Novel ultrasonic bone densitometry based on two longitudinal waves: significant correlation with pQCT measurement values and age-related changes in trabecular bone density, cortical thickness, and elastic modulus of trabecular bone in a normal Japanese population.

UNLABELLED: A reference database for trabecular bone density, cortical thickness, and elastic modulus of trabecular bone for a novel ultrasonic bone densitometry system (LD-100) based on two longitudinal waves (fast and slow) was determined over a wide age range in a normal Japanese population. INTRODUCTION: A novel ultrasonic bone densitometry system (LD-100 system) was applied to create a reference database for trabecular bone density (TBD), cortical thickness (CoTh), and elastic modulus of trabecular bone (EMTb) for this device over a wide age range in a normal Japanese population. METHODS: In a comparative study between LD-100 and peripheral quantitative computed tomography (pQCT) systems, 52 individuals were examined by both systems at the same radius simultaneously. To create a reference database, a total of 2,380 healthy subjects (1,179 men, 1,201 women), ages 18-99 years, were examined using the LD-100 system. RESULTS: Highly significant correlations between the LD-100 and pQCT systems were found in TBD (r = 0.877, p < 0.001) and CoTh (r = 0.723, p < 0.001). For the reference database, peak values of TBD, CoTh, and EMTb were observed at 30-34 years (255.09 mg/cm(3)), 20-24 years (5.23 mm), and 20-24 years (4.09 GPa) in men, and at 25-29 years (209.24 mg/cm(3)), 25-29 years (3.98 mm), and 20-24 years (3.33 GPa) in women, respectively. The TBD fell significantly (p < 0.05) beginning at 55-59 years in both sexes, with a relatively rapid decrease in women. The CoTh showed a significant decrease beginning at 40-44 years in men and 50-54 years in women. The EMTb showed a significant decrease beginning at 40-44 years in men and 55-59 years in women. CONCLUSIONS: The LD-100 system is a useful bone densitometry device and the database of age-related changes in TBD, CoTh, and EMTb established in this study will provide fundamental data for future studies related to bone status.

Body composition and vertebral fracture risk in female patients treated with glucocorticoid.

INTRODUCTION: Glucocorticoid (GC) causes bone loss and an increase in bone fragility. However, fracture risk was found to be only partly explained by bone mineral density in GC-treated patients (GC patients). Although GC causes a change in the distribution of fat in the body, the relationship between body composition and fracture risk in GC patients remains unknown. METHODS: The present study examined the relationship between the presence or absence of vertebral fractures and various indices, including body composition, in 92 premenopausal GC patients, 122 postmenopausal GC patients and 122 postmenopausal age-matched control subjects. Dual-energy X-ray absorptiometry was employed to analyze body composition. RESULTS: Percentage lean body mass (LBM), % fat and % trunk fat were not significantly different between postmenopausal GC patients and the control women. When groups with and without vertebral fractures were compared, % LBM and % fat were significantly higher and lower in groups with vertebral fractures, respectively, in postmenopausal GC patients, but not in the postmenopausal control women, although % trunk fat was not significantly different between groups with and without vertebral fractures. Femoral neck BMD was negatively correlated with % LBM and positively correlated with % fat. In premenopausal GC patients, % trunk fat was significantly higher in the fracture group, although % LBM and % fat were not significantly different between groups with and without vertebral fractures. CONCLUSION: The present study revealed that body composition is related to vertebral fracture risk in GC-treated patients. Lower % fat can be included in the determination of vertebral fractures in postmenopausal GC-treated patients. The influence of body composition on vertebral fracture risk may be different between the pre- and postmenopausal state in GC patients.

Characterization and mechanism of action of a reactivating factor for adenosylcobalamin-dependent glycerol dehydratase.

Adenosylcobalamin-dependent glycerol dehydratase undergoes mechanism-based inactivation by its physiological substrate glycerol. We identified two genes (gdrAB) of Klebsiella pneumoniae for a glycerol dehydratase-reactivating factor (Tobimatsu, T., Kajiura, H., Yunoki, M., Azuma, M., and Toraya, T. (1999) J. Bacteriol. 181, 4110-4113). Recombinant GdrA and GdrB proteins formed a tight complex of (GdrA)(2)(GdrB)(2), which is a putative reactivating factor. The purified factor reactivated the glycerol-inactivated and O(2)-inactivated glycerol dehydratases as well as activated the enzyme-cyanocobalamin complex in vitro in the presence of ATP, Mg(2+), and adenosylcobalamin. The factor mediated the exchange of the enzyme-bound, adenine-lacking cobalamins for free, adenine-containing cobalamins in the presence of ATP and Mg(2+) through intermediate formation of apoenzyme. The factor showed extremely low ATP-hydrolyzing activity and formed a tight complex with apoenzyme in the presence of ADP. Incubation of the enzyme-cyanocobalamin complex with the reactivating factor in the presence of ADP brought about release of the enzyme-bound cobalamin. The resulting tight inactive complex of apoenzyme with the factor dissociated upon incubation with ATP, forming functional apoenzyme and a low affinity form of factor. Thus, it was established that the reactivation of the inactivated holoenzymes takes place in two steps: ADP-dependent cobalamin release and ATP-dependent dissociation of the apoenzyme-factor complex. We propose that the glycerol dehydratase-reactivating factor is a molecular chaperone that participates in reactivation of the inactivated enzymes.

Specificities of reactivating factors for adenosylcobalamin-dependent diol dehydratase and glycerol dehydratase.

Adenosylcobalamin-dependent glycerol and diol dehydratases undergo inactivation by the physiological substrate glycerol during catalysis. In the permeabilized cells of Klebsiella pneumoniae, Klebsiella oxytoca, and recombinant Escherichia coli, glycerol-inactivated glycerol dehydratase and diol dehydratase are reactivated by their respective reactivating factors in the presence of ATP, Mg2+, and adenosylcobalamin. Both of the reactivating factors consist of two subunits. To examine the specificities of the reactivating factors, their genes or their hybrid genes were co-expressed with dehydratase genes in E. coli cells in various combinations. The reactivating factor of K. oxytoca for diol dehydratase efficiently cross-reactivated the inactivated glycerol dehydratase, whereas the reactivating factor of K. pneumoniae for glycerol dehydratase hardly cross-reactivated the inactivated diol dehydratase. Both of the two hybrid reactivating factors rapidly reactivated the inactivated glycerol dehydratase. In contrast, the hybrid reactivating factor containing the large subunit of the glycerol dehydratase reactivating factor hardly reactivated the inactivated diol dehydratase. These results indicate that the glycerol dehydratase reactivating factor is much more specific for the dehydratase partner than the diol dehydratase reactivating factor and that a large subunit of the reactivating factors principally determines the specificity for a dehydratase.

Extremely low activity of methionine synthase in vitamin B-12-deficient rats may be related to effects on coenzyme stabilization rather than to changes in coenzyme induction.

Severely vitamin B-12 (B-12)-deficient rats were produced by feeding a B-12-deficient diet. The status of B-12 deficiency was confirmed by an increase in urinary methylmalonate excretion and decreases in liver B-12 concentrations and cobalamin-dependent methionine synthase activity. Rat liver methionine synthase existed almost exclusively as the holoenzyme. In B-12-deficient rats, the level of methionine synthase protein was lower, although the mRNA level was not significantly different from that of control rats. When methylcobalamin, the coenzyme for methionine synthase, was administered to the B-12-deficient rats, growth, liver B-12 concentrations and urinary excretion of methylmalonate were reversed although not always to control (B-12-sufficient) levels in a short period. During this recovery process, methionine synthase activity and its protein level increased, whereas the mRNA level was unaffected. We reported previously that rat apomethionine synthase is very unstable and is stabilized by forming a complex with methylcobalamin. Thus, the extremely low activity of methionine synthase in B-12-deficient rats may be related to effects on "coenzyme stabilization" (stabilization of the enzyme by cobalamin binding) rather than to changes in "coenzyme induction."

Heterologous high level expression, purification, and enzymological properties of recombinant rat cobalamin-dependent methionine synthase.

Rat methionine synthase was expressed chiefly as apoenzyme in recombinant baculovirus-infected insect cells (Yamada, K., Tobimatsu, T., and Toraya, T. (1998) Biosci. Biotech. Biochem. 62, 2155-2160). The apoenzyme produced was very unstable, and therefore, after complexation with methylcobalamin, the functional holoenzyme was purified to homogeneity. The specific activity and apparent K(m) values for substrates were in good agreement with those obtained with purified rat liver enzyme. The electronic spectrum of the purified recombinant enzyme resembled that of cob(II)alamin and changed to a methylcobalamin-like one upon incubation of the enzyme with titanium(III) and S-adenosylmethionine. The rate of oxidative inactivation of the enzyme in the absence of S-adenosylmethionine was slower with a stronger reducing agent like titanium(III). The nucleotide moiety, especially the phosphodiester group, was shown to play an important role in the binding of the coenzyme to apoprotein and thus for catalysis. Upon incubation with the apoenzyme in the absence of a reducing agent, cyano- and aquacobalamin were not effective or were effective only slightly in reconstituting holoenzyme. Ethyl- and propylcobalamin formed inactive complexes with apoenzyme, which were converted to holoenzyme by photolytic activation. Adenosylcobalamin was not able to form a complex with apoenzyme, which was convertible to holoenzyme by photoirradiation.

A new mode of B12 binding and the direct participation of a potassium ion in enzyme catalysis: X-ray structure of diol dehydratase.

BACKGROUND: Diol dehydratase is an enzyme that catalyzes the adenosylcobalamin (coenzyme B12) dependent conversion of 1,2-diols to the corresponding aldehydes. The reaction initiated by homolytic cleavage of the cobalt-carbon bond of the coenzyme proceeds by a radical mechanism. The enzyme is an alpha2beta2gamma2 heterooligomer and has an absolute requirement for a potassium ion for catalytic activity. The crystal structure analysis of a diol dehydratase-cyanocobalamin complex was carried out in order to help understand the mechanism of action of this enzyme. RESULTS: The three-dimensional structure of diol dehydratase in complex with cyanocobalamin was determined at 2.2 A resolution. The enzyme exists as a dimer of heterotrimers (alphabetagamma)2. The cobalamin molecule is bound between the alpha and beta subunits in the 'base-on' mode, that is, 5,6-dimethylbenzimidazole of the nucleotide moiety coordinates to the cobalt atom in the lower axial position. The alpha subunit includes a (beta/alpha)8 barrel. The substrate, 1,2-propanediol, and an essential potassium ion are deeply buried inside the barrel. The two hydroxyl groups of the substrate coordinate directly to the potassium ion. CONCLUSIONS: This is the first crystallographic indication of the 'base-on' mode of cobalamin binding. An unusually long cobalt-base bond seems to favor homolytic cleavage of the cobalt-carbon bond and therefore to favor radical enzyme catalysis. Reactive radical intermediates can be protected from side reactions by spatial isolation inside the barrel. On the basis of unique direct interactions between the potassium ion and the two hydroxyl groups of the substrate, direct participation of a potassium ion in enzyme catalysis is strongly suggested.

Folylpoly-gamma-glutamate synthetase: generation of isozymes and the role in one carbon metabolism and antifolate cytotoxicity.

A single human gene encodes both mitochondrial and cytosolic isoforms of the enzyme. The major mRNA species in human cells encodes the mitochondrial isoform but alternate translation initiation at a downstream in-frame ATG also generates the cytosolic isoform. Cytosolic FPGS may also be generated by use of alternate transcription initiation start sites 3' to the start ATG of the mitochondrial FPGS. Three additional human FPGS mRNAs differing in exon 1 have been identified. One of these is a major species in HEP-G2 cells and other tissue culture cells, and can encode a protein lacking the first 8 amino acids of cytosolic FPGS. A protein of the predicted size is observed in coupled transcription/translation systems. However, expression of this protein in E. coli does not generate an active enzyme. Mutagenesis studies indicate that Tyr-3 of the missing N terminal residues is required for enzyme activity. The major cellular folate pools are in the cytosol and mitochondria and FPGS activity is normally distributed in both compartments. Mitochondrial FPGS activity is required for mitochondrial folate accumulation, and cells lacking this isozyme are auxotrophic for glycine. Overexpression of cytosolic FPGS does not complement the lack of mitochondrial activity. Cells expressing FPGS activity solely in the mitochondria are glycine prototrophs, but also possess cytosolic folylpolyglutamates and are prototrophic for thymidine and purines, products of cytosolic one carbon metabolism. Although cytosolic folylpolyglutamates cannot enter the mitochondrion, mitochondrial folylpolyglutamates are released intact into the cytosolic compartment. Cellular accumulation of some antifolates and their cytotoxic efficacy is highly responsive to the level of FPGS activity. Polyglutamylation of methotrexate (MTX) has little affect on its affinity for dihydrofolate reductase, its target enzyme, but does affect the cellular accumulation of the drug. The sensitivity of model cells, expressing a range of FPGS activities similar to that observed in leukemia blasts, to MTX varied over four orders of magnitude. MTX toxicity was dependent on cytosolic FPGS activity as this drug does not enter the mitochondria, and cells expressing very high levels of FPGS solely in the mitochondria were resistant to MTX. The cytotoxic efficacy of other folate antagonists that are transported into the mitochondria was enhanced by mitochondrial FPGS activity, even when their loci of inhibition was a cytosolic enzyme. Mitochondrial metabolism of these drugs increased cytosolic drug levels. Compartmentalization of antifolate metabolism has to be considered in evaluating mechanisms for increased drug cytotoxicity and for the development of acquired resistance to these agents.

Identification and expression of the genes encoding a reactivating factor for adenosylcobalamin-dependent glycerol dehydratase.

Adenosylcobalamin-dependent glycerol dehydratase undergoes inactivation by glycerol, the physiological substrate, during catalysis. In permeabilized cells of Klebsiella pneumoniae, the inactivated enzyme is reactivated in the presence of ATP, Mg2+, and adenosylcobalamin. We identified the two open reading frames as the genes for a reactivating factor for glycerol dehydratase and designated them gdrA and gdrB. The reactivation of the inactivated glycerol dehydratase by the gene products was confirmed in permeabilized recombinant Escherichia coli cells coexpressing GdrA and GdrB proteins with glycerol dehydratase.

Crystallization and preliminary x-ray study of two crystal forms of Klebsiella oxytoca diol dehydratase-cyanocobalamin complex.

Two crystal forms of Klebsiella oxytoca diol dehydratase complexed with cyanocobalamin have been obtained and preliminary crystallographic experiments have been performed. The crystals belong to two different space groups, depending on the crystallization conditions. One crystal (form I) belongs to space group P212121 with unit-cell parameters a = 76.2, b = 122.3, c = 209. 6 A, and diffracts to 2.2 A resolution using an X-ray beam from a synchrotron radiation source. The other crystal (form II) belongs to space group P21 with unit-cell parameters a = 75.4, b = 132.7, c = 298.8 A, beta = 91.9 degrees, and diffracts to 3.0 A resolution. For the purpose of structure determination, a heavy-atom derivative search was carried out and some mercuric derivatives were found to be promising. Structure analysis by the multiple isomorphous replacement method is now under way.

Molecular cloning, sequencing and characterization of the genes for adenosylcobalamin-dependent diol dehydratase of Klebsiella pneumoniae.

Klebsiella pneumoniae and some of the other Enterobacteriaceae form both diol dehydratase and glycerol dehydratase in response to growth substrates. To compare these enzymes produced by the same bacterium, the pdd genes of K. pneumoniae encoding adenosylcobalamin-dependent diol dehydratase were cloned and sequenced. The sequential three open reading frames (pddA, pddB, and pddC genes) encoded polypeptides of 554, 228, and 174 amino acid residues with predicted molecular weights of 60,379(alpha), 24,401(beta), and 19,489(gamma), respectively. The deduced amino acid sequences of the subunits were 84-100% and 54-71% identical with those reported for diol dehydratases and glycerol dehydratases, respectively.

Evidence for axial coordination of 5,6-dimethylbenzimidazole to the cobalt atom of adenosylcobalamin bound to diol dehydratase.

It was demonstrated by electron paramagnetic resonance (EPR) spectroscopy that organic radical intermediates disappeared and cob(II)alamin accumulated upon suicide inactivation of diol dehydratase by 2-methyl-1,2-propanediol. The resulting EPR spectra showed that the eight hyperfine lines due to the divalent cobalt atom of cob(II)alamin further split into triplets by the superhyperfine coupling to the 14N nucleus. Essentially the same superhyperfine splitting of the octet into triplets was observed with [14N]- and [15N]apoenzyme. When the adenosyl form of [14N2]- and [15N2]imidazolyl analogues of the coenzyme [Toraya, T., and Ishida, A. (1991) J. Biol. Chem. 266, 5430-5437] was used with unlabeled apoenzyme, the octet showed superhyperfine splitting into triplets and doublets, respectively. Therefore, it was concluded that cobalamin is bound to this enzyme with 5,6-dimethylbenzimidazole coordinating to the cobalt atom. This conclusion is consistent with the fact that the consensus sequence forming part of a cobalamin-binding motif, conserved in methionine synthase and some of the other cobalamin enzymes, was not found in the deduced amino acid sequences of the subunits of diol dehydratase. Adenosylcobinamide methyl phosphate, a coenzyme analogue lacking the nucleotide moiety, underwent cleavage of the cobalt-carbon bond upon binding to the enzyme in the presence of substrate, forming a cob(II)inamide derivative without nitrogenous base coordination, as judged by EPR and optical spectroscopy. Therefore, this analogue may be a useful probe for determining whether the replacement of the 5, 6-dimethylbenzimidazole ligand by a histidine residue takes place upon binding of cobalamin to proteins.

Cloning, sequencing, and heterologous expression of rat methionine synthase cDNA.

Methionine synthase catalyzes cobalamin-dependent methyl transfer reaction from 5-methyltetrahydrofolate to homocysteine, forming methionine. Rat methonine synthase cDNA was cloned and analyzed by RT-PCR, 3'- and 5'-RACE techniques. The cDNA consists of a 0.3-kb upstream untranslated region, a 3.8-kb coding region, and a 0.4-kb downstream untranslated region. The open reading frame encoded a polypeptide of 1,253 amino acid residues with a calculated molecular weight of 139,162. This molecular weight was in good agreement with the observed one (143,000) of the purified rat liver enzyme. The deduced amino acid sequence was 53, 92, and 64% identical with those of the Escherichia coli, human, and presumptive Caenorhabditis elegans enzymes, respectively. All the fingerprint sequences, forming parts of the cobalamin- and S-adenosylmethionine-binding sites, were completely conserved in the rat methionine synthase. A high-level expression of catalytically active enzyme in insect cells was done by infection with a baculovirus containing the rat methionine synthase cDNA.

Heterologous expression, purification, and properties of diol dehydratase, an adenosylcobalamin-dependent enzyme of Klebsiella oxytoca.

Recombinant adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca overexpressed in Escherichia coli was purified to homogeneity. The enzyme has a low solubility and was extracted from the crude membrane fraction with 1% Brij 35 in a high recovery. Subsequent chromatography on DEAE-cellulose resulted in 4.9-fold purification of the enzyme in an overall yield of 65%. The enzyme thus obtained showed specific activity comparable to that of the wild-type enzyme of K. oxytoca. The apparent molecular weight determined by nondenaturing gel electrophoresis on a gradient gel was 220,000. The enzyme consists of equimolar amounts of the three subunits with apparent Mr of 60,000 (alpha), 30,000 (beta), and 19,000 (gamma). Therefore, the subunit structure of the enzyme is most likely alpha2beta2gamma2. The recombinant enzyme was also separated into components F and S upon DEAE-cellulose chromatography in the absence of substrate. Components F and S were identified as the beta subunit and alpha2gamma2 complex, respectively. Apparent Km for adenosylcobalamin, 1,2-propanediol, glycerol, and 1,2-ethanediol were 0.83 microM, 0.08 mM, 0.73 mM, and 0.56 mM, respectively. The three genes encoding the subunits of diol dehydratase were overexpressed individually or in various combinations in Escherichia coli. The alpha and gamma subunits mutually required each other for correct folding forming the soluble, active alpha2gamma2 complex (component S). Expression of the beta subunit in a soluble, active form (component F) was promoted by coexpression with both the alpha and gamma subunits, probably by coexistence with component S. These lines of evidence indicate that each subunit mutually affects the folding of the others in this heterooligomer enzyme.

A protein factor is essential for in situ reactivation of glycerol-inactivated adenosylcobalamin-dependent diol dehydratase.

The adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca undergoes suicidal inactivation by glycerol during catalysis involving irreversible dissociation of the Co-C bond of the coenzyme. The glycerol-inactivated holoenzyme in permeabilized cells (in situ) of E. coli harboring a plasmid containing the diol dehydratase genes and their flanking regions was rapidly reactivated in the presence of free AdoCbl, ATP, and Mg2+. beta,gamma-Methylene ATP was not able to replace ATP. Inactive complexes of the enzyme with aqCbl, CN-Cbl, and PeCbl were activated in situ in the presence of AdoCbl, ATP, and Mg2+, but the complex with AdePeCbl was not. These results suggest that the inactivated holoenzyme is reactivated in situ in the presence of ATP and Mg2+ by exchange of the inactivated coenzyme lacking the adenine moiety for free intact AdoCbl. The in situ reactivation was also observed when an analog lacking the alpha-ribose moiety of the nucleotide loop was used as coenzyme. The results with a recombinant E. coli strains carrying a deletion mutant plasmid demonstrate that certain protein(s) encoded by the 3'-flanking region of the diol dehydratase genes are essential for the in situ reactivation of inactivated diol dehydratase.

Purification and some properties of cobalamin-dependent methionine synthase from rat liver.

Cobalamin-dependent methionine synthase was purified from rat liver. The enzyme activity was separated into two peaks upon Mono-Q column chromatography. Peaks I and II of the enzyme, eluted in this order, were purified 18,000- and 44,000-fold in overall yields of 0.7 and 1.8%, respectively. Peak II methionine synthase, the major fraction, was homogeneous as judged by SDS-polyacrylamide gel electrophoresis. The enzyme was a large monomeric protein with an apparent molecular weight of 143,000 Da. Interconversion of the enzyme between the two peaks was not observed during purification procedures. The enzyme required S-adenosylmethionine and a reducing system for activity. Apparent K(m) values of the peak II enzyme for 5-methyltetrahydrofolate and homocysteine were 75 and 1.7 microM, respectively.

Characterization, sequencing, and expression of the genes encoding a reactivating factor for glycerol-inactivated adenosylcobalamin-dependent diol dehydratase.

Diol dehydratase undergoes suicide inactivation by glycerol during catalysis involving irreversible cleavage of the Co-C bond of adenosylcobalamin. In permeabilized Klebsiella oxytoca and Klebsiella pneumoniae cells, the glycerol-inactivated holoenzyme or the enzyme-cyanocobalamin complex is rapidly activated by the exchange of the inactivated coenzyme or cyanocobalamin for free adenosylcobalamin in the presence of ATP and Mg2+ (Honda, S., Toraya, T., and Fukui, S. (1980) J. Bacteriol. 143, 1458-1465; Ushio, K., Honda, S., Toraya, T., and Fukui, S. (1982) J. Nutr. Sci. Vitaminol. 28, 225-236). Permeabilized Escherichia coli cells co-expressing the diol dehydratase genes with two open reading frames in the 3'-flanking region were capable of reactivating glycerol-inactivated diol dehydratase as well as activating the enzyme-cyanocobalamin complex in situ in the presence of free adenosylcobalamin, ATP, and Mg2+. These open reading frames, designated as ddrA and ddrB genes, were identified as the genes of a putative reactivating factor for inactivated diol dehydratase. The genes encoded polypeptides consisting of 610 and 125 amino acid residues with predicted molecular weights of 64,266 and 13,620, respectively. Co-expression of the open reading frame in the 5'-flanking region was stimulatory but not obligatory for conferring the reactivating activity upon E. coli. Thus, the product of this gene was considered not an essential component of the reactivating factor.

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.

Molecular cloning, sequencing, and expression of the genes encoding adenosylcobalamin-dependent diol dehydrase of Klebsiella oxytoca.

The pdd genes encoding adenosylcobalamin-dependent diol dehydrase of Klebsiella oxytoca were cloned by using a synthetic oligodeoxyribonucleotide as a hybridization probe followed by measuring the enzyme activity of each clone. Five clones of Escherichia coli exhibited diol dehydrase activity. At least one of them was shown to express diol dehydrase genes under control of their own promoter. Sequence analysis of the DNA fragments found in common in the inserts of these five clones and the flanking regions revealed four open reading frames separated by 10-18 base pairs. The sequential three open reading frames from the second to the fourth (pddA, pddB, and pddC genes) encoded polypeptides of 554, 224, and 173 amino acid residues with predicted molecular weights of 60,348 (alpha), 24,113 (beta), and 19,173 (gamma), respectively. Overexpression of these three genes in E. coli produced more than 50-fold higher level of functional apodiol dehydrase than that in K. oxytoca. The recombinant enzyme was indistinguishable from the wild-type one of K. oxytoca by the criteria of polyacrylamide gel electrophoretic and immunochemical properties. It was thus concluded that these three gene products are the subunits of functional diol dehydrase. Comparisons of the deduced amino acid sequences of the three subunits with other proteins failed to reveal any apparent homology.