far for a radical transfer to occur (8). After rotation, the C1 and C2 carbons of the substrate are 3.128Å and 4.091Å away, respectively (8). The pro-S hydrogen atom is now located on the same side as the adenosyl group and very close to the C5’, while the pro-R hydrogen is positioned in a position far from the reaction. The major structural changes of the enzyme upon substrate binding are located in the beta subunit and the region of the alpha subunit that interacts with the gamma subunit (7).
The entire dehydration reaction of the substrate is constant at a pH range between 6.0-10.0 (8). One of the most important critical residues is the histidine residue at 143 in the alpha subunit. The protonated and deprotonated state of this residue fluctuate the strength of the hydrogen-bond interaction with the O2 atom of the substrate. When His143 is protonated, the “catitonic imidazolium” ion strong attracts the substrate O2 oxygen atom, with a distance of 1.667Å apart (8). When His-143 is unprotonated (His-143), the hydrogen connected to the nitrogen on the imidazole group is weakly attracted the O2 oxygen atom, at 1.926Å. The OH migration of the substrate is presumably started when the imidazole ion of the protonated His-143. The proton transfer then occurs between substrate and the histidine residue, which produces a water molecule and aldehyde radical species (8). The carboxyl group on Glu-170 reduces the activation barrier of the OH group migration and stabilizes the aldehyde radical species by temporally accepting a proton. The C-O and O-H bond on C2 are heterolytically cleaved, producing a water molecule. The water molecule then attacks the C1 atom to recombine with the aldehyde radial species to reproduce the protonated form of histidine 143 and OH group to produce the 1,1-diol radical intermediate. This intermediate is 15.8 kcal/mol more stable than the aldehyde radical intermediate (8), but the unprotonated from of His-143 (Hie-143) is actually energetically produces a more favorable overall intermediate product. The aldehyde from the aldehyde radical intermediate is likely to occur without the OH migration in the protonated form of His-143, since the C2 atom of the aldehyde radical species is in close contact with the ribose moiety. The same mechanism with the deprotonated His-143 occurs but has no hydrogen bonding interaction between the substrate and residue occurs. Instead the activation of the OH group is due to the pull of Glu-170 and pushed by the deprotonated His-143 residue (8).
Glycerol dehydratase is an isofunctional enzyme that is almost identical in both structure and function to diol dehydratase, with a Z-score of 67.7 and and E-value of 0.0 (11). The Z score was obtained from Dali server, which compares proteins based on their quaternary or 3D structure, not simply the function. Both enzymes exist as a dimer of the alpha, beta, gamma heterotrimer, and each subunit of glycerol dehydratase is 71%, 58%, and 54% identical to diol dehydratase, respectively (9). Each enzyme however, performs two completely different physiological roles in glycerol metabolism (10). The two enzymes are distinguishable by the monovalent cation selectivity and substrate binding specificity. Glycerol dehydratase (PDB ID: 1IWP) has a higher binding affinity for glycerol. Glycerol dehydratase takes prevalence when bacteria are performing metabolism in anaerobic conditions. The glycerol substrate is more reduced than 1,2-propanediol and is thus more favorable for the when done anaerobically.
Glycerol and diol dehydratase are both substrate sensitive, but diol dehydratase is particularly susceptible to inactivation if glycerol is bound to its binding site. The Co-C bond is irreversibly cleaved, which then forms 5’-deoxyadenosine and converts the cobalmin coenzyme into OH-Cbl (2). This cobalmin species remains tight bound to the enzyme and irreversibly inactivates the function. Ser-122 on glycerol dehydratase has a hydroxyl group that is hydrogen bound to the corrin ring (9). In diol dehydratase, that corresponding residue is Pro-122, which cannot form a hydrogen bond with the very important corrin ring. The five amino acid residues in glycerol dehydratase make shorter bond lengths with the Ado-Cbl complex, rather than the longer bonds in diol dehydratase, which only has four amino acid residues. This difference in bonding allows the adenosylcobalamin group to be more tightly bound to the enzyme.