The intracelluar passage of B12 (or cobalamin), a rare and reactive organometallic cofactor, is fraught with perils as it navigates its way to only two enzymes in humans that rely on it: cytoplasmic methionine synthase (MS) and mitochondrial methylmalonyl-CoA mutase (MCM). A squad of dedicated chaperones guards against inadvertant loss of B12 via its dilution into solution. The existence of B12 chaperones was first hinted at by clinical genetics studies on patients with inborn errors of B12 metabolism, which lead to isolated or combined homocystinuria and methylmalonic aciduria, and can present early (birth to a few months) or late (years), depending on the severity of the biochemical deficit. The P.I's laboratory has been at the forefront of deciphering functions as the genes encoding B12 chaperones have been identified, and has furnished a wealth of kinetic, spectroscopic and structural insights that are consistently interwoven with clinical data and characterization of patient mutations. In the next cycle, we propose to elucidate how redox-linked coordination chemistry is used as an exquisite and recurring strategy for controlling the chemical reactivity of B12 as it is processed, and for enabling its mobility, as it is transferred between active sites. Specifically, we will address the following aims. (i) Elucidate the cytoplasmic pathway comprising CblC, that displays a ?jack of all trades? style chemical versatility as it processes varied incoming B12 derivatives to a common cob(II)alamin intermediate, and CblD, an elusive protein bearing strong structural resemblance to CblC, but lacking its ability to bind B12. The cytoplasmic pathway terminates in MS and we will address how B12 is loaded from a novel CblD-thiolato-Co(II)-CblC intermediate that we have discovered, and assess the dependence of this transfer process on yet another chaperone, MS reductase. (ii) Elucidate the mitochondrial pathway comprising the enzyme, adenosyltransferase, which doubles as an escort, synthesizing the active 5-deoxyadenosylcobalamin form of the cofactor and transferring it to MCM in a process that is gated by CblA, a GTPase. We will build on our discovery of an unprecedented strategy for cofactor retention involving sacrificial cobalt-carbon bond homolysis that is triggered when MCM acceptor sites are unavailable, and elucidate the rationale for equilibrating MCM-G-protein oligomeric complexes that are nucleotide sensitive. (iii) Elucidate the intersections between B12 and itaconate, an immunomodulatory molecule that is linked to the recently de-orphaned citramalyl-CoA lyase. Itaconyl-CoA (the dehydrated form of citramalyl-CoA) is a potent inhibitor of human MCM. We will determine the underlying mechanism of B12 deficiency when citramalyl-CoA lyase is missing (as in ~3-6% of some populations) and whether mycobacterial MCM represents an additional target of itaconate for shutting down pathogenic cholesterol-dependent energy metabolism.

Public Health Relevance

Vitamin B12 is an essential nutrient obtained from the diet and navigates an intricate intracellular pathway for assimilation and delivery to find its way to only two known client enzymes. Our laboratory has been at the forefront of elucidating the functions of the protein handlers in the B12 trafficking pathway. In the next cycle, we will illuminate the complex regulation of trafficking proteins that tailor and sequester this cofactor, how these processes are disrupted by disease-causing mutations, and how B12 metabolism in host and pathogen, are targeted by the immunomodulator, itaconate.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK045776-30
Application #
10086462
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Sechi, Salvatore
Project Start
1998-02-01
Project End
2023-01-31
Budget Start
2021-02-01
Budget End
2022-01-31
Support Year
30
Fiscal Year
2021
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Biochemistry
Type
Schools of Medicine
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Banerjee, Ruma (2018) Introduction to the Thematic Minireview Series: Redox metabolism and signaling. J Biol Chem 293:7488-7489
Ruetz, Markus; Shanmuganathan, Aranganathan; Gherasim, Carmen et al. (2017) Antivitamin B12 Inhibition of the Human B12 -Processing Enzyme CblC: Crystal Structure of an Inactive Ternary Complex with Glutathione as the Cosubstrate. Angew Chem Int Ed Engl 56:7387-7392
Li, Zhu; Kitanishi, Kenichi; Twahir, Umar T et al. (2017) Cofactor Editing by the G-protein Metallochaperone Domain Regulates the Radical B12 Enzyme IcmF. J Biol Chem 292:3977-3987
Campanello, Gregory C; Lofgren, Michael; Yokom, Adam L et al. (2017) Switch I-dependent allosteric signaling in a G-protein chaperone-B12 enzyme complex. J Biol Chem 292:17617-17625
Li, Zhu; Shanmuganathan, Aranganathan; Ruetz, Markus et al. (2017) Coordination chemistry controls the thiol oxidase activity of the B12-trafficking protein CblC. J Biol Chem 292:9733-9744
Shen, Hongying; Campanello, Gregory C; Flicker, Daniel et al. (2017) The Human Knockout Gene CLYBL Connects Itaconate to Vitamin B12. Cell 171:771-782.e11
Banerjee, Ruma (2017) Introduction to the Thematic Minireview Series: Redox metabolism and signaling. J Biol Chem 292:16802-16803
Banerjee, Ruma (2017) Introduction to the Thematic Minireview Series: Host-microbiome metabolic interplay. J Biol Chem 292:8544-8545
Banerjee, Ruma (2016) Introduction to the Thematic Minireview Series on Intrinsically Disordered Proteins. J Biol Chem 291:6679-80
Benjdia, Alhosna; Pierre, St├ęphane; Gherasim, Carmen et al. (2015) The thiostrepton A tryptophan methyltransferase TsrM catalyses a cob(II)alamin-dependent methyl transfer reaction. Nat Commun 6:8377

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