B9 and B12

A theme of this post is that B12 (cobalamin Cbl) and and B9/folic acid are partners in many metabolic pathways that involve transfer of single carbons, or methyl groups, from one molecule to another. Cbl and THF are cofactors in pathways involving a mind boggling number of enzymes, any one of which may be inactive or sub optimally active. Patients with mutations in genes that code for these enzymes may have deficits in “methylation” and “remethylation” of genes, proteins, and small molecules. Metabolism of odd chain fatty acids and branched chain amino acids might be compromised. An unexpected discovery in writing this post is that the lysosomes are key in acquisition of Cbl/B12. Thus, we can hypothesize that lysosomal storage disease patients might also have issues with one carbon transfer pathways.

How do these B vitamins with totally different structures interact to transfer single carbons from one site to another?

A large study of patients with remethylation defects [1]

1. Deficiency of the enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR) leads to impaired production of 5-methylenetetrahydrofolate, resulting in decreased Methionine Synthase capacity.
2. Remethylation also depends on the cofactor methylcobalamin, which requires cobalamin (Cbl) to get inside the cell. A lot of things can go wrong with the enzymes that transport Cbl from our GI tracts to inside our cells.

Organ systems impacted

The first thought was that these deficits would affect organ systems that rely on metabolism of branched chain amino acids and odd chain fatty acids the most, not the brain…Then there is pernicious anemia caused by B12/Cbl deficiency. Folate deficiency can also cause anemia.

treatments

jj

The authors really did not say why the branched chain amino acid valine was also given as a treatment. in four of the patients with Cbl pathway protein defects. Elevated homosyteine in the blood is generally considered a risk factor for cardiovascular disease.

outcomes

The effects of the individual treatments, e.g. OH-Cbl & carnitine, in one cblC defect patient, was not reported. We do see that treatments seem to be decreasing the homocysteine in the blood.

This study is being presented simply because it is kind of encouraging that dietary interventions can decrease just one outcome variable of a rather large assortment of genetic defects.

Vitamin B₁₂, folate [2]

The Froese review gave an excellent if not bewildering overview of enzymes involved in B12, aka cobalamin, processing. If there can be a take home, it is that our bodies exert an extreme amount of effort in acquiring Cbl and that many things can go wrong in this process. This post will make a modest attempt to highlight key points from the Froese review.

Introduction

Many readers probably realize that Vitamin B₁₂ (cobalamin, Cbl) is not produced in humans. It is required for just two very essential enzymes:

• cytosolic methionine synthase (MS, EC 2.1.1.13) MS uses the methylated form of Cbl to transfer its methyl group to remethylate homosyteine to form methione. This pathwat is needed for de novo purine synthesis.
• mitochondrial methylmalonyl-CoA mutase (MUT, EC 5.4.99.2). MUT regulates the entry of branched chain amino acids and odd carbon fatty acids into the TCA cycle. This includes propionate.

structure

Haplocorrin potentially being needed to protect Cbl in the stomach is interesting in terms of use of CBl supplements. On the other hand, intrinsic factor is secreted by the parietal cells of the stomach and is required for CBl being handed off to the renal albumin transporter cubilin. Lysosomes are required for dissociation of CBl from IF. The ATP driven MRP1 is required for CBl exit from the enterocyte into the blood. In the blood CBl may be complexed to haptocorrin or trans cobalamin.

3-5 entering the cell, going to the mitochondia, cytosol

The Froese review gives a very good discussion of the various transport proteins that target Cbl to the mitochondria vs cytosol. What was not discussed was whether NADH/NADPH and ATP, indicators of mitochondria (TCA and electron transport chain) function influence Cbl targeting. A trip to Uniprot.org often reveals many post translational modification sites. This post will not be diving down into the rabbit hole of post translational modifications of any of the proteins mentioned in the Froese review.

The question not addressed in this cartoon is what happens to branched chain amino acids. odd chain fatty acids, and cholesterol side chains that are not able to enter the TCA cycle when Cbl is not around. Are these part of the mTor signalling?

6 folic acid isoforms and one carbon transfer

The froese review discussed the different forms of folic acid/B6. They tied systolic vs mitochondria of THF to that of Cbl to the entry of odd chain fatty acids (two plus one carbon) and branched chain amino acids into the TCA cycle. However, an important clue may come from the finding that mitochondrial one-carbon oxidation accounts for approximately 50% of the NADPH produced in the cell according to one citation in teh Froese review. a massive reducing and energy source. From this has stemmed the hypothesis that one-carbon oxidation is localized to the mitochondria in order to uncouple it from glycolysis, which might be blocked by the depleted NAD⁺ levels that would arise should one-carbon oxidation take place in the cytosol

8. Allosteric control

This is the title of section 8. Allosteric is defined as “Of or involving a change in the shape and activity of an enzyme that results from molecular binding with a regulatory substance at a site other than the enzymatically active one.” The American Heritage® Dictionary of the English Language, 5th Edition. We will not go down this rabbit hole.

Froese and coauthors re-introduced MMACHC, an enzyme calbalamin reductase. The following are from th UnProt link and link to PubMed publications. The rest of this section came from the UniProt link.

• Cyanocobalamin is the inactive form of vitamin B12 found in the diet. Once the cyanide group is removed B12 becomes active. (PubMed:19801555).
• Cobalamin (vitamin B12) cytosolic chaperone that catalyzes the reductive decyanation of cyanocob(III)alamin (cyanocobalamin, CNCbl) to yield cob(II)alamin and cyanide, using FAD or FMN as cofactors and NADPH as cosubstrate … according to unsupervised AI. It would appear that the reducing agents are flexible (PubMed:18779575, PubMed:19700356, PubMed:21697092, PubMed:25809485).
• The reductase forms a complex with the lysosomal transporter ABCD4 and its chaperone LMBRD1, to transport cobalamin across the lysosomal membrane into the cytosol (PubMed:25535791).
• The processing of cobalamin in the cytosol occurs in a multi protein complex composed of at least MMACHC, MMADHC, MTRR (methionine synthase reductase) and MTR (methionine synthase) which may contribute to shuttle safely and efficiently cobalamin towards MTR in order to produce methionine (PubMed:21071249, PubMed:27771510).
• Cysteine and homocysteine cannot substitute for glutathione in this reaction (PubMed:19801555).

The role of the lysosome, key word search…

• Cubam (heterodimer of amnionless and cubilin), bound tointrinsic factor/B12, IF-Cbl, complex is endocytosed and targeted to the lysosome where the cubam receptor is degraded.
• In the blood, Cbl may be bound to either of two proteins—haptocorrin or transcobalamin (TC). Our cells take up the Cbl-TC complex and send he endocytic vesicles to the lysosomes where the protein receptor and TC are degraded.
• ATP is required to transport Cbl out of the lysosomes with specialized proteins.
  

So many rabbit holes not explored

This post has just examined two publications regarding B6 and B12 pathways and dietary interventions. We have ignored single nucleotide polymorphisms and outright mutations in the many genes that code for proteins in these pathways. Then we have post translational modifications like serine, threonine, tyrosine phosphorylation, lysine acetylation, glutathionylation, and so one.

References

1. Huemer M, Diodato D, Martinelli D, Olivieri G, Blom H, Gleich F, Kölker S, Kožich V, Morris AA, Seifert B, Froese DS, Baumgartner MR, Dionisi-Vici C; EHOD consortium; Martin CA, Baethmann M, Ballhausen D, Blasco-Alonso J, Boy N, Bueno M, Burgos Peláez R, Cerone R, Chabrol B, Chapman KA, Couce ML, Crushell E, Dalmau Serra J, Diogo L, Ficicioglu C, García Jimenez MC, García Silva MT, Gaspar AM, Gautschi M, González-Lamuño D, Gouveia S, Grünewald S, Hendriksz C, Janssen MCH, Jesina P, Koch J, Konstantopoulou V, Lavigne C, Lund AM, Martins EG, Meavilla Olivas S, Mention K, Mochel F, Mundy H, Murphy E, Paquay S, Pedrón-Giner C, Ruiz Gómez MA, Santra S, Schiff M, Schwartz IV, Scholl-Bürgi S, Servais A, Skouma A, Tran C, Vives Piñera I, Walter J, Weisfeld-Adams J. Phenotype, treatment practice and outcome in the cobalamin-dependent remethylation disorders and MTHFR deficiency: Data from the E-HOD registry. J Inherit Metab Dis. 2019 Mar;42(2):333-352. Sci-Hub free paper
2. Froese DS, Fowler B, Baumgartner MR. Vitamin B₁₂ , folate, and the methionine remethylation cycle-biochemistry, pathways, and regulation. J Inherit Metab Dis. 2019 Jul;42(4):673-685. free paper