Enzymes for Research, Diagnostic and Industrial Use


Official Full Name
Glycerol dehydrogenase is an enzyme in the oxidoreductase family that utilizes the NAD+ to catalyze the oxidation of glycerol to form glycerone (dihydroxyacetone).
EC; NAD+-linked glycerol dehydrogenase; glycerol:NAD+ 2-oxidoreductase; GDH; GlDH; GlyDH; 9028-14-2; glycerin dehydrogenase

EC No.
CAS No.9028-14-2
CAS No.9028-14-2
SourceEnterobacter ae...
CAS No.9028-14-2
SourceCellulomonas sp...
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Related Protocols
glycerol dehydrogenase -Enzymatic Assay Protocol
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Many microorganisms utilize glycerol as a source of carbon through coupled oxidative and reductive pathways in anaerobic conditions. Glycerol dehydrogenase (glycerol:NAD+ 2-oxidoreductase, GlyDH, GDH, EC is an enzyme that catalyzes the dehydrogenation of glycerol to form dihydroxyacetone (1,3-dihydroxypropanone) with concomitant reduction of NAD+ to NADH. The dihydroxyacetone is then phosphorylated by dihydroxyacetone kinase and enters the glycolytic pathway for further degradation.


Glycerol dehydrogenases are members of a diverse group of polyol dehydrogenases. Polyol dehydrogenases can be classified into three protein families. The first family is the Zn2+-dependent, “medium-chain” alcohol dehydrogenases, which all have a subunit size that contains approximately 400 residues, containing horse-liver alcohol dehydrogenase and human β1β1 alcohol dehydrogenase. The second family consists of the “short-chain” enzymes, based on a subunit of approximately 250 residues, including alcohol dehydrogenases from Drosophila, ribitol dehydrogenases, mammalian 11β- and 17β-hydroxysteroid dehydrogenases, and β-ketoreductase. The third family is the “iron-containing” alcohol and glycerol dehydrogenases, includes polyol dehydrogenases isolated from bacteria and yeast. Enzymes in this family appear to require a divalent metal ion for catalysis and to distinguish them from the medium-chain polyol dehydrogenase family, which also requires a divalent metal ion.


The subunit of glycerol dehydrogenase has approximate dimensions 60 × 40 × 40 Å and consists of a single polypeptide chain of 370 residues. The N-terminal α/β domain consists of a parallel 6-stranded β sheet, flanked by 4 α helices and an additional β strand (β1). All β strands are connected via α helices, except strands β6 and β9, which are separated by a β hairpin (strands β7, β8). The C-terminal domain comprises two subdomains, each one formed from a bundle of α helices. Helices α6, α7, and α8 form a long antiparallel up-and-down helix bundle with a left-handed superhelical twist of approximately 60° around the common axis of the bundle. Helices α9–α14 form an antiparallel Greek key helix bundle composed of helices α9, α10, α11, α13, and α14.

GDHFigure 1. The GlyDH Subunit from B. stearothermophilus. (Ruzheinikov S N. et al. 2001)

Analysis of the structure shows that a deep cleft is formed between the N- and C-terminal domains, with the Greek key helix bundle and N-terminal domain forming opposing faces of the cleft and the α6–α8 bundle forming the floor of the cleft. The Zn2+ ion is bound deep in the cleft and is tetrahedrally coordinated through ion-dipole interaction with amino acid residues Asp173, His256, and His274 and one water molecule.


Glycerol dehydrogenases catalyze the chemical reaction: glycerol + NAD+ « glycerone + NADH + H+. While the precise mechanism of the enzyme has not yet been characterized, kinetic studies support that the catalysis is comparable to those of other alcohol dehydrogenases. 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.

GDHFigure 2. Mechanism of glycerol dehydrogenase.

Industrial Application

Glycerol is commonly used in many industries, such as pharmaceuticals, food, and cosmetics industry. The formation of crude glycerol has increased as the byproduct of increasing biodiesel production. Therefore, it has been expensive to purify and utilize crude glycerol with increased production in these industries. Researchers of biotechnology are interested in finding new economical ways to utilize low-grade glycerol products. As a catalyst for the conversion of glycerol to glycerone, glycerol dehydrogenase is one such enzyme being investigated for this industrial purpose.


  1. Ruzheinikov S N, Burke J, Sedelnikova S, et al. Glycerol Dehydrogenase: Structure, Specificity, and Mechanism of a Family III Polyol Dehydrogenase. Structure (Cambridge), 2001, 9(9):789-802.

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