Cbl-dependent Enzymes Research Articles

Elucidating the mechanism of cob(I)alamin mediated methylation reactions by alkyl halides: SN2 or radical mechanism?

Methyl transfer reactions mediated by cobalamins (Cbls) have been considered as one of the most important biologically relevant molecular transformations for many enzymatic reactions catalyzed by Cbl-dependent enzymes. The exact mechanism of methyl transfer reactions involving Cbls is still poorly understood. To investigate the mechanistic details of Cbl mediated methylations by alkyl halides, density functional theory (DFT) along with the polarizable continuum model (PCM/water) for solvation has been applied. Two different mechanisms have been examined, namely SN2 and radical-based electron transfer (ET) to elucidate the methyl transfer reaction. The calculations have suggested that the methyl transfer from methyl halides proceeds through SN2 nucleophilic displacement. However, with more bulky alkyl substrate, namely isopropyl and tert-butyl halides the reaction followed the ET-based radical pathway which is associated with an ET from diradical form of cob(I)alamin to alkyl halides. Our proposed mechanism for alkyl transfer reaction corroborates with the experimental findings, which reported a mechanistic switch from a two-electron (SN2-type) to a one-electron mechanism for sterically demanding alkyl halides. The present theoretical contribution provides more in-depth insight into the methyl transfer reaction catalyzed by corrinoid-dependent methyltransferases.

The clinical presentation of cobalamin-related disorders: From acquired deficiencies to inborn errors of absorption and intracellular pathways.

This review gives an overview of clinical characteristics, treatment and outcome of nutritional and acquired cobalamin (Cbl; synonym: vitamin B12) deficiencies, inborn errors of Cbl absorption and intracellular trafficking, as well as methylenetetrahydrofolate dehydrogenase (MTHFD1) and methylene tetrahydrofolate reductase (MTHFR) deficiencies, which impair Cbl-dependent remethylation. Acquired and inborn Cbl-related disorders and MTHFR deficiency cause multisystem, often severe disease. Failure to thrive, neurocognitive or psychiatric symptoms, eye disease, bone marrow alterations, microangiopathy and thromboembolic events are characteristic. The recently identified MTHFD1 defect additionally presents with severe immune deficiency. Deficient Cbl-dependent enzymes cause reduced methylation capacity and metabolite toxicity. Further net-effects of perturbed Cbl function or reduced Cbl supply causing oxidative stress, altered cytokine regulation or immune functions are discussed.

Molecular basis of cobalamin-dependent RNA modification.

Queuosine (Q) was discovered in the wobble position of a transfer RNA (tRNA) 47 years ago, yet the final biosynthetic enzyme responsible for Q-maturation, epoxyqueuosine (oQ) reductase (QueG), was only recently identified. QueG is a cobalamin (Cbl)-dependent, [4Fe-4S] cluster-containing protein that produces the hypermodified nucleoside Q in situ on four tRNAs. To understand how QueG is able to perform epoxide reduction, an unprecedented reaction for a Cbl-dependent enzyme, we have determined a series of high resolution structures of QueG from Bacillus subtilis. Our structure of QueG bound to a tRNATyr anticodon stem loop shows how this enzyme uses a HEAT-like domain to recognize the appropriate anticodons and position the hypermodified nucleoside into the enzyme active site. We find Q bound directly above the Cbl, consistent with a reaction mechanism that involves the formation of a covalent Cbl-tRNA intermediate. Using protein film electrochemistry, we show that two [4Fe-4S] clusters adjacent to the Cbl have redox potentials in the range expected for Cbl reduction, suggesting how Cbl can be activated for nucleophilic attack on oQ. Together, these structural and electrochemical data inform our understanding of Cbl dependent nucleic acid modification.

Relevance of vitamins, homocysteine and other metabolites in neuropsychiatric disorders.

Indistinguishable hematologic abnormalities are seen in most patients with cobalamin (Cbl, vitamin B12) or folate deficiency. Approximately one third of Cbl-deficient patients develop a wide variety of non-focal neuropsychiatric abnormalities that are not seen in folate deficiency. Serum levels of homocysteine are elevated to a similar degree in Cbl-deficient patients with and without neuropsychiatric abnormalities, and in folate-deficient patients. Serum levels of eight other metabolites including methylmalonic acid also fail to elucidate the biochemical basis for the neuropsychiatric abnormalities. Levels of homocysteine and methylmalonic acid are often only slightly elevated in Cbl-deficient patients who have significant neuropsychiatric defects. Moderate elevations of homocysteine and methylmalonic acid occur in 20%-30% of various elderly populations (mean age 80) and may play a role in the similar neuropsychiatric abnormalities that occur increasingly with aging. Taken together, these studies suggest that an important unknown Cbl-dependent enzyme, metabolic abnormality, environmental factor, or genetic factor may play a major role in the pathophysiology of the neuropsychiatric abnormalities caused by Cbl deficiency.

In Vitro and in Vivo Inactivation of Transcobalamin II Receptor by Its Antiserum

Rabbits injected with pure human placental transcobalamin II-receptor (TC II-R) failed to thrive with no apparent tissue or organ damage, but a 2-fold elevation of the metabolites, homocysteine, methylmalonic acid, and the ligand, transcobalamin II, in their plasma. Exogenously added transcobalamin II-[57Co]cyanocobalamin bound very poorly (2-5%) to the affected rabbit liver, kidney, and intestinal total or intestinal basolateral membrane extracts relative to the binding by membrane extracts from normal rabbit tissues. The activity was restored to normal values following a wash of affected rabbit tissue membranes with pH 3 buffer containing 200 mM potassium thiocyanate. Immunoblot analysis of normal and affected rabbit kidney and liver total membranes revealed similar amounts of 124-kDa TC II-R dimer protein. The neutralized and dialyzed extract from the affected rabbit membranes inhibited the binding of the ligand to pure TC II-R and the harvested affected rabbit serum inhibited the uptake of TC II-[57Co]cobalamin (Cbl) from the basolateral side of human intestinal epithelial (Caco-2) cells and decreased the utilization of [57Co]Cbl as coenzymes by the Cbl-dependent enzymes. The loss of exogenously added ligand binding or the binding of 125I-protein A occurred with the intestinal basolateral, but not the apical membranes. Based on these results, we suggest that circulatory antibodies to TC II-R cause its in vivo functional inactivation, suppress Cbl uptake by multiple tissues, and thus cause severe Cbl deficiency and the noted failure to thrive.

Metabolic abnormalities in cobalamin (vitamin B12) and folate deficiency.

Mammalian cells contain two Cbl-dependent enzymes, L-methylmalonyl-CoA mutase and methionine synthase. The former requires adenosyl-Cbl and catalyzes the conversion of L-methylmalonyl-CoA to succinyl-CoA. The latter requires CH3-Cbl and catalyzes the conversion of 5-CH3-tetrahydrofolate and homocysteine to tetrahydrofolate and methionine, respectively. Biochemical abnormalities related to a decrease in the activity of methionine synthase are thought to be responsible for the indistinguishable hematologic abnormalities seen in both Cbl and folate deficiency. The biochemical basis for the neuropsychiatric abnormalities seen in Cbl deficiency, but not in folate deficiency, is not known although hypotheses have been proposed that implicate one or the other of the two Cbl-dependent enzymes. Recent studies have shown that levels of serum methylmalonic acid, 2-methylcitric acids I and II, total homocysteine, and cystathionine are elevated in most patients with Cbl deficiency and that total homocysteine, cystathionine, N,N-dimethylglycine, and N-methylglycine are elevated in most patients with folate deficiency. Analysis of these metabolic abnormalities in various patient groups fails to support hypotheses that either L-methylmalonyl-CoA mutase or methionine synthase alone are responsible for the neuropsychiatric abnormalities. We suggest that they may result from a third, unknown mammalian Cbl-dependent enzyme or from a combined deficiency of both Cbl-dependent enzymes together with an unknown genetic or environmental factor.

Inhibition of cobalamin-dependent enzymes by cobalamin analogues in rats.

To determine which parts of the cobalamin (cbl) molecule are required for enzyme activity and which parts, if altered, might inhibit cbl-dependent enzyme activity, we synthesized 16 cbl analogues and administered them to nutritionally normal rats. The cbl analogues, with either modifications of the propionamide side chains of the A-, B-, and C-rings, the acetamide side chain of the B-ring, or the nucleotide moiety, were administered to rats by continuous 14-d subcutaneous infusion. Infusion of cbl-stimulated, cbl-dependent activity. Changes in any part of the cbl molecule always abolished stimulation and, in some cases, caused potent inhibition of both cbl-dependent enzymes. The most inhibitory analogues, OH-cbl[c-lactam], a B-ring analogue, and OH-cbl[e-dimethylamide] and OH-cbl[e-methylamide], two C-ring analogues, decreased mean liver holo-L-methylmalonyl-coenzyme A mutase activity to 65% of control values and increased serum methylmalonic acid concentrations to as high as 3,200% of the control values. Liver methionine synthetase activity was decreased to approximately 20% of the control and mean serum total homocysteine concentrations were increased to 340% of control. A similar level of inhibition was demonstrated in rats who were exposed to 28 d of inhaled nitrous oxide or a prolonged period of dietary cbl deficiency. The inhibitory cbl analogues, nitrous oxide, and diet deficiency all depleted liver cbl. The naturally occurring cbl analogues with modifications of the nucleotide moiety had no effects. We conclude that all parts of the cbl molecule are necessary for in vivo cbl-dependent enzyme activity and that modifications of the side chains of the B and C rings are associated with potent in vivo inhibition of cbl-dependent enzyme activity.

Function of vitamin B12 in the central nervous system as revealed by congenital defects

The 13 cases of methylcobalamin (MeCbl) deficiency presenting in early infancy have all been developmentally delayed, and the majority have had seizures, hypotonia, lethargy, and microcephaly. The CNS injury appears to occur during the first 6 months of postnatal life. The same symptoms are seen in acquired cobalamin (Cbl) deficiency in the same age group. MRI performed at age 18-19 months and after 13-14 months of large amounts of Cbl, in two cases showed delayed myelination, most pronounced in the cerebrum. Isolated MeCbl deficiency is the consequence of cblE and G mutations where the lesion is of a single Cbl-dependent enzyme, the methyltransferase. One effect of a deficiency of MeCbl, and of the associated failure of the methionine synthase reaction, is, therefore, an impairment of myelination of the brain of the newborn. The slow, but usually incomplete, improvement in psychomotor status after years of treatment with Cbl may be related to the eventual myelination. However, the hypotonia, lethargy, and impaired responsiveness react to treatment with Cbl within 24-48 hours, which suggests an expression of MeCbl deficiency on the CNS distinct from the delayed myelination. Although there is much to be learned, it is now clear that a normally functioning Cbl-dependent methyl transferase is required for development and function of the human brain.

Purification and some properties of cytosolic cobalamin-binding protein in Euglena gracilis.

In Euglena gracilis SM-ZK, a bleached mutant of E. gracilis z, the cobalamin- (Cbl-)binding activity was distributed in cytosol (49.2%), mitochondria (20.3%) and microsomal fraction (20.4%). The cytosolic Cbl-binding activity gave two major peaks in isoelectric focusing. The Cbl-binding protein with pI 3.2 was purified 6500-fold in a yield of 19.9%, and that with pI 4.7 5800-fold in a yield of 11.9%. The monomeric Mr values of both Cbl-binding proteins were about 66,000. The Cbl-binding activity of both proteins showed a very low pH-dependency, and thiol groups and metal ions were not concerned with the Cbl-binding activities. The Ks values of the Cbl-binding proteins with pI 3.8 and 4.7 for CN-Cbl were 1.0 and 2.0 nM respectively. The Cbl-binding protein with pI 3.8 was shown to be immunologically identical with the protein with pI 4.7 by double-immunodiffusion experiments against antibody to the protein with pI 3.8. The two cytosolic Cbl-binding proteins did not show the activities of Cbl-dependent enzymes in E. gracilis, N5-methyltetrahydrofolate:homocysteine methyltransferase, methylmalonyl-CoA mutase and ribonucleotide reductase, suggesting that the two cytosolic Cbl-binding proteins play a physiological role as intracellular Cbl carriers.

Recognition of two intracellular cobalamin binding proteins and their identification as methylmalonyl-CoA mutase and methionine synthetase.

The granulocyte R-type cobalamin binding protein delivers cobalamin (Cbl) exclusively to hepatocytes, and transcobalamin II delivers Cbl to various mammalian cells. Both protein-Cbl complexes enter cells by pinocytosis, and the protein moieties are rapidly degraded in lysosomes. The liberated Cbl is subsequently bound to a high-molecular-weight intracellular cobalamin binding protein (ICB). The nature of ICB-Cbl is unknown but appears important because ICB-[57Co]Cbl is missing from cultured fibroblasts of a group of patients whose cells take up CN-[57Co]Cbl normally but do not convert it to either of its coenzyme forms. We have examined supernatants of sonicated rabbit livers and have found that 65% of the total endogenous Cbl elutes from Sephadex G-150 as ICB-Cbl and that this fraction also contains the two mammalian Cbl-dependent enzymes, methylmalonyl-CoA mutase (methylmalonyl-CoA CoA-carbonylmutase;EC 5.4.99.2) and methionine synthetase (tetrahydropteroylglutamate methyltransferase; 5-methyltetrahydropteroyl-L-glutamate:L-homocysteine-S-methyltransferase; EC 2.1.1.13). Gradient elution from DEAE-Sephadex reveals that 90--95% of the ICB--Cbl elutes with methylmalonyl-CoA mutase and 5--10% elutes with methionine synthetase. ICB--[57Co]Cbl first appears 2 hr after the intravenous injection of CN[57Co]Cbl bound to granulocyte R-type protein. This ICB-[57Co]Cbl is associated with either methylmalonyl-CoA mutase or methionine synthetase although the latter appears to be formed at a relatively faster rate. Our studies indicate that mammalian cells contain two ICBs, that these proteins are methylmalonyl-CoA mutase and methionine synthetase, and that the primary abnormality in the group of patients mentioned above lies at a step that is common to the formation of both Cbl coenzymes and that precedes the stable binding of Cbl to both methylmalonyl-CoA mutase and methionine synthetase.