The reactivity of the cobalt-carbon bond in cobalamins is the key to their chemical versatility, supporting both methyl transfer and isomerization reactions. During evolution of higher eukaryotes that utilize vitamin B 12 , the high reactivity of the cofactor coupled with its low abundance pressured development of an efficient system for uptake, assimilation, and delivery of the cofactor to client B 12 -dependent enzymes. Although most proteins suspected to be involved in B 12 trafficking were discovered by 2009, the recent identification of a new protein reveals that the quest for elucidating the intracellular B 12 highway is still far from complete. Herein, we review the biochemistry of cobalamin trafficking.Cofactors are variously deployed in nature to stabilize macromolecular structures, expand catalytic functionality, transport gases, transduce signals, and function as sensors. Due to their relative rarity and/or reactivity, cells have evolved strategies for sequestering and regulating the movement of cofactors from their point of entry into the cell to their point of docking in target proteins (1). A subset of cofactors, i.e. the vitamins, is obtained in a precursor form from the diet. Reactions catalyzing the assimilation of inactive cofactors into their active forms are integral to their trafficking pathways. Similarly, elaboration of metals into clusters often occurs on chaperones that subsequently transfer the cofactor to target proteins. The interprotein transfer of metals can occur via ligand exchange reactions that are driven by differences in metal coordination geometry and affinity between the donor and acceptor proteins (2, 3). Seclusion of cofactors in chaperones during assembly/processing into their active forms minimizes unwanted side reactions, whereas guided delivery averts dilution and promotes specificity of cofactor docking.In contrast to our understanding of cellular strategies used for trafficking metals (4 -6) and metal clusters (7), significantly less is known about strategies for shepherding organic and organometallic cofactors to target proteins. This picture has been changing, however, with the convergence of clinical genetics and biochemical approaches that are beginning to illuminate an elaborate pathway for assimilation and delivery of dietary vitamin B 12 or cobalt-containing cobalamin, a complex organometallic cofactor (8 -10). Much less is known about how the tetrapyrrolic cousins of B 12 , e.g. iron protoporphyrin (heme), nickel corphin (F430), and magnesium chlorin (chlorophyll), are guided to specific destinations.In this minireview, we describe a model for mammalian cobalamin trafficking, which includes strategies for conversion of inactive precursors to the active cofactor forms methylcobalamin (MeCbl) 3 and 5Ј-deoxyadenosylcobalamin (AdoCbl; coenzyme B 12 ) and discuss the human diseases that result from impairments along the trafficking highway. We posit that the navigation strategy for B 12 , in which a rare, reactive, and high value cofactor is sequestered and targ...

Fidelity during cofactor assembly is essential for the proper functioning of metalloenzymes and is ensured by specific chaperones. MeaB, a G-protein chaperone for the coenzyme B12-dependent radical enzyme, methylmalonyl-CoA mutase (MCM), utilizes the energy of GTP binding and/or hydrolysis to regulate cofactor loading into MCM, protect MCM from inactivation, and rescue MCM inactivated during turnover. Typically, G-proteins signal to client proteins using the conformationally mobile switch I and II loops. Crystallographic snapshots of MeaB reported herein reveal a novel switch III element, which exhibits substantial conformational plasticity. Using alanine-scanning mutagenesis, we demonstrate that the switch III motif is critical for bidirectional signal transmission of the GTPase activating protein activity of MCM and the chaperone functions of MeaB in the MeaB:MCM complex. Mutations in the switch III loop identified in patients corrupt this inter-protein communication and lead to methylmalonic aciduria, an inborn error of metabolism.

Protein O-fucosyltransferase-1 (POFUT1), which transfers fucose residues to acceptor sites on serine and threonine residues of epidermal growth factor-like repeats of recipient proteins, is essential for Notch signal transduction in mammals. Here, we examine the consequences of POFUT1 loss on the oncogenic signaling associated with certain leukemia-associated mutations of human Notch1, report the structures of human POFUT1 in free and GDP-fucose bound states, and assess the effects of Dowling-Degos mutations on human POFUT1 function. CRISPR-mediated knockout of POFUT1 in U2OS cells suppresses both normal Notch1 signaling, and the ligand-independent signaling associated with leukemogenic mutations of Notch1. Normal and oncogenic signaling are rescued by wild-type POFUT1 but rescue is impaired by an active-site R240A mutation. The overall structure of the human enzyme closely resembles that of the Caenorhabditis elegans protein, with an overall backbone RMSD of 0.93 Å, despite primary sequence identity of only 39% in the mature protein. GDP-fucose binding to the human enzyme induces limited backbone conformational movement, though the side chains of R43 and D244 reorient to make direct contact with the fucose moiety in the complex. The reported Dowling-Degos mutations of POFUT1, except for M262T, fail to rescue Notch1 signaling efficiently in the CRISPR-engineered POFUT1-/- background. Together, these studies identify POFUT1 as a potential target for cancers driven by Notch1 mutations and provide a structural roadmap for its inhibition.

G-proteins regulate various processes ranging from DNA replication and protein synthesis to cytoskeletal dynamics and cofactor assimilation and serve as models for uncovering strategies deployed for allosteric signal transduction. MeaB is a multifunctional G-protein chaperone, which gates loading of the active 5'-deoxyadenosylcobalamin cofactor onto methylmalonyl-CoA mutase (MCM) and precludes loading of inactive cofactor forms. MeaB also safeguards MCM, which uses radical chemistry, against inactivation and rescues MCM inactivated during catalytic turnover by using the GTP-binding energy to offload inactive cofactor. The conserved switch I and II signaling motifs used by G-proteins are predicted to mediate allosteric regulation in response to nucleotide binding and hydrolysis in MeaB. Herein, we targeted conserved residues in the MeaB switch I motif to interrogate the function of this loop. Unexpectedly, the switch I mutations had only modest effects on GTP binding and on GTPase activity and did not perturb stability of the MCM-MeaB complex. However, these mutations disrupted multiple MeaB chaperone functions, including cofactor editing, loading, and offloading. Hence, although residues in the switch I motif are not essential for catalysis, they are important for allosteric regulation. Furthermore, single-particle EM analysis revealed, for the first time, the overall architecture of the MCM-MeaB complex, which exhibits a 2:1 stoichiometry. These EM studies also demonstrate that the complex exhibits considerable conformational flexibility. In conclusion, the switch I element does not significantly stabilize the MCM-MeaB complex or influence the affinity of MeaB for GTP but is required for transducing signals between MeaB and MCM.

Background: MeaB is a G-protein chaperone of methylmalonyl-CoA mutase (MCM). Results: Mutations in the canonical switch II motif disrupt signaling in the MeaB-MCM complex. Conclusion:The switch II loop is autoinhibitory for the intrinsic GTPase activity of MeaB. Significance: Signaling in the MeaB-MCM complex is achieved via nucleotide-dependent conformational coupling between switches II and III.