Glycerol Dehydrase Research Articles

Determination of vitamin B12 using the enzyme glycerol dehydrase

Glycerol dehydrase is an enzyme that catalyzes dehydration of glycerol into β‐propionaldehyde. It requires 5′‐deoxyadenosylcobalamin, one of the forms of vitamin B12, as a coenzyme. The enzyme is inactivated in vitro by all forms of vitamin B12 stoichiometrically. The objective of this study was to determine vitamin B12 content by utilizing the inactivation of the enzyme by vitamin B12. After various examinations, an excellent standard curve was obtained up to 1 pmol vitamin B12 using 14 mU of the enzyme per tube. Glycerol dehydrase does not respond to vitamin B12 if it is bound to haptocorrin, a vitamin B12‐binding protein. This necessitates a procedure for extraction of vitamin B12 from samples before assay. The enzyme was less inactivated by 5′‐deoxyadenosylcobalamin than any other form of vitamin B12. However, this did not matter because all forms of vitamin B12 were converted into cyanocobalamin during the extraction procedure cited above, which was performed in a buffer containing potassium cyanide.

Cloning, Sequencing, and High Level Expression of the Genes Encoding Adenosylcobalamin-dependent Glycerol Dehydrase of Klebsiella pneumoniae

The gld genes encoding adenosylcobalamin-dependent glycerol dehydrase of Klebsiella pneumoniae were cloned by cross-hybridization with a DNA fragment of Klebsiella oxytoca diol dehydrase genes. Since the Escherichia coli clones isolated did not show appreciable enzyme activity, plasmids for high level expression of cloned genes were constructed. The enzyme expressed in E. coli was indistinguishable from the wild-type glycerol dehydrase of K. pneumoniae by the criteria of polyacrylamide gel electrophoretic, immunochemical, and catalytic properties. It was also shown that the recombinant functional enzyme consists of Mr 61,000, 22,000, and 16, 000 subunits. Sequence analysis of the genes revealed four open reading frames separated by 2-12 bases. The sequential three open reading frames from the first to the third (gldA, gldB, and gldC genes) encoded polypeptides of 555, 194, and 141 amino acid residues with predicted molecular weights of 60,659(alpha), 21,355(beta), and 16,104(gamma), respectively. High level expression of these three genes in E. coli produced more than 14-fold higher level of fully active apoenzyme than that in K. pneumoniae. It was thus concluded that these are the genes encoding the subunits of glycerol dehydrase. The deduced amino acid sequences of the three subunits were 71, 58, and 54% identical with those of the alpha, beta, and gamma subunits of diol dehydrase, respectively, but failed to show any apparent homology with other proteins.

Production of 3-Hydroxypropionaldehyde from Glycerol

3-Hydroxypropionaldehyde is a precursor to acrolein, which can be used as an intermediate for making acrylic acid and a variety of other useful industrial chemicals. Conversion of glycerol, a renewable resource, to 3-hydroxypropionaldehyde was attempted via action of glycerol dehydrase isolated from Lactobacillus sp. strain NRRL B-1720. This method, however, was unsatisfactory because enzyme activity was lost within 60 to 90 min after the reaction initiation. Fermentation of glycerol by whole cells of Klebsiella pneumoniae NRRL B-199 in the presence of optimal semicarbazide hydrochloride proved more effective. Using this technique, glycerol solutions of 30 g/liter yielded 3-hydroxypropionaldehyde solutions of 13.1 g/liter. Thus, a conversion efficiency equal to 55% of the theoretical maximum was realized.

A physical explanation of the EPR spectrum observed during catalysis by enzymes utilizing coenzyme B

We have proposed that the “doublet” EPR spectra observed during catalysis by a number of coenzyme B 1,2-requiring enzymes arises from a weak electrostatic exchange interaction between an organic free radical and low spin Co(II), B 1,2r. By varying the magnitude of the exchange coupling we have quite accurately simulateed the published EPR spectra from the enzyme systems: diol dehydrase, glycerol dehydrase, ribonucleotide reductase, and ethanolamine ammonia lyase. A dipolar model was shown to be incompatible with the observed properties of these systems.

Detection of intermediates during the conversion of propane-1,2-diol to propionaldehyde by glycerol dehydrase, a coenzyme B12-dependent reaction

Detection of intermediates during the conversion of propane-1,2-diol to propionaldehyde by glycerol dehydrase, a coenzyme B12-dependent reaction

Studies on the thermal dissociation of glycerol dehydrase

It has previously been shown that the interaction of the apoenzyme of glycerol dehydrase with Li or Na ions produced a reversible dissociation into two different, separately inactive protein subunits, A and B. On the other hand the formation of the complex AB · B 12OH between hydroxocobalamin and the associated apoenzyme AB, strengthens the binding of A to B so that effective dissociating agents for the apoenzyme, such as Li, Na, or EDTA, do not cause dissociation. Dissociation of AB · B 12OH can be achieved, however, if the complex is heated to over 60 °. Heating produces an active subunit B, an inactivated subunit A, and a B 12 derivative. Li and Na ions promote this thermal dissociation while K, Rb, Cs, and NH 4 ions inhibit it, in descending order. When the separated subunits are heated individually; subunit A is very heat labile and is somewhat stabilized against heating by high concentrations (of the order of 2 m) of Na +, Rb +, K +, and Cs +, but not by Li + at any concentration tried; while subunit B is stabilized against heat by K + and is made more labile to heat by Na + or Li +. Potassium ions inhibit heat-mediated dissociation of AB · B 12OH complex. Potassium ions may thus aid the association, while sodium or lithium ions may promote dissociation, by virtue of their effects on the conformation of subunit B in the AB · B 12OH complex.

Vitamin B 12.

In spite of the considerable progress made in recent years toward the understanding of the chemistry and biological function of the cobalt-containing B(12) group of compounds, much of the information still is more descriptive than definitive in nature. In general terms, it is known that the free vitamin forms can function as methyl group carriers and that the 5'-deoxyadenosyl or coenzyme forms serve as hydrogen carriers; but the mechanism of these processes is not understood in detail. More systematic studies of the pure chemistry of these complex molecules containing a carbon-cobalt covalent bond are needed before the biochemist can interpret many of his observations on the enzyme-catalyzed reactions. Even in relatively simple solutions it is difficult to ascertain the state of oxidation of several of the vitamin forms, and these problems are compounded when the reactive thiol compounds and complex proteins of the biological systems also are present. For example, both vitamin B(12r) (the Co(2+) form) and corresponding analogs are known to disproportionate in solution to B(12s) (Co(1+)) and B(12a) (Co(3+)) under a variety of mild conditions (12, 57). This means that in the biological systems it is exceedingly difficult to ascertain the chemical nature of many B(12) intermediates and reaction products. The role of the protein moiety of the various B(12)-linked enzymes in the catalytic processes is little known as is, also, the mode of binding of the B(12) derivative to the protein. These types of questions perhaps can be answered eventually by the crystallographers, whose art is becoming increasingly sophisticated. Note added after preparation of manuscript. In contrast to the values given in Table 4 for the molecular weights of the two dissimilar protein moieties of glycerol dehydrase, a recent report (57a), gives a value of 188,000 for the molecular weight of a stable, catalytically inactive complex of 1 mole of hydroxocobalamin and 1 mole of the apoenzyme complex of glycerol dehydrase. The latter is presumed to contain one equivalent of each of the two dissimilar protein subunits. The original estimate of 240,000 as the molecular weight of the unstable sulfhydryl protein moiety (39) was undoubtedly made on partially aggregated material.

Purification and Properties of Glycerol Dehydrase

Abstract Glycerol dehydrase, a coenzyme B12-dependent enzyme, from Aerobacter aerogenes, has been purified to a stable, inactive, homogeneous complex with hydroxocobalamin. The inactive complex can be converted to the active holoenzyme by replacement of hydroxocobalamin with coenzyme B12 in the presence of Mg++ and SO3=. The molecular weight of the hydroxocobalamin apoenzyme complex is 188,000 and 1 mole of hydroxocobalamin is bound to 1 mole of the apoenzyme. There is a significant difference between the spectra of the dissociated and the protein-bound hydroxocobalamin. The greater stability of the protein hydroxocobalamin complex as compared to the apoenzyme was shown by various methods. Complete dissociation of the apoenzyme is caused by low pH, Na+, or EDTA, whereas the hydroxocobalamin apoenzyme complex does not dissociate under the same conditions. EDTA markedly inhibits the activity of the enzyme whereas other chelating agents have little or no inhibitory effect. Partial reversal of the inhibitory effect of EDTA is achieved by increased concentrations of glycerol or by adding an excess of either subunit, A or B. The experimental evidence suggests that the effect of EDTA on the apoenzyme does not involve a divalent cation.

SOME PROPERTIES OF THE GLYCEROL DEHYDRASE SYSTEM FROM A SPECIES OF LACTOBACILLUS.

Annals of the New York Academy of SciencesVolume 112, Issue 2 p. 706-712 SOME PROPERTIES OF THE GLYCEROL DEHYDRASE SYSTEM FROM A SPECIES OF LACTOBACILLUS K. L. Smiley, K. L. Smiley Hiram Walker & Sons, Inc., Peoria, Ill. Northern Utilization Research Laboratory, United States Department of Agriculture, Peoria, Ill.Search for more papers by this authorMurray Sobolov, Murray Sobolov Hiram Walker & Sons, Inc., Peoria, Ill. Hiram Walker & Sons Limited, Walkerville, Ontario, Canada.Search for more papers by this author K. L. Smiley, K. L. Smiley Hiram Walker & Sons, Inc., Peoria, Ill. Northern Utilization Research Laboratory, United States Department of Agriculture, Peoria, Ill.Search for more papers by this authorMurray Sobolov, Murray Sobolov Hiram Walker & Sons, Inc., Peoria, Ill. Hiram Walker & Sons Limited, Walkerville, Ontario, Canada.Search for more papers by this author First published: April 1964 https://doi.org/10.1111/j.1749-6632.1964.tb45048.xCitations: 6 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume112, Issue2Vitamin B12 CoenzymesApril 1964Pages 706-712 RelatedInformation

A cobamide-requiring glycerol dehydrase from an acrolein-forming lactobacillus

A glycerol dehydrase has been isolated from Lactobacillus 208-A which requires a cobamide coenzyme and a univalent cation for activity. The enzyme is measured by the colorimetric determination of the acrolein formed from β-hydroxypropionaldehyde produced from glycerol by the dehydrase. Vitamin B 12 inhibits the dehydrase. The degree of inhibition by vitamin B 12 is greater when it contacts the apoenzyme before the cobamide coenzyme. The toxicity of mercurial compounds can be relieved by cobamide coenzymes or by 2,3-dimercaptopropanol (BAL). This dehydrase has an optimum pH of 5.8–6.0.