Vitamin B12 metabolism

Any supplemental form of cobalamin is reduced to core cobalamin

Vitamin B12 is often sold as a supplement in what is referred to as its “active form,” which is typically either adenosylcobalamin or methylcobalamin. However, when these forms are taken orally or topically, they are not truly active. They undergo processing, during which the alkyl group is cleaved off, and a new methyl or adenosyl group is reattached to the cobalamin:

all supplemental or food-derived B12 forms are reduced to a core cobalamin molecule, which converts to the intracellular active forms: MeCbl and AdCbl, in a ratio not influenced by the form of B12 ingested. The methyl and adenosyl components of supplemental MeCbl and AdCbl are cleaved inside cells and are not used in the synthesis of intracellular MeCbl and AdCbl, respectively. However, the overall bioavailability of each form of supplemental B12 may be influenced by many factors such as gastrointestinal pathologies, age, and genetics. (R6)

B12 processing consumes Glutathione and SAMe

Intracellular processing of cobalamin (vitamin B12) requires glutathione and SAMe before it can be used as a cofactor by the target enzymes.

After cobalamin is stored in lysosomes, it must be processed by the enzymatic complex MMACHC+MMADHC, which does the following:

1. Removes of the methyl-, adenosyl-, cyano- ligands using glutathione molecule - it acts as a glutathione transferase.
2. Adds a methyl group taken from SAMe, or adenosyl ligand taken from ATP.
3. Transports the resulting alkyl-cobalamin to the branch point where it can go to one of the two target enzymes (MMUT or MTR).

First two functions are performed by MMACHC and transportation is performed by MMADHC. (R1, R2)

Removed methyl- or adenosyl- ligand is attached to glutathione, forming S-methyl-glutathione or S-Adenosyl-glutathione respectively.

That means a molecule of GSH will be spent to export the alkyl-group.

Another two molecules of GSH are used for handling cobalamin itself:

These studies support a chemical mechanism of thiol oxidation in which glutathionyl-cobalamin (GSCbl) is formed as an intermediate and GSH-dependent deglutathionylation of GSCbl yields GSSG. (R8)

Therefore, the total cost of cobalamin processing is at least 3 molecules of GSH per one molecule of cobalamin. While one GSH is lost for the cell, and other two became GSSG (which can be reduced back to 2 GSH by Glutathione Reductase).

In addition to GSH utilization, there is also hydrogen peroxide produced (see below).

It also means that there is not much of difference between orally taken supplements methyl-cobalamin and adenosyl-cobalamin because both will be processed in the same way, and they are not used in the original form by the enzymes. Cobalamin part will be separated, and an active form will be formed in the next step.

The second step in the process requires the source of either methyl- or adenosyl- ligand.

That means SAMe will be spent to produce methylcobalamin. This is important for people with seriously lowered methylation potential.

Hydrogen peroxide is created

It appears that MMACHC generates H₂O₂ in the process of cobalamin processing:

The thiol oxidase activity of ce_CblC was initially monitored during the dealkylation of AdoCbl by GSH by monitoring O2 consumption as well as GSSG and H2O2 generation. (R8)

Processing of the Adenosyl- form is 30-67 times slower than of methyl- form

There is a study that evaluated how fast both forms are processed by MMACHC and it turned out that adenosyl form’s processing time is 67 higher than of methyl form:

The catalytic turnover numbers for the dealkylation of methylcobalamin and 5'-deoxyadenosylcobalamin by MMACHC are 11.7 +/- 0.2 and 0.174 +/- 0.006 h(-1) at 20 degrees C, respectively. (R2)

Another study (R8) provides different rates, but the overall picture is the same - adenosyl form is processed much slower than a methyl-form, and cyanocobalamin is processed 293 times slower than meB12:

Form	Processing rate	Rate of GSSG production
GSCbl	108 ± 12	102 ± 9
MeCbl	13.5 ± 0.4	83 ± 9
OH2Cbl	0.46 ± 0.02	21 ± 2
AdoCbl	0.19 ± 0.03	19 ± 2
CNCbl	0.046 ± 0.03	2.9 ± 0.1

In these results, adoB12 is processed 30 times slower than meB12.

Comparative utilization of hydroxy-, methyl- and adenosyl- forms

Another study provides comparison of utilization of the three forms of cobalamin. By utilization the authors mean ability to process hydroxy form into two active forms in the cell lines, and they measured the % of the form inside the cell (R4):

Intracellular % of the form: Methyl: 60.7 ±3.0 Adenosyl: 13.4 ± 6.8 Hydroxy: 25.9 ± 5.3

It means, when OH-Cbl was supplied to the cell line, it was observed that 60.7% of intracellular cobalamin was methyl-Cbl and 13.4% was ado-Cbl, while 25.9% remained as OH-Cbl.

Fate of S-methyl-glutathione

S-methyl-glutathione can be processed by the enzyme Glutathione hydrolase 6 (GGT6), but it’s located outside of the cell on the membrane (ref?).

That means it has to be exported outside, leading to a loss of glutathione molecule inside the cell.

GGT6 uses water molecule to convert S-methyl-glutathione into L-Glutamate and S-methyl-L-cysteinylglycine (R3).

That leads to increased extracellular L-Glutamate unless it’s taken up by the cell rapidly.

S-methyl-L-cysteinylglycine is further broken down by Cysteinylglycine dipeptidase (DPEP1) probably into S-Methyl-cysteine and glycine:

This selectivity has allowed Tate and coworkers to estimate that about 65, 60, and 95% of S-methyl-cysteinylglycine hydrolyzing activities of the rat renal, jejunal, and epididymal membranes are due to cysteinylglycine dipeptidase (R5)

Lysosomal Separation of cobalamin from Transcobalamin II

Once Transcobalamin II is taken up by the cell via the Transcobalamin Receptor, it ends up in a lysosome, where cobalamin is cleaved off from the TC II.

LMBD1 is a transporter that exports cobalamin from the lysosome:

we identified a candidate gene on chromosome 6q13, LMBRD1, encoding LMBD1, a lysosomal membrane protein with homology to lipocalin membrane receptor LIMR.

We identified five different frameshift mutations in LMBRD1 resulting in loss of LMBD1 function, with 18 of the 24 disease chromosomes carrying the same mutation embedded in a common 1.34-Mb haplotype. Transfection of fibroblasts of individuals with cblF with wild-type LMBD1 rescued cobalamin coenzyme synthesis and function. (R11)

MS-MSR-MMACHC complex

MSR (MTRR) has an essential role in the enzymatic complex of Methionine synthase.

• It assists in the formation of MS+methylcobalamin in the presence of NADPH
• It stabilizes apoMS (MS without cobalamin)
• It reduces aquacobalamin to cob(II)alamin in the presence of NAPDH

The effects of hMSR on the formation of hMS holoenzyme also were examined by using crude extracts of baculovirus-infected insect cells containing hMS apoenzyme (apoMS).

In the presence of MSR and NADPH, holoenzyme formation from apoMS and methylcobalamin was significantly enhanced.

The observed stimulation is shown to be due to stabilization of human apoMS in the presence of MSR. Apoenzyme alone is quite unstable at 37 degrees C.

MSR also is able to reduce aquacobalamin to cob(II)alamin in the presence of NADPH, and this reduction leads to stimulation of the conversion of apoMS and aquacobalamin to MS holoenzyme.

Based on these findings, we propose that MSR serves as a special chaperone for hMS and as an aquacobalamin reductase, rather than acting solely in the reductive activation of MS. (R7)

Lysosomal processing of TCN2 depends on NPRL2 and mTORC1

NPRL2 gene encodes GATOR1 complex (Uniprot).

The methionine synthase enzyme is dependent on cobalamin (vitamin-B12), which is carried in plasma bound to the cobalamin-transport protein, called TCN2, and transported into cells by the endocytic-lysosomal pathway. …. Collectively, these data strongly suggest that loss of NPRL2 produces cobalamin-deficiency, and thus methionine-deficiency, perhaps due to defective lysosomal-dependent TCN2 processing (R9)

Loss of NPRL2 leads to low methionine and high 5-Me-THF amounts in embryos, suggesting a “folate-trap” that can occur by cobalamin-deficiency. We further observe that lysosomal processing of cobalamin is downstream of NPRL2 and establish mTORC1 as a regulator of cobalamin-processing and the cobalamin-dependent enzyme, methionine synthase. … Collectively, our findings reveal that NPRL2, a negative regulator of mTORC1, controls cobalamin availability, methionine homeostasis, and re-methylation potential, which are important metabolic pathways for hematopoiesis. (R9)

Disease associated with GATOR1 - Epilepsy, familial focal, with variable foci 2 (FFEVF2)

An autosomal dominant form of epilepsy characterized by focal seizures arising from different cortical regions, including the temporal, frontal, parietal, and occipital lobes. Seizure types commonly include temporal lobe epilepsy, frontal lobe epilepsy, and nocturnal frontal lobe epilepsy. Some patients may have intellectual disability or autism spectrum disorders. Seizure onset usually occurs in the first or second decades, although later onset has been reported, and there is phenotypic variability within families. A subset of patients have structural brain abnormalities. Penetrance of the disorder is incomplete.

GATOR1 (NPRL2)

• Deletion of NPRL2 results in increased type II fiber composition in soleus muscle
• NPRL2 is necessary to repress mTORC1 in soleus muscle during fasting
• Loss of NPRL2 increases pyruvate conversion to lactate and reduces pyruvate entry into TCA cycle
• NPRL2 coordinates glucose and amino acid metabolism (R10)

ZNF143 regulates MMACHC

ZNF143 is a transcription factor that interacts with HCFC1, which regulates MMACHC:

Patient cells accumulated transcobalamin-bound-Cbl, a complex which usually dissociates in the lysosome to release free Cbl. Whole exome sequencing identified putative disease-causing variants c.851T>G (p.L284*) and c.1019C>T (p.T340I) in **transcription factor ZNF143**.

Proximity biotinylation analysis confirmed the interaction between ZNF143 and HCFC1, a protein that regulates expression of the Cbl trafficking enzyme MMACHC. qRT-PCR analysis revealed low MMACHC expression levels both in patient fibroblasts, and in control fibroblasts incubated with ZNF143 siRNA. (R12)

ZNF143

Transcriptional activator. Activates the gene for selenocysteine tRNA (tRNAsec). Binds to the SPH motif of small nuclear RNA (snRNA) gene promoters. (Uniprot)

Different strategy of reactivation of oxidized cobalamin

Normally, MTR would methylate oxidized cobalamin to restore the function of Methionine Synthase. But in neuronal cells oxidised cobalamin dissociates from the MS enzyme and replaced by methylcobalamin:

Neuronal cells have a uniquely different strategy for reactivating methionine synthase, which is tightly dependent upon GSH status. Whereas SAM-dependent methylation of oxidized cobalamin occurs in most cell types, in neuronal cells oxidized cobalamin dissociates from the enzyme and is replaced by methylcobalamin, allowing reactivation.

However, methylcobalamin synthesis proceeds through an intermediate step that requires GSH, so methionine synthase will remain inactive longer when GSH levels are below normal (Fig. 1).

This relationship ensures that methylation activity in neurons, including dopamine-stimulated PLM described below, will be restricted under conditions of oxidative stress and also ensures that growth factor-induced cysteine uptake will exert a powerful influence over methylation. (R13, page 190)

Insertion of adoCbl by MMAA into MUT depends on GTP

MMAA regulates the incorporation of the cofactor adenosylcobalamin (AdoCbl), generated from the MMAB adenosyltransferase, into the destination enzyme methylmalonyl-CoA mutase (MUT).

This function of MMAA depends on its GTPase activity, which is stimulated by an interaction with MUT. (R14)

MMAA - Methylmalonic aciduria type A protein, mitochondrial

1. GTPase, binds and hydrolyzes GTP (R, R, R, R).
2. Involved in intracellular vitamin B12 metabolism, mediates the transport of cobalamin (Cbl) into mitochondria for the final steps of adenosylcobalamin (AdoCbl) synthesis (R, R).
3. Functions as a G-protein chaperone that assists AdoCbl cofactor delivery from MMAB to the methylmalonyl-CoA mutase (MMUT) (R, R).
4. Plays a dual role as both a protectase and a reactivase for MMUT (R, R).
5. Protects MMUT from progressive inactivation by oxidation by decreasing the rate of the formation of the oxidized inactive cofactor hydroxocobalamin (OH2Cbl) (R, R).
6. Additionally acts a reactivase by promoting the replacement of OH2Cbl by the active cofactor AdoCbl, restoring the activity of MMUT in the presence and hydrolysis of GTP

(Uniprot)