Glycerol dehydrogenase
Glycerol dehydrogenase is an enzyme in the oxidoreductase family that utilizes the NAD+ to catalyze the oxidation of glycerol to form glycerone.
This enzyme is an oxidoreductase, specifically a metal-dependent alcohol dehydrogenase that plays a role in anaerobic glycerol metabolism and has been isolated from a number of bacteria, including Enterobacter aerogenes, ''Klebsiella aerogenes, Streptococcus faecalis, Erwinia aeroidea, Bacillus megaterium, and Bacillus stearothermophilus. However, most studies of glycerol dehydrogenase have been performed in Bacillus stearothermophilus, '' due to its thermostability and the following structural and functional information will, therefore, refer primarily to the characterization of the enzyme in this bacterium.
Structure
Glycerol dehydrogenase is a homooctamer composed of eight identical monomer subunits made up of a single polypeptide chain of 370 amino acids. Each subunit contains 9 beta sheets and 14 alpha helices within two distinct domains. The deep cleft formed between these two domains serves as the enzyme's active site. This active site consists of one bound metal ion, one NAD+ nicotinamide ring binding site, and a [Enzyme Enzyme substrate (biology)|substrate (biology)|substrate] binding site.Research into the structure of B. stearothermophilus shows that the active site contains a divalent cation—zinc ion, Zn2+. This zinc ion forms tetrahedral dipole interactions between the amino acid residues Asp173, His256, and His274 as well as a water molecule.
The NAD+ binding site, resembling the Rossmann fold within the N-terminal domain, extends from the surface of the enzyme to the cleft containing the active site. The nicotinamide ring binds in a pocket of the cleft consisting of the residues Asp100, Asp123, Ala124, Ser127, Leu129, Val131, Asp173, His174, and Phe247.
Finally, the substrate binding site consists of the residues Asp123, His256, His274 as well as a water molecule.
Function
Encoded by the gene gldA, the enzyme glycerol dehydrogenase, GlyDH catalyzes the oxidation of glycerol to glycerone. Unlike more common pathways utilizing glycerol, GlyDH effectively oxidizes glycerol in anaerobic metabolic pathways under ATP-independent conditions. In addition, GlyDH selectively oxidizes the C2 hydroxyl group to form a ketone rather than a terminal hydroxyl group to form an aldehyde.Mechanism
While the precise mechanism of this specific enzyme has not yet been characterized, kinetic studies support that GlyDH catalysis of the chemical reactionis comparable to those of other alcohol dehydrogenases. Therefore, the following mechanism offers a reasonable representation of glycerol oxidation by NAD+.
After NAD+ is bound to the enzyme, glycerol substrate binds to the active site in such a way as to have two coordinated interactions between two adjacent hydroxyl groups and the neighboring zinc ion. GlyDH then catalyzes the base-assisted deprotonation of the C2 hydroxyl group, forming an alkoxide. The zinc atom further serves to stabilize the negative charge on the alkoxide intermediate before the excess electron density around the charged oxygen atom shifts to form a double bond with the C2 carbon atom. Hydride is subsequently removed from the secondary carbon and acts as a nucleophile in electron transfer to the NAD+ nicotinamide ring. As a result, the H+ removed by the base is released as a proton into the surrounding solution; followed by the release of the product glycerone, then NADH by GlyDH.