Overall reaction of glycerol to glycerone with NAD+ as catalyzed by glycerol dehydrogenase
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,[3]Klebsiella aerogenes,[4]Streptococcus faecalis,[5]Erwinia aeroidea,[6]Bacillus megaterium,[7] and Bacillus stearothermophilus. However, most studies of glycerol dehydrogenase have been performed in Bacillus stearothermophilus,(B. 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.[8]
Structure
Glycerol dehydrogenase is a homooctamer composed of eight identical monomer subunits made up of a single polypeptide chain of 370 amino acids (molecular weight 42,000 Da). Each subunit contains 9 beta sheets and 14 alpha helices within two distinct domains (N-terminal, residues 1-162 and C-terminal, residues 163–370). 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 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 (the active region of NAD+) 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.[9]
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 (a useful mechanism in the breakdown of glycerol in bacteria). In addition, GlyDH selectively oxidizes the C2 hydroxyl group to form a ketone rather than a terminal hydroxyl group to form an aldehyde.[10]
Mechanism
While the precise mechanism of this specific enzyme has not yet been characterized, kinetic studies support that GlyDH catalysis of the chemical reaction
glycerol + NAD+ glycerone + NADH + H+
is comparable to those of other alcohol dehydrogenases. Therefore, the following mechanism offers a reasonable representation of glycerol oxidation by NAD+.
Mechanism of glycerol dehydrogenase
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.[11]
Industrial implications
As a result of increasing biodiesel production, formation of the byproduct, crude glycerol, has also increased. While glycerol is commonly used in food, pharmaceuticals, cosmetics, and other industries, increased production of crude glycerol has become very expensive to purify and utilize in these industries. Because of this, researchers are interested in finding new economical ways to utilize low-grade glycerol products. Biotechnology is one such technique: using particular enzymes to break down crude glycerol to form products such as 1,3-propanediol, 1,2-propanediol, succinic acid, dihydroxyacetone (glycerone), hydrogen, polyglycerols, and polyesters. As a catalyst for the conversion of glycerol to glycerone, glycerol dehydrogenase is one such enzyme being investigated for this industrial purpose.[12]
A dehydrogenase is an enzyme belonging to the group of oxidoreductases that oxidizes a substrate by reducing an electron acceptor, usually NAD+/NADP+ or a flavin coenzyme such as FAD or FMN. Like all catalysts, they catalyze reverse as well as forward reactions, and in some cases this has physiological significance: for example, alcohol dehydrogenase catalyzes the oxidation of ethanol to acetaldehyde in animals, but in yeast it catalyzes the production of ethanol from acetaldehyde.
Alcohol dehydrogenases (ADH) (EC 1.1.1.1) are a group of dehydrogenase enzymes that occur in many organisms and facilitate the interconversion between alcohols and aldehydes or ketones with the reduction of nicotinamide adenine dinucleotide (NAD+) to NADH. In humans and many other animals, they serve to break down alcohols that are otherwise toxic, and they also participate in the generation of useful aldehyde, ketone, or alcohol groups during the biosynthesis of various metabolites. In yeast, plants, and many bacteria, some alcohol dehydrogenases catalyze the opposite reaction as part of fermentation to ensure a constant supply of NAD+.
Nicotinamide adenine dinucleotide (NAD) is a coenzyme central to metabolism. Found in all living cells, NAD is called a dinucleotide because it consists of two nucleotides joined through their phosphate groups. One nucleotide contains an adenine nucleobase and the other, nicotinamide. NAD exists in two forms: an oxidized and reduced form, abbreviated as NAD+ and NADH (H for hydrogen), respectively.
In enzymology, a sorbitol-6-phosphate dehydrogenase (EC 1.1.1.140) is an enzyme that catalyzes the chemical reaction
In enzymology, a D-arabinitol 4-dehydrogenase (EC 1.1.1.11) is an enzyme that catalyzes the chemical reaction
In enzymology, a D-malate dehydrogenase (decarboxylating) (EC 1.1.1.83) is an enzyme that catalyzes the chemical reaction
In enzymology, a sn-glycerol-1-phosphate dehydrogenase (EC 1.1.1.261) is an enzyme that catalyzes the chemical reaction
In enzymology, a glycerol 2-dehydrogenase (NADP+) (EC 1.1.1.156) is an enzyme that catalyzes the chemical reaction
In enzymology, a glycerol-3-phosphate dehydrogenase (NAD+) (EC 1.1.1.8) is an enzyme that catalyzes the chemical reaction
In enzymology, histidinol dehydrogenase (HIS4) (HDH) (EC 1.1.1.23) is an enzyme that catalyzes the chemical reaction
In enzymology, an inositol 2-dehydrogenase (EC 1.1.1.18) is an enzyme that catalyzes the chemical reaction
In enzymology, a retinol dehydrogenase (RDH) (EC 1.1.1.105) is an enzyme that catalyzes the chemical reaction
In enzymology, a ribitol 2-dehydrogenase (EC 1.1.1.56) is an enzyme that catalyzes the chemical reaction
In enzymology, a 1,3-propanediol dehydrogenase (EC 1.1.1.202) is an enzyme that catalyzes the chemical reaction
In enzymology, a glycerol-3-phosphate dehydrogenase [NAD(P)+] (EC 1.1.1.94) is an enzyme that catalyzes the chemical reaction
In enzymology, a 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase (EC 1.3.1.28) is an enzyme that catalyzes the chemical reaction
In enzymology, a methylmalonate-semialdehyde dehydrogenase (acylating) (EC 1.2.1.27) is an enzyme that catalyzes the chemical reaction
Azobenzene reductase also known as azoreductase (EC 1.7.1.6) is an enzyme that catalyzes the chemical reaction:
In molecular biology, the ELFV dehydrogenase family of enzymes include glutamate, leucine, phenylalanine and valine dehydrogenases. These enzymes are structurally and functionally related. They contain a Gly-rich region containing a conserved Lys residue, which has been implicated in the catalytic activity, in each case a reversible oxidative deamination reaction.
Diacetyl reductase ((S)-acetoin forming) (EC 1.1.1.304, (S)-acetoin dehydrogenase) is an enzyme with systematic name (S)-acetoin:NAD+ oxidoreductase. This enzyme catalyses the following chemical reaction
References
Notes
↑ Mallinder, Phillip R.; Pritchard, Andrew; Moir, Anne (1992). "Cloning and characterization of a gene from Bacillus stearothermophilus var. non-diastaticus encoding a glycerol dehydrogenase". Gene. 110 (1): 9–16. doi:10.1016/0378-1119(92)90438-U. ISSN0378-1119. PMID1339360.
↑ Burton, Robert Main; Kaplan, Nathan O. (1953). "A DPN Specific Glycerol Dehydrogenase from Aerobacter Aerogenes1". Journal of the American Chemical Society. 75 (4): 1005–1006. doi:10.1021/ja01100a520. ISSN0002-7863.
↑ Lin ECC; Magasanik B (1960). "The activation of glycerol dehydrogenase from Aerobacter aerogenes by monovalent cations". J. Biol. Chem. 235: 1820–1823. PMID14417009.
↑ Leichus, Betty N.; Blanchard, John S. (1994). "Isotopic Analysis of the Reaction Catalyzed by Glycerol Dehydrogenase". Biochemistry. 33 (48): 14642–14649. doi:10.1021/bi00252a033. ISSN0006-2960. PMID7981227.
Asnis RE, Brodie AF (1953). "A glycerol dehydrogenase from Escherichia coli". J. Biol. Chem. 203 (1): 153–9. PMID13069498.
Burton RM; Kaplan NO (1953). "A DPN specific glycerol dehydrogenase from Aerobacter aerogenes". J. Am. Chem. Soc. 75 (4): 1005–1007. doi:10.1021/ja01100a520.
Lin ECC; Magasanik B (1960). "The activation of glycerol dehydrogenase from Aerobacter aerogenes by monovalent cations". J. Biol. Chem. 235: 1820–1823. PMID14417009.
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