Cobalamins and cobalamin-dependent enzymes in Candida utilis.

Candida utilis has been shown to contain 4.7 pmol of cobalamin per g of wet cell paste. Purification of the cobalamin showed it to be a mixture of methylcobalamin and adenosylcobalamin. Two cobalamin-dependent enzyme systems have been found in the yeast: methylcobalamin-dependent methionine biosynthesis and leucine 2,3-aminomutase. The cobalamin extracted from the yeast is as effective as authentic adenosylcobalamin in stimulating leucine 2,3-aminomutase.     

Glial cells as a model for the role of cobalamin in the nervous system: impaired synthesis of cobalamin coenzymes in cultured human astrocytes following short-term cobalamin-deprivation.

The conversion of cyanocobalamin to adenosyl- and methylcobalamin is impaired in cobalamin-deficient cultured human glial cells. In contrast cultured human skin fibroblasts retained their ability to synthesize coenzyme forms when grown in cobalamin-deficient medium. Cells were pre-conditioned by growing in cobalamin-deficient media for six weeks and then subcultured in medium containing either free or transcobalamin II-bound 57Co-cyanocobalamin. Although both coenzyme levels were low in cobalamin-deficient glial cells, the decrease in methylcobalamin was more marked than that of adenosylcobalamin. Methionine synthase and Cb1 reductase activities were markedly decreased in cobalamin-deficient glial cells but were unchanged in fibroblasts cultured in cobalamin-deficient medium. Our data suggest that in glial cells, cobalamin coenzyme synthesis and function is exquisitely sensitive to short-term cobalamin deprivation. Glial cells apparently synthesize and secrete transcobalamin II since antibodies directed against the transport protein inhibit the uptake of free cobalamin.     

Leucine 2,3-aminomutase, an enzyme of leucine catabolism.

The initial step in the fermentation of leucine to acetate, isobutyrate, and ammonia by Clostridium sporogenes is the B12 coenzyme-dependent conversion of alpha-leucine to beta-leucine (3-amino-4-methylpentanoate). The amino group migration reaction, catalyzed by leucine 2,3-aminomutase, is reversible and is inhibited by intrinsic factor. The enzyme activity has been found in several clostridia, in rat, sheep, rhesus, and African green monkey livers, and in human leukocytes.     

A Single Rainbow Trout Cobalamin-binding Protein Stands in for Three Human Binders

Cobalamin uptake and transport in mammals are mediated by three cobalamin-binding proteins: haptocorrin, intrinsic factor, and transcobalamin. The nature of cobalamin-binding proteins in lower vertebrates remains to be elucidated. The aim of this study was to characterize the cobalamin-binding proteins of the rainbow trout (Oncorhynchus mykiss) and to compare their properties with those of the three human cobalamin-binding proteins. High cobalamin-binding capacity was found in trout stomach (210 pmol/g), roe (400 pmol/g), roe fluid (390 nmol/liter), and plasma (2500 nmol/liter). In all cases, it appeared to be the same protein based on analysis of partial sequences and immunological responses. The trout cobalamin-binding protein was purified from roe fluid, sequenced, and further characterized. Like haptocorrin, the trout cobalamin-binding protein was stable at low pH and had a high binding affinity for the cobalamin analog cobinamide. Like haptocorrin and transcobalamin, the trout cobalamin-binding protein was present in plasma and recognized ligands with altered nucleotide moiety. Like intrinsic factors, the trout cobalamin-binding protein was present in the stomach and resisted degradation by trypsin and chymotrypsin. It also resembled intrinsic factor in the composition of conserved residues in the primary cobalamin-binding site in the C terminus. The trout cobalamin-binding protein was glycosylated and displayed spectral properties comparable with those of haptocorrin and intrinsic factor. In conclusion, only one soluble cobalamin-binding protein was identified in the rainbow trout, a protein that structurally behaves like an intermediate between the three human cobalamin-binding proteins.     

The cobalamin-binding protein in zebrafish is an intermediate between the three cobalamin-binding proteins in human

In humans, three soluble extracellular cobalamin-binding proteins; transcobalamin (TC), intrinsic factor (IF), and haptocorrin (HC), are involved in the uptake and transport of cobalamin. In this study, we investigate a cobalamin-binding protein from zebrafish (Danio rerio) and summarize current knowledge concerning the phylogenetic evolution of kindred proteins. We identified a cobalamin binding capacity in zebrafish protein extracts (8.2 pmol/fish) and ambient water (13.5 pmol/fish) associated with a single protein. The protein showed resistance toward degradation by trypsin and chymotrypsin (like human IF, but unlike human HC and TC). The cobalamin analogue, cobinamide, bound weaker to the zebrafish cobalamin binder than to human HC, but stronger than to human TC and IF. Affinity for another analogue, adenosyl-pseudo-cobalamin was low compared with human HC and TC, but high compared with human IF. The absorbance spectrum of the purified protein in complex with hydroxo-cobalamin resembled those of human HC and IF, but not TC. We searched available databases to further explore the phylogenies of the three cobalamin-binding proteins in higher vertebrates. Apparently, TC-like proteins are the oldest evolutionary derivatives followed by IF and HC (the latter being present only in reptiles and most but not all mammals). Our findings suggest that the only cobalamin-binding protein in zebrafish is an intermediate between the three human cobalamin binders. These findings support the hypothesis about a common ancestral gene for all cobalamin-binding proteins in higher vertebrates.     

A Conformational Sampling Model for Radical Catalysis in Pyridoxal Phosphate- and Cobalamin-dependent Enzymes

Cobalamin-dependent enzymes enhance the rate of C–Co bond cleavage by up to ∼10¹²-fold to generate cob(II)alamin and a transient adenosyl radical. In the case of the pyridoxal 5′-phosphate (PLP) and cobalamin-dependent enzymes lysine 5,6-aminomutase and ornithine 4,5 aminomutase (OAM), it has been proposed that a large scale domain reorientation of the cobalamin-binding domain is linked to radical catalysis. Here, OAM variants were designed to perturb the interface between the cobalamin-binding domain and the PLP-binding TIM barrel domain. Steady-state and single turnover kinetic studies of these variants, combined with pulsed electron-electron double resonance measurements of spin-labeled OAM were used to provide direct evidence for a dynamic interface between the cobalamin and PLP-binding domains. Our data suggest that following ligand binding-induced cleavage of the Lys⁶²⁹-PLP covalent bond, dynamic motion of the cobalamin-binding domain leads to conformational sampling of the available space. This supports radical catalysis through transient formation of a catalytically competent active state. Crucially, it appears that the formation of the state containing both a substrate/product radical and Co(II) does not restrict cobalamin domain motion. A similar conformational sampling mechanism has been proposed to support rapid electron transfer in a number of dynamic redox systems.     

The S subunit of D-ornithine aminomutase from Clostridium sticklandii is responsible for the allosteric regulation in D-alpha-lysine aminomutase

Ornithine and lysine are degraded in quite a similar way in Clostridium sticklandii. Both pathways involve adenosylcobalamin-dependent enzymes, d-ornithine 4,5-aminomutase and lysine 5,6-aminomutase. According to previous reports, lysine 5,6-aminomutase is an ATP-dependent allosteric enzyme with many different activators and inhibitors. However, recent studies indicate that ATP does not have a regulatory effect on the recombinant enzyme. To monitor the activity of lysine aminomutase, a novel capillary electrophoresis-based assay method was developed. The present results demonstrate that the S subunit of d-ornithine aminomutase, OraS, is capable of forming a complex with KamDE of lysine 5,6-aminomutase and restores the enzyme's ATP-dependent allosteric regulation. Not only does ATP lower the K(m) of the KamDE-OraS complex for adenosylcobalamin and pyridoxal phosphate, but also OraS protein alone lowers the K(m) of KamDE for adenosylcobalamin and pyridoxal phosphate. The activity of reconstituted enzyme can also be activated by ammonium ion as reported by Morley and Stadtman.     

Adenosylcobalamin and cob(II)alamin as prosthetic groups of 2-methyleneglutarate mutase from Clostridium barkeri.

The ultraviolet/visible spectrum of the pure pink-orange 2-methyleneglutarate mutase from Clostridium barkeri between 300-600 nm showed the presence of cobalamins; notably the peaks at 470 and 528 nm were indicative of oxygen-stable cob(II)alamin and adenosylcobalamin (coenzyme B12), respectively. Using the absorption coefficients of the isosbestic points at 340, 393 and 489 nm, the total cobalamin content was estimated as 3.7 +/- 0.3 mol/mol tetrameric enzyme (m = 300 kDa). Denaturation with 8 M urea in the presence of 2 mM dithiothreitol followed by gel chromatography and renaturation afforded an inactive enzyme which contained 40-50% of the initially bound cobalamin. This preparation could be reactivated to 95-100% by addition of adenosylcobalamin. The cobalamins were removed to 85% from the mutase by denaturation with 8 M urea in the presence of 1 M cyanide (pH 12) with irreversible loss of activity. 2-Methyleneglutarate mutase was inactivated by incubation with aquo-, cyano- or methylcobalamin; up to 50% of the activity was recovered by addition of adenosylcobalamin. Upon incubation of the mutase with [5'-3H]adenosylcobalamin about 30% of the total cobalamin was exchanged by the tritium-labelled cofactor without loss of activity. During aerobic catalysis the enzyme became sensitive towards oxygen which was accompanied by loss of activity and formation of aquocobalamin from adenosylcobalamin. EPR spectroscopy demonstrated the presence of 0.8 mol base-on cob(II)alamin/mol enzyme. Upon addition of 2-methyleneglutarate a second EPR signal of about equal intensity at g = 2.13 arose. The question of whether the oxygen-stable cob(II)alamin participates in catalysis or its complex with the enzyme represents an inactive form is currently under investigation.     

Glucose determination in samples taken by microdialysis by peroxidase-catalyzed luminol chemiluminescence

An automatic, luminometric assay of glucose in samples of the extracellular water space obtained by microdialysis is described. The assay involves oxidation by glucose oxidase (EC 1.1.3.4) and mutarotation of glucose by aldose mutarotase (EC 5.1.3.3.). The H2O2 formed is subsequently determined in a reaction catalyzed by horseradish peroxidase (EC 1.11.1.7) using luminol as electron donor. The assay is linear between 0.01 and 1 nmol in the cuvette. The detection limit, defined as 3 standard deviations of the reagent blank, was 0.008 mumol/liter in the cuvette. A complete oxidation of glucose is obtained within 4 min and 25 samples are automatically assayed within 75 min. Addition of microdialysate sample obtained from human adipose tissue in vivo did not interfere with the standard curves. Glucose added to microdialysate resulted in a complete recovery compared to a H2O2 standard. Analytical interference from different factors was investigated. No interference was observed up to the following concentrations: 5 mumol/liter epinephrine, 1 mumol/liter norepinephrine, 100 mumol/liter insulin, 500 mumol/liter pyruvate, 50 mmol/liter lactate, and 1 mumol/liter ascorbate. The glucose values with the present method correlated strongly (r = 0.984) with values obtained using a routine method involving glucose oxidase and peroxidase.     

Solid-phase immunoassay for the vitamin B12-binding protein transcobalamin II in human serum

A solid-phase radio immunoassay was developed for total immunoreactive transcobalamin II (TC II). Rabbit antihuman TC II antiserum (which recognizes both apo- and holo-TC II), was immobilized by covalent binding to acrylamide-acrylic acid copolymer beads. A normal mean and SD for immunoreactive TC II in serum was determined in 130 healthy adult individuals and found to be 1150 ± 250 ng/liter cobalamin equivalent. Mean holo-TC II (N = 30), estimated by substraction of apo-TC II from total TC II, was 137 ng/liter bound cobalamin (or 12% of total TC II). Three patients with lack of functional TC II had immunoreactive TC II levels between 22 and 39% of normal mean, which demonstrated that the solid-phase bound antiserum recognized deficient TC II molecules, whereas the same antiserum in its soluble form did not. Eight out of nine individuals, recognized as heterozygous for TC II deficiency, had TC II levels below the normal range, on the order of 50% of the normal mean. The stability of immunoreactive TC II was strongly enhanced by the presence of an unknown serum factor not corresponding to serum albumin.     

Interference by methylcobalamin analogues with synthesis of cobalamin coenzymes in human lymphocytes in vitro.

1. 72 h uptake of cyano[57Co]cobalamin and formation of 57Co-labelled methylcobalamin, adenosylcobalamin and hydroxocobalamin has been estimated with and without the addition of methylcobalamin analogues in phytohaemagglutinin-stimulated lymphocytes from healthy human subjects. 2. Difluorochloromethylcobalamin reduced cell uptake of cyanocobalamin and caused a disproportionate reduction in synthesis of adenosylcobalamin. 3. Methylcobalamin-palladium trichloride reduced cell uptake of cyanobalamin more effectively than did difluorochloromethylcobalamin and reduced the formation of methylcobalamin, adenosylcobalamin and hydroxocobalamin in proportion. 4. The results suggest that in addition to inhibiting uptake of cyanocobalamin, one or both compounds may have interfered directly with the mechanism of synthesis of the cobalamin coenzymes.     

The role of S-adenosylmethionine in the lysine 2,3-aminomutase reaction

The interconversion of L-lysine and L-3,6-diamino-hexanoate (L-beta-lysine) catalyzed by lysine 2,3-aminomutase is known to be stimulated by added S-adenosylmethionine (Chirpich, T. P., Zappia, V., Costilow, R. N., and Barker, H. A. (1970) J. Biol. Chem. 245, 1778-1789). In this paper we show that enzyme activated by S-[2,8,5'-3H]adenosylmethionine catalyzes the conversion of L-lysine to the equilibrium mixture of L-lysine and L-beta-lysine with incorporation of high levels of tritium into both isomers. The tritium levels in the isomers reflect the equilibrium constant for their interconversion, 84% in the L-beta-lysine and 16% in L-lysine compared with Keq = 5.3 +/- 0.3 in the direction of the formation of L-beta-lysine at pH 7.7 and 30 degrees C. No significant tritium is incorporated into lysine from S-[2,8-3H]adenosylmethionine or S-adenosyl[methyl-3H] methionine under comparable conditions. Therefore, the tritium incorporated into lysine in the former reaction arises from the 5'-position of the 5'-deoxyadenosyl group in S-adenosylmethionine. These experiments implicate the 5'-deoxyadenosyl portion of S-adenosylmethionine in the hydrogen transfer mechanism of this reaction, perhaps in a role analogous to that played by the 5'-deoxyadenosyl moiety of deoxyadenosyl cobalamin in coenzyme B12-dependent rearrangements.     

Isolation and characterization of methylmalonyl-CoA mutase from human placenta

Methylmalonyl-CoA mutase, one of two known cobalamin-dependent enzymes present in mammalian tissues, has been isolated from 2.5 kg of human placenta utilizing affinity chromatography on 5'-deoxyadenosylcobalamin-Sepharose as the major purification step. The enzyme gives a single band on polyacrylamide disc gel electrophoresis. The Mr of the enzyme is 145,000 and it has two subunits of Mr = 72,000. Amino acid analysis reveals major differences from other human cobalamin-binding proteins. Based on x-ray fluorescence, the enzyme has 2 mol of cobalamin bound/mol of enzyme. In contrast to purified cobalamin transport proteins, most of the cobalamin bound to the enzyme is not released by boiling at low pH in the presence of KCN, or dialysis against 7.5 M guanidine containing 0.2 M dithiothreitol, or both, suggesting the possibility that cobalamin may be covalently attached to the purified enzyme. Both precipitating antibodies and antibodies that inhibit enzyme activity have been raised in a chicken.     

Cobalamin neuropathy. Is S-adenosylhomocysteine toxicity a factor?

Cobalamin neuropathy was produced in cape fruit bats (Rousettus aegyptiacus) by a cobalamin-free diet combined with intermittent exposure to nitrous oxide, which inactivates cobalamin. There were no significant differences in S-adenosylmethionine/S-adenosylhomocysteine ratios in the central nervous system of cobalamin-deficient and cobalamin-replete bats. Taken with other data there are no grounds of support for a hypothesis that cobalamin neuropathy is the result of impaired methylation, however produced.     

Activation of a Cl- current by hypotonic volume increase in human endothelial cells

We have used whole-cell and perforated patches to study ionic currents induced by hypotonic extracellular solutions (HTS, 185 mOsm instead of 290 mOsm) in endothelial cells from human umbilical veins. These currents activated within 30-50 s after application of HTS, reached a maximum value after approximately 50-150 s and recovered completely after re-exposing the cells to normal osmolarity. They slowly inactivated at potentials positive to +50 mV. The same current was also activated by breaking into endothelial cells with a hypertonic pipette solution (377 mOsm instead of 290 mOsm). The reversal potential of these volume-induced currents using different extracellular and intracellular Cl- concentrations was always close to the Cl(-)- equilibrium potential. These currents are therefore mainly carried by Cl-. DIDS only weakly blocked the current (KI = 120 microM), while another Cl(-)-channel blocker, DCDPC (20 microM) was ineffective. We were unable to record single channel activity in cell-attached patches but we always observed an increased current variance during HTS. From the mean current-variance relation of the whole-cell current records, we determined a single channel conductance of 1.1 pS. The size and kinetics of the current were not correlated with the concomitant changes in intracellular calcium. Furthermore, the currents could still be activated in the presence of 10 mmol/liter intracellular EGTA and are thus Ca2+ independent. A similar current was also activated with iso-osmotic pipette solutions containing 300 mumol/liter GTP gamma S. Neomycin (1 mmol/liter), a blocker of PLC, did not prevent activation of this current. TPA (4 mumol/liter) was also ineffective in modulation of this current. The HTS-induced current was completely blocked by 10 mumol/liter pBPB, a PLA2 inhibitor. NDGA (4 mumol/liter) and indomethacin (5 mumol/liter), blockers of lipoxygenase and cyclo- oxygenase respectively, did however not affect the current induced by hypotonic solutions. The effects of arachidonic acid (10 mumol/liter) were variable. In 12 out of 40 cells it either directly activated a Cl- current or potentiated the current activated by HTS. The membrane current was decreased at all potentials in 18 cells, and was not affected in 10 cells. The HTS-induced currents may therefore be modulated by cleavage products of PLA2, but not by messengers downstream of arachidonic acid. Loading the cells with a segment of the heat stable protein kinase A inhibitor PKI (5-24) did not prevent activation of the HTS-induced current.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cloning, sequencing, heterologous expression, purification, and characterization of adenosylcobalamin-dependent D-lysine 5, 6-aminomutase from Clostridium sticklandii

D-Lysine 5,6-aminomutase from Clostridium sticklandii catalyzes the 1,2-shift of the epsilon-amino group of D-lysine and reverse migration of C5(H). The two genes encoding 5,6-aminomutase have been cloned, sequenced, and expressed in Escherchia coli. They are adjacent on the Clostridial chromosome and encode polypeptides of 57. 3 and 29.2 kilodaltons. The predicted amino acid sequence includes a conserved base-off 5'-deoxyadenosylcobalamin binding motif and a 3-cysteine cluster in the small subunit, as well as a P-loop sequence in the large subunit. Activity of the recombinant enzyme exceeds that of the 5,6-aminomutase purified from C. sticklandii by 6-fold, presumably due to the absence of bound, inactive corrinoids in the recombinant enzyme. The K(m) values for adenosylcobalamin and pyridoxal 5'-phosphate are 6.6 and 1.0 microM, respectively. ATP does not have a regulatory effect on the recombinant protein. The rapid turnover associated inactivation reported for the enzyme purified from Clostridium is also seen with the recombinant form. Aminomutase activity does not depend on structural or catalytic metal ions. Electron paramagnetic resonance experiments with [(15)N-dimethylbenz-imidazole]adenosylcobalamin demonstrate base-off binding, consistent with other B(12)-dependent enzymes that break unactivated C-H bonds.     

Separation by high-performance liquid chromatography of alpha- and beta-amino acids: application to assays of lysine 2,3-aminomutase and leucine 2,3-aminomutase

Several pairs of alpha- and beta-amino acids were well separated as the ortho-phthalaldehyde derivatives by reversed phase HPLC. These included alpha- and beta-lysine, leucine, tyrosine, serine, aminoisobutyric acid, and beta-hydroxyvaline and its positional isomer. The separation was applicable to assays of lysine 2,3-aminomutase and leucine 2,3-aminomutase. However, for unknown reasons, no leucine 2,3-aminomutase activity could be found in rat liver homogenate.     

MTHFR Polymorphisms Involved in Vitamin B12 Deficiency Associated with Atrophic Gastritis

Genetic polymorphisms affecting methylentetrahydrofolate reductase (MTHFR) activity may influence hematological and neurological dysfunction in cobalamin-deficient patients. We studied the prevalence of C677T and A1298C polymorphisms by analyzing genomic DNA in 30 cobalamin-deficient patients. No significant difference was found in 677 and 1298 genotype distribution with respect to hematological parameters, B₁₂ and folate levels, and neurological symptoms. The two MTHFR polymorphisms were not protective against anemia or neurological dysfunction in patients with cobalamin deficiency; however, we found evidence of a significant increase in atrophic gastritis in the 677TT group (P = 0.009) but not for the 1298CC genotype. Based on observations that inadequate cobalamin intake and reduced MTHFR activity might be significant risk factors for gastric cancer, and the increased risk of gastric cancer shown in patients affected by atrophic gastritis, we speculate that concomitant atrophic gastritis and impaired MTHFR function could have a role in the development of gastric cancer.     

Dihydroflavin-driven Adenosylation of 4-Coordinate Co(II) Corrinoids: ARE COBALAMIN REDUCTASES ENZYMES OR ELECTRON TRANSFER PROTEINS?*

The identity of the source of the biological reductant needed to convert cobalamin to its biologically active form adenosylcobalamin has remained elusive. Here we show that free or protein-bound dihydroflavins can serve as the reductant of Co²⁺Cbl bound in the active site of PduO-type ATP-dependent corrinoid adenosyltransferase enzymes. Free dihydroflavins (dihydroriboflavin, FMNH₂, and FADH₂) effectively drove the adenosylation of Co²⁺Cbl by the human and bacterial PduO-type enzymes at very low concentrations (1 μm). These data show that adenosyltransferase enzymes lower the thermodynamic barrier of the Co²⁺ → Co⁺ reduction needed for the formation of the unique organometalic Co–C bond of adenosylcobalamin. Collectively, our in vivo and in vitro data suggest that cobalamin reductases identified thus far are most likely electron transfer proteins, not enzymes.