Vitamin B12 (cobalamin, Cbl) is a nutrient essential to human health. Due to its complex structure and dual cofactor forms, Cbl undergoes a complicated series of absorptive and processing steps before serving as cofactor for the enzymes methylmalonyl-CoA mutase and methionine synthase. Methylmalonyl-CoA mutase is required for the catabolism of certain (branched-chain) amino acids into an anaplerotic substrate in the mitochondrion, and dysfunction of the enzyme itself or in production of its cofactor adenosyl-Cbl result in an inability to successfully undergo protein catabolism with concomitant mitochondrial energy disruption. Methionine synthase catalyzes the methyl-Cbl dependent (re)methylation of homocysteine to methionine within the methionine cycle; a reaction required to produce this essential amino acid and generate S-adenosylmethionine, the most important cellular methyl-donor. Disruption of methionine synthase has wide-ranging implications for all methylation-dependent reactions, including epigenetic modification, but also for the intracellular folate pathway, since methionine synthase uses 5-methyltetrahydrofolate as a one-carbon donor. Folate-bound one-carbon units are also required for deoxythymidine monophosphate and de novo purine synthesis; therefore, the flow of single carbon units to each of these pathways must be regulated based on cellular needs. This review provides an overview on Cbl metabolism with a brief description of absorption and intracellular metabolic pathways. It also provides a description of folate-mediated one-carbon metabolism and its intersection with Cbl at the methionine cycle. Finally, a summary of recent advances in understanding of how both pathways are regulated is presented.

https://www.zora.uzh.ch/id/eprint/181018/1/Froese_et_al-2019-Journal_of_Inherited_Metabolic_Disease.pdf

Vitamin B12 , folate, and the methionine remethylation cycle-biochemistry, pathways, and regulation.

Remethylation defects are rare inherited disorders in which impaired remethylation of homocysteine to methionine leads to accumulation of homocysteine and perturbation of numerous methylation reactions. To summarise clinical and biochemical characteristics of these severe disorders and to provide guidelines on diagnosis and management. Review, evaluation and discussion of the medical literature (Medline, Cochrane databases) by a panel of experts on these rare diseases following the GRADE approach. We strongly recommend measuring plasma total homocysteine in any patient presenting with the combination of neurological and/or visual and/or haematological symptoms, subacute spinal cord degeneration, atypical haemolytic uraemic syndrome or unexplained vascular thrombosis. We strongly recommend to initiate treatment with parenteral hydroxocobalamin without delay in any suspected remethylation disorder; it significantly improves survival and incidence of severe complications. We strongly recommend betaine treatment in individuals with MTHFR deficiency; it improves the outcome and prevents disease when given early.

https://link.springer.com/content/pdf/10.1007%2Fs10545-016-9991-4.pdf

Guidelines for diagnosis and management of the cobalamin-related remethylation disorders cblC, cblD, cblE, cblF, cblG, cblJ and MTHFR deficiency

Lysosomes are cytoplasmic organelles that contain a variety of different hydrolases. A genetic deficiency in the enzymatic activity of one of these hydrolases will lead to the accumulation of the material meant for lysosomal degradation. Examples include glycogen in the case of Pompe disease, glycosaminoglycans in the case of the mucopolysaccharidoses, glycoproteins in the cases of the oligosaccharidoses, and sphingolipids in the cases of Niemann-Pick disease types A and B, Gaucher disease, Tay-Sachs disease, Krabbe disease, and metachromatic leukodystrophy. Sometimes, the lysosomal storage can be caused not by the enzymatic deficiency of one of the hydrolases, but by the deficiency of an activator protein, as occurs in the AB variant of GM2 gangliosidosis. Still other times, the accumulated lysosomal material results from failed egress of a small molecule as a consequence of a deficient transporter, as in cystinosis or Salla disease. In the last couple of decades, enzyme replacement therapy has become available for a number of lysosomal storage diseases. Examples include imiglucerase, taliglucerase and velaglucerase for Gaucher disease, laronidase for Hurler disease, idursulfase for Hunter disease, elosulfase for Morquio disease, galsulfase for Maroteaux-Lamy disease, alglucosidase alfa for Pompe disease, and agalsidase alfa and beta for Fabry disease. In addition, substrate reduction therapy has been approved for certain disorders, such as eliglustat for Gaucher disease. The advent of treatment options for some of these disorders has led to newborn screening pilot studies, and ultimately to the addition of Pompe disease and Hurler disease to the Recommended Uniform Screening Panel (RUSP) in 2015 and 2016, respectively.

/pdf/lysosomal-storage-diseases-6vo2fsgm74.pdf

Lysosomal storage diseases

Navigating the B12 Road: Assimilation, Delivery, and Disorders of Cobalamin

The transport of specific molecules across lipid membranes is an essential function of all living organisms. The processes are usually mediated by specific transporters. One of the largest transporter families is the ATP-binding cassette (ABC) family. More than 40 ABC transporters have been identified in human, which are divided into 7 subfamilies (ABCA to ABCG) based on their gene structure, amino acid sequence, domain organization, and phylogenetic analysis. Of them, at least 11 ABC transporters including P-glycoprotein (P-GP/ABCB1), multidrug resistance-associated proteins (MRPs/ABCCs), and breast cancer resistance protein (BCRP/ABCG2) are involved in multidrug resistance (MDR) development. These ABC transporters are expressed in various tissues such as the liver, intestine, kidney, and brain, playing important roles in absorption, distribution, and excretion of drugs. Some ABC transporters are also involved in diverse cellular processes such as maintenance of osmotic homeostasis, antigen processing, cell division, immunity, cholesterol, and lipid trafficking. Several human diseases such as cystic fibrosis, sitosterolemia, Tangier disease, intrahepatic cholestasis, and retinal degeneration are associated with mutations in corresponding transporters. This chapter will describe function and expression of several ABC transporters (such as P-GP, BCRP, and MRPs), their substrates and inhibitors, as well as their clinical significance.

https://link.springer.com/content/pdf/10.1007%2F978-981-13-7647-4_2.pdf

ABC Family Transporters.

Subcellular location of MMACHC and MMADHC, two human proteins central to intracellular vitamin B(12) metabolism.

Iron acquisition at the outer membrane (OM) of Gram-negative bacteria is powered by the proton motive force (PMF) of the cytoplasmic membrane (CM), harnessed by the CM-embedded complex of ExbB, ExbD, and TonB. Its stoichiometry, ensemble structural features, and mechanism of action are unknown. By panning combinatorial phage libraries, periplasmic regions of dimerization between ExbD and TonB were predicted. Using overexpression of full-length His 6 -tagged exbB-exbD and S-tagged tonB , we purified detergent-solubilized complexes of ExbB-ExbD-TonB from Escherichia coli. Protein-detergent complexes of ∼230 kDa with a hydrodynamic radius of ∼6.0 nm were similar to previously purified ExbB 4 -ExbD 2 complexes. Significantly, they differed in electronegativity by native agarose gel electrophoresis. The stoichiometry was determined to be ExbB 4 -ExbD 1 -TonB 1 . Single-particle electron microscopy agrees with this stoichiometry. Two-dimensional averaging supported the phage display predictions, showing two forms of ExbD-TonB periplasmic heterodimerization: extensive and distal. Three-dimensional (3D) particle classification showed three representative conformations of ExbB 4 -ExbD 1 -TonB 1 . Based on our structural data, we propose a model in which ExbD shuttles a proton across the CM via an ExbB interprotein rearrangement. Proton translocation would be coupled to ExbD-mediated collapse of extended TonB in complex with ligand-loaded receptors in the OM, followed by repositioning of TonB through extensive dimerization with ExbD. Here we present the first report for purification of the ExbB-ExbD-TonB complex, molar ratios within the complex (4:1:1), and structural biology that provides insights into 3D organization. IMPORTANCE Receptors in the OM of Gram-negative bacteria allow entry of iron-bound siderophores that are necessary for pathogenicity. Numerous iron-acquisition strategies rely upon a ubiquitous and unique protein for energization: TonB. Complexed with ExbB and ExbD, the Ton system links the PMF to OM transport. Blocking iron uptake by targeting a vital nanomachine holds promise in therapeutics. Despite much research, the stoichiometry, structural arrangement, and molecular mechanism of the CM-embedded ExbB-ExbD-TonB complex remain unreported. Here we demonstrate in vitro evidence of ExbB 4 -ExbD 1 -TonB 1 complexes. Using 3D EM, we reconstructed the complex in three conformational states that show variable ExbD-TonB heterodimerization. Our structural observations form the basis of a model for TonB-mediated iron acquisition.

Membrane Protein Complex ExbB4-ExbD1-TonB1 from Escherichia coli Demonstrates Conformational Plasticity

Structural features of recombinant MMADHC isoforms and their interactions with MMACHC, proteins of mammalian vitamin B12 metabolism.

Mutations in human LMBRD1 and ABCD4 prevent lysosomal export of vitamin B(12) to the cytoplasm, impairing the vitamin B(12)-dependent enzymes methionine synthase and methylmalonyl-CoA mutase. The gene products of LMBRD1 and ABCD4 are implicated in vitamin B(12) transport at the lysosomal membrane and are proposed to act in complex. To address the mechanism for lysosomal vitamin B(12) transport, we report the novel recombinant production of LMBD1 and ABCD4 for detailed biophysical analyses. Using blue native PAGE, chemical crosslinking, and size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), we show that both detergent-solubilized LMBD1 and detergent-solubilized ABCD4 form homodimers. To examine the functional binding properties of these proteins, label-free surface plasmon resonance (SPR) provides direct in vitro evidence that: (i) LMBD1 and ABCD4 interact with low nanomolar affinity; and (ii) the cytoplasmic vitamin B(12)-processing protein MMACHC also interacts with LMBD1 and ABCD4 with low nanomolar affinity. Accordingly, we propose a model whereby membrane-bound LMBD1 and ABCD4 facilitate the vectorial delivery of lysosomal vitamin B(12) to cytoplasmic MMACHC, thus preventing cofactor dilution to the cytoplasmic milieu and protecting against inactivating side reactions.

Justin C. Deme

Papers

Subcellular location of MMACHC and MMADHC, two human proteins central to intracellular vitamin B(12) metabolism.

Membrane Protein Complex ExbB4-ExbD1-TonB1 from Escherichia coli Demonstrates Conformational Plasticity

Structural features of recombinant MMADHC isoforms and their interactions with MMACHC, proteins of mammalian vitamin B12 metabolism.

Purification and interaction analyses of two human lysosomal vitamin B12 transporters: LMBD1 and ABCD4