Structure of metallochaperone in complex with the cobalamin-binding domain of its target mutase provides insight into cofactor delivery.

G-protein metallochaperone MeaB in bacteria [methylmalonic aciduria type A (MMAA) in humans] is responsible for facilitating the delivery of adenosylcobalamin (AdoCbl) to methylmalonyl-CoA mutase (MCM), the only AdoCbl-dependent enzyme in humans. Genetic defects in the switch III region of MMAA lead to the genetic disorder methylmalonic aciduria in which the body is unable to process certain lipids. Here, we present a crystal structure of Methylobacterium extorquens MeaB bound to a nonhydrolyzable guanosine triphosphate (GTP) analog guanosine-5'-[(ß,γ)-methyleno]triphosphate (GMPPCP) with the Cbl-binding domain of its target mutase enzyme (MeMCMcbl). This structure provides an explanation for the stimulation of the GTP hydrolyase activity of MeaB afforded by target protein binding. We find that upon MCMcbl association, one protomer of the MeaB dimer rotates ~180°, such that the inactive state of MeaB is converted to an active state in which the nucleotide substrate is now surrounded by catalytic residues. Importantly, it is the switch III region that undergoes the largest change, rearranging to make direct contacts with the terminal phosphate of GMPPCP. These structural data additionally provide insights into the molecular basis by which this metallochaperone contributes to AdoCbl delivery without directly binding the cofactor. Our data suggest a model in which GTP-bound MeaB stabilizes a conformation of MCM that is open for AdoCbl insertion, and GTP hydrolysis, as signaled by switch III residues, allows MCM to close and trap its cofactor. Substitutions of switch III residues destabilize the active state of MeaB through loss of protein:nucleotide and protein:protein interactions at the dimer interface, thus uncoupling GTP hydrolysis from AdoCbl delivery.

Bivalent molecular mimicry by ADP protects metal redox state and promotes coenzyme B12 repair.

Control over transition metal redox state is essential for metalloprotein function and can be achieved via coordination chemistry and/or sequestration from bulk solvent. Human methylmalonyl-Coenzyme A (CoA) mutase (MCM) catalyzes the isomerization of methylmalonyl-CoA to succinyl-CoA using 5'-deoxyadenosylcobalamin (AdoCbl) as a metallocofactor. During catalysis, the occasional escape of the 5'-deoxyadenosine (dAdo) moiety leaves the cob(II)alamin intermediate stranded and prone to hyperoxidation to hydroxocobalamin, which is recalcitrant to repair. In this study, we have identified the use of bivalent molecular mimicry by ADP, coopting the 5'-deoxyadenosine and diphosphate moieties in the cofactor and substrate, respectively, to protect against cob(II)alamin overoxidation on MCM. Crystallographic and electron paramagnetic resonance (EPR) data reveal that ADP exerts control over the metal oxidation state by inducing a conformational change that seals off solvent access, rather than by switching five-coordinate cob(II)alamin to the more air stable four-coordinate state. Subsequent binding of methylmalonyl-CoA (or CoA) promotes cob(II)alamin off-loading from MCM to adenosyltransferase for repair. This study identifies an unconventional strategy for controlling metal redox state by an abundant metabolite to plug active site access, which is key to preserving and recycling a rare, but essential, metal cofactor.

A Noble Metal Substitution Leads to B12 Cofactor Mimicry by a Rhodibalamin.

In mammals, cobalamin is an essential cofactor that is delivered by a multitude of chaperones in an elaborate trafficking pathway to two client enzymes, methionine synthase and methylmalonyl-CoA mutase (MMUT). Rhodibalamins, the rhodium analogs of cobalamins, have been described as antimetabolites due to their ability to inhibit bacterial growth. In this study, we have examined the reactivity of adenosylrhodibalamin (AdoRhbl) with two key human chaperones, MMACHC (also known as CblC) and adenosyltransferase (MMAB, also known as ATR), and with the human and Mycobacterium tuberculosis MMUT. We demonstrate that while AdoRhbl binds tightly to all four proteins, the Rh-carbon bond is resistant to homolytic (on MMAB and MMUT) as well as heterolytic (on MMACHC) rupture. On the other hand, MMAB catalyzes Rh-carbon bond formation, converting rhodi(I)balamin in the presence of ATP to AdoRhbl. We report the first crystal structure of a rhodibalamin (AdoRhbl) bound to a B12 protein, i.e., MMAB, in the presence of triphosphate, which shows a weakened but intact Rh-carbon bond. The structure provides insights into how MMAB cleaves the corresponding Co-carbon bond in a sacrificial homolytic reaction that purportedly functions as a cofactor sequestration strategy. Collectively, the study demonstrates that while the noble metal substitution of cobalt by rhodium sets up structural mimicry, it compromises chemistry, which could be exploited for targeting human and bacterial B12 chaperones and enzymes.

Structural insight into G-protein chaperone-mediated maturation of a bacterial adenosylcobalamin-dependent mutase.

G-protein metallochaperones are essential for the proper maturation of numerous metalloenzymes. The G-protein chaperone MMAA in humans (MeaB in bacteria) uses GTP hydrolysis to facilitate the delivery of adenosylcobalamin (AdoCbl) to AdoCbl-dependent methylmalonyl-CoA mutase, an essential metabolic enzyme. This G-protein chaperone also facilitates the removal of damaged cobalamin (Cbl) for repair. Although most chaperones are standalone proteins, isobutyryl-CoA mutase fused (IcmF) has a G-protein domain covalently attached to its target mutase. We previously showed that dimeric MeaB undergoes a 180° rotation to reach a state capable of GTP hydrolysis (an active G-protein state), in which so-called switch III residues of one protomer contact the G-nucleotide of the other protomer. However, it was unclear whether other G-protein chaperones also adopted this conformation. Here, we show that the G-protein domain in a fused system forms a similar active conformation, requiring IcmF oligomerization. IcmF oligomerizes both upon Cbl damage and in the presence of the nonhydrolyzable GTP analog, guanosine-5'-[(ß,γ)-methyleno]triphosphate, forming supramolecular complexes observable by mass photometry and EM. Cryo-EM structural analysis reveals that the second protomer of the G-protein intermolecular dimer props open the mutase active site using residues of switch III as a wedge, allowing for AdoCbl insertion or damaged Cbl removal. With the series of structural snapshots now available, we now describe here the molecular basis of G-protein-assisted AdoCbl-dependent mutase maturation, explaining how GTP binding prepares a mutase for cofactor delivery and how GTP hydrolysis allows the mutase to capture the cofactor.

Redox-Linked Coordination Chemistry Directs Vitamin B12 Trafficking.

Metals are partners for an estimated one-third of the proteome and vary in complexity from mononuclear centers to organometallic cofactors. Vitamin B12 or cobalamin represents the epitome of this complexity and is the product of an assembly line comprising some 30 enzymes. Unable to biosynthesize cobalamin, mammals rely on dietary provision of this essential cofactor, which is needed by just two enzymes, one each in the cytoplasm (methionine synthase) and the mitochondrion (methylmalonyl-CoA mutase). Brilliant clinical genetics studies on patients with inborn errors of cobalamin metabolism spanning several decades had identified at least seven genetic loci in addition to the two encoding B12 enzymes. While cells are known to house a cadre of chaperones dedicated to metal trafficking pathways that contain metal reactivity and confer targeting specificity, the seemingly supernumerary chaperones in the B12 pathway had raised obvious questions as to the rationale for their existence.With the discovery of the genes underlying cobalamin disorders, our laboratory has been at the forefront of ascribing functions to B12 chaperones and elucidating the intricate redox-linked coordination chemistry and protein-linked cofactor conformational dynamics that orchestrate the processing and translocation of cargo along the trafficking pathway. These studies have uncovered novel chemistry that exploits the innate chemical versatility of alkylcobalamins, i.e., the ability to form and dismantle the cobalt-carbon bond using homolytic or heterolytic chemistry. In addition, they have revealed the practical utility of the dimethylbenzimidazole tail, an appendage unique to cobalamins and absent in the structural cousins, porphyrin, chlorin, and corphin, as an instrument for facilitating cofactor transfer between active sites.In this Account, we navigate the chemistry of the B12 trafficking pathway from its point of entry into cells, through lysosomes, and into the cytoplasm, where incoming cobalamin derivatives with a diversity of upper ligands are denuded by the ß-ligand transferase activity of CblC to the common cob(II)alamin intermediate. The broad reaction and lax substrate specificity of CblC also enables conversion of cyanocobalamin (technically, vitamin B12, i.e., the form of the cofactor in one-a-day supplements), to cob(II)alamin. CblD then hitches up with CblC via a unique Co-sulfur bond to cob(II)alamin at a bifurcation point, leading to the cytoplasmic methylcobalamin or mitochondrial 5'-deoxyadenosylcobalamin branch. Mutations at loci upstream of the junction point typically affect both branches, leading to homocystinuria and methylmalonic aciduria, whereas mutations in downstream loci lead to one or the other disease. Elucidation of the biochemical penalties associated with individual mutations is providing molecular insights into the clinical data and, in some instances, identifying which cobalamin derivative(s) might be therapeutically beneficial.Our studies on B12 trafficking are revealing strategies for cofactor sequestration and mobilization from low- to high-affinity and low- to high-coordination-number sites, which in turn are regulated by protein dynamics that constructs ergonomic cofactor binding pockets. While these B12 lessons might be broadly relevant to other metal trafficking pathways, much remains to be learned. This Account concludes by identifying some of the major gaps and challenges that are needed to complete our understanding of B12 trafficking.

Structure-Based Demystification of Radical Catalysis by a Coenzyme B12 Dependent Enzyme-Crystallographic Study of Glutamate Mutase with Cofactor Homologues.

Catalysis by radical enzymes dependent on coenzyme B12 (AdoCbl) relies on the reactive primary 5'-deoxy-5'adenosyl radical, which originates from reversible Co-C bond homolysis of AdoCbl. This bond homolysis is accelerated roughly 1012 -fold upon binding the enzyme substrate. The structural basis for this activation is still strikingly enigmatic. As revealed here, a displaced firm adenosine binding cavity in substrate-loaded glutamate mutase (GM) causes a structural misfit for intact AdoCbl that is relieved by the homolytic Co-C bond cleavage. Strategically interacting adjacent adenosine- and substrate-binding protein cavities provide a tight caged radical reaction space, controlling the entire radical path. The GM active site is perfectly structured for promoting radical catalysis, including "negative catalysis", a paradigm for AdoCbl-dependent mutases.

Switch I-dependent allosteric signaling in a G-protein chaperone-B12 enzyme complex.

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.

The entropic contributions in vitamin B12 enzymes still reflect the electrostatic paradigm.

The catalytic power of enzymes containing coenzyme B12 has been, in some respects, the "last bastion" for the strain hypothesis. Our previous study of this system established by a careful sampling that the major part of the catalytic effect is due to the electrostatic interaction between the ribose of the ado group and the protein and that the strain contribution is very small. This finding has not been sufficiently appreciated due to misunderstandings of the power of the empirical valence bond (EVB) calculations and the need of sufficient sampling. Furthermore, some interesting new experiments point toward entropic effects as the source of the catalytic power, casting doubt on the validity of the electrostatic idea, at least, in the case of B12 enzymes. Here, we focus on the observation of the entropic effects and on analyzing their origin. We clarify that our EVB approach evaluates free energies rather than enthalpies and demonstrate by using the restraint release (RR) approach that the observed entropic contribution to the activation barrier is of electrostatic origin. Our study illustrates the power of the RR approach by evaluating the entropic contributions to catalysis and provides further support to our paradigm for the origin of the catalytic power of B12 enzymes. Overall, our study provides major support to our electrostatic preorganization idea and also highlights the basic requirements from ab initio quantum mechanics/molecular mechanics calculations of activation free energies of enzymatic reactions.

Visualization of a radical B12 enzyme with its G-protein chaperone.

G-protein metallochaperones ensure fidelity during cofactor assembly for a variety of metalloproteins, including adenosylcobalamin (AdoCbl)-dependent methylmalonyl-CoA mutase and hydrogenase, and thus have both medical and biofuel development applications. Here, we present crystal structures of IcmF, a natural fusion protein of AdoCbl-dependent isobutyryl-CoA mutase and its corresponding G-protein chaperone, which reveal the molecular architecture of a G-protein metallochaperone in complex with its target protein. These structures show that conserved G-protein elements become ordered upon target protein association, creating the molecular pathways that both sense and report on the cofactor loading state. Structures determined of both apo- and holo-forms of IcmF depict both open and closed enzyme states, in which the cofactor-binding domain is alternatively positioned for cofactor loading and for catalysis. Notably, the G protein moves as a unit with the cofactor-binding domain, providing a visualization of how a chaperone assists in the sequestering of a precious cofactor inside an enzyme active site.

Molecular Genetic Characterization of 151 Mut-Type Methylmalonic Aciduria Patients and Identification of 41 Novel Mutations in MUT.

Isolated methylmalonic aciduria (MMA) is an autosomal-recessive disorder of propionate metabolism that is most commonly caused by mutations in the methylmalonyl-CoA mutase (MUT) gene (mut-type MMA). We investigated a cohort of 151 patients, classifying 114 patients as mut(0) and 32 as mut(-) (five not defined). As per the definition, mut(-) patients showed a higher propionate incorporation ratio in vitro, which was correlated to a considerably later age of onset compared with mut(0) patients. In all patients, we found a total of 110 different mutations, of which 41 were novel. While the missense alleles p.Asn219Tyr, p.Arg369His, and p.Arg694Trp recurred in >10 alleles, 47 mutations were identified only once, suggesting many patients carry private mutations. Deficient alleles in the mut(-) subclass were almost exclusively caused by missense mutations, found disproportionately in the C-terminal cofactor binding domain. On the contrary, only half of the mut(0) mutations were of the missense type. Western blot analysis revealed reduced MUT protein for all 34 cell lines (27 mut(0) , seven mut(-) ) tested, suggesting protein instability as a major mechanism of deficiency in mut-type MMA. This large-scale evaluation helps to characterize the landscape of MUT mutations and their relationship to dysfunction and disease.

Structural basis of the stereospecificity of bacterial B12-dependent 2-hydroxyisobutyryl-CoA mutase.

Bacterial coenzyme B12-dependent 2-hydroxyisobutyryl-CoA mutase (HCM) is a radical enzyme catalyzing the stereospecific interconversion of (S)-3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA. It consists of two subunits, HcmA and HcmB. To characterize the determinants of substrate specificity, we have analyzed the crystal structure of HCM from Aquincola tertiaricarbonis in complex with coenzyme B12 and the substrates (S)-3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA in alternative binding. When compared with the well studied structure of bacterial and mitochondrial B12-dependent methylmalonyl-CoA mutase (MCM), HCM has a highly conserved domain architecture. However, inspection of the substrate binding site identified amino acid residues not present in MCM, namely HcmA Ile(A90) and Asp(A117). Asp(A117) determines the orientation of the hydroxyl group of the acyl-CoA esters by H-bond formation, thus determining stereospecificity of catalysis. Accordingly, HcmA D117A and D117V mutations resulted in significantly increased activity toward (R)-3-hydroxybutyryl-CoA. Besides interconversion of hydroxylated acyl-CoA esters, wild-type HCM as well as HcmA I90V and I90A mutant enzymes could also isomerize pivalyl- and isovaleryl-CoA, albeit at >10 times lower rates than the favorite substrate (S)-3-hydroxybutyryl-CoA. The nonconservative mutation HcmA D117V, however, resulted in an enzyme showing high activity toward pivalyl-CoA. Structural requirements for binding and isomerization of highly branched acyl-CoA substrates such as 2-hydroxyisobutyryl- and pivalyl-CoA, possessing tertiary and quaternary carbon atoms, respectively, are discussed.

A switch III motif relays signaling between a B12 enzyme and its G-protein chaperone.

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), uses the energy of GTP binding, hydrolysis or both to regulate cofactor loading into MCM, protect MCM from inactivation and rescue MCM that is 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 new switch III element that has 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 interprotein communication and lead to methylmalonic aciduria, an inborn error of metabolism.

Functional and structural characteristics of methylmalonyl-CoA mutase from Pyrococcus horikoshii.

Methylmalonyl-CoA mutase (MCM) requires 5'-deoxyadenosylcobalamin (AdoCbl) as a cofactor and is widely distributed in organisms from bacteria and animals. Although genes encoding putative MCMs are present in many archaea, they are separately encoded in large and small subunits. The large and small subunits of archaeal MCM are similar to the catalytic and AdoCbl-binding domains of human MCM, respectively. In Pyrococcus horikoshii OT3, putative genes PH1306 and PH0275 encode the large and small subunits, respectively. Because information on archaeal MCM is extremely restricted, we examined the functional and structural characteristics of P. horikoshii MCM. Reconstitution experiments using recombinant PH0275 and PH1306 showed that these proteins assemble in equimolar ratios and form of heterotetrameric complexes in the presence of AdoCbl. Subsequent immunoprecipitation experiments using anti-PH0275 and anti-PH1306 antibodies suggested that PH0275 and PH1306 form a complex in P. horikoshii cells in the presence of AdoCbl.

A mechanochemical switch to control radical intermediates.

B12-dependent enzymes employ radical species with exceptional prowess to catalyze some of the most chemically challenging, thermodynamically unfavorable reactions. However, dealing with highly reactive intermediates is an extremely demanding task, requiring sophisticated control strategies to prevent unwanted side reactions. Using hybrid quantum mechanical/molecular mechanical simulations, we follow the full catalytic cycle of an AdoB12-dependent enzyme and present the details of a mechanism that utilizes a highly effective mechanochemical switch. When the switch is "off", the 5'-deoxyadenosyl radical moiety is stabilized by releasing the internal strain of an enzyme-imposed conformation. Turning the switch "on," the enzyme environment becomes the driving force to impose a distinct conformation of the 5'-deoxyadenosyl radical to avoid deleterious radical transfer. This mechanochemical switch illustrates the elaborate way in which enzymes attain selectivity of extremely chemically challenging reactions.

Navigating the B(12) road: assimilation, delivery, and disorders of cobalamin.

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 B12, 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 B12-dependent enzymes. Although most proteins suspected to be involved in B12 trafficking were discovered by 2009, the recent identification of a new protein reveals that the quest for elucidating the intracellular B12 highway is still far from complete. Herein, we review the biochemistry of cobalamin trafficking.

Autoinhibition and signaling by the switch II motif in the G-protein chaperone of a radical B12 enzyme.

MeaB is an accessory GTPase protein involved in the assembly, protection, and reactivation of 5'-deoxyadenosyl cobalamin-dependent methylmalonyl-CoA mutase (MCM). Mutations in the human ortholog of MeaB result in methylmalonic aciduria, an inborn error of metabolism. G-proteins typically utilize conserved switch I and II motifs for signaling to effector proteins via conformational changes elicited by nucleotide binding and hydrolysis. Our recent discovery that MeaB utilizes an unusual switch III region for bidirectional signaling with MCM raised questions about the roles of the switch I and II motifs in MeaB. In this study, we addressed the functions of conserved switch II residues by performing alanine-scanning mutagenesis. Our results demonstrate that the GTPase activity of MeaB is autoinhibited by switch II and that this loop is important for coupling nucleotide-sensitive conformational changes in switch III to elicit the multiple chaperone functions of MeaB. Furthermore, we report the structure of MeaB·GDP crystallized in the presence of AlFx(-) to form the putative transition state analog, GDP·AlF4(-). The resulting crystal structure and its comparison with related G-proteins support the conclusion that the catalytic site of MeaB is incomplete in the absence of the GTPase-activating protein MCM and therefore unable to stabilize the transition state analog. Favoring an inactive conformation in the absence of the client MCM protein might represent a strategy for suppressing the intrinsic GTPase activity of MeaB in which the switch II loop plays an important role.

Mutagenesis of a conserved glutamate reveals the contribution of electrostatic energy to adenosylcobalamin co-C bond homolysis in ornithine 4,5-aminomutase and methylmalonyl-CoA mutase.

Binding of substrate to ornithine 4,5-aminomutase (OAM) and methylmalonyl-CoA mutase (MCM) leads to the formation of an electrostatic interaction between a conserved glutamate side chain and the adenosyl ribose of the adenosylcobalamin (AdoCbl) cofactor. The contribution of this residue (Glu338 in OAM from Clostridium sticklandii and Glu392 in human MCM) to AdoCbl Co-C bond labilization and catalysis was evaluated by substituting the residue with a glutamine, aspartate, or alanine. The OAM variants, E338Q, E338D, and E338A, showed 90-, 380-, and 670-fold reductions in catalytic turnover and 20-, 60-, and 220-fold reductions in k(cat)/K(m), respectively. Likewise, the MCM variants, E392Q, E392D, and E392A, showed 16-, 330-, and 12-fold reductions in k(cat), respectively. Binding of substrate to OAM is unaffected by the single-amino acid mutation as stopped-flow absorbance spectroscopy showed that the rates of external aldimine formation in the OAM variants were similar to that of the native enzyme. The decrease in the level of catalysis is instead linked to impaired Co-C bond rupture, as UV-visible spectroscopy did not show detectable AdoCbl homolysis upon binding of the physiological substrate, d-ornithine. AdoCbl homolysis was also not detected in the MCM mutants, as it was for the native enzyme. We conclude from these results that a gradual weakening of the electrostatic energy between the protein and the ribose leads to a progressive increase in the activation energy barrier for Co-C bond homolysis, thereby pointing to a key role for the conserved polar glutamate residue in controlling the initial generation of radical species.

Biochemistry of B12-cofactors in human metabolism.

Vitamin B12, the "antipernicious anaemia factor", is a crystallisable cobalt-complex, which belongs to a group of unique "complete" corrinoids, named cobalamins (Cbl). In humans, instead of the "vitamin", two organometallic B12-forms are coenzymes in two metabolically important enzymes: Methyl-cobalamin, the cofactor of methionine synthase, and coenzyme B12 (adenosyl-cobalamin), the cofactor of methylmalonyl-CoA mutase. The cytoplasmatic methionine synthase catalyzes the transfer of a methyl group from N-methyl-tetrahydrofolate to homocysteine to yield methionine and to liberate tetrahydrofolate. In the mitochondrial methylmalonyl-CoA mutase a radical process transforms methylmalonyl-CoA (a remains e.g. from uneven numbered fatty acids) into succinyl-CoA, for further metabolic use. In addition, in the human mitochondria an adenosyl-transferase incorporates the organometallic group of coenzyme B12. In all these enzymes, the bound B12-derivatives engage (or are formed) in exceptional organometallic enzymatic reactions. This chapter recapitulates the physiological chemistry of vitamin B12, relevant in the context of the metabolic transformation of B12-derivatives into the relevant coenzyme forms and their use in B12-dependent enzymes.

The elusive 5'-deoxyadenosyl radical in coenzyme-B12-mediated reactions.

Vitamin B(12) and its biologically active counterparts possess the only examples of carbon-cobalt bonds in living systems. The role of such motifs as radical reservoirs has potential application in future catalytic and electronic nanodevices. To fully understand radical generation in coenzyme B(12) (dAdoCbl)-dependent enzymes, however, major obstacles still need to be overcome. In this work, we have used Car-Parrinello molecular dynamics (CPMD) simulations, in a mixed quantum mechanics/molecular mechanics (QM/MM) framework, to investigate the initial stages of the methylmalonyl-CoA-mutase-catalyzed reaction. We demonstrate that the 5'-deoxyadenosyl radical (dAdo(•)) exists as a distinct entity in this reaction, consistent with the results of extensive experimental and some previous theoretical studies. We report free energy calculations and first-principles trajectories that help understand how B(12) enzymes catalyze coenzyme activation and control highly reactive radical intermediates.

Microarray based mutational analysis of patients with methylmalonic acidemia: identification of 10 novel mutations.

Methylmalonic acidemia is an autosomal recessive metabolic disorder affecting the propionate oxidation pathway in the catabolism of several amino acids, odd-chain fatty acids, and cholesterol. Methylmalonic acidemia is characterized by elevated levels of methylmalonic acid in the blood and urine. Mutations in the MUT gene, encoding methylmalonyl-CoA mutase carries out isomerization of L-methylmalonyl-CoA to succinyl-CoA, cause methylmalonic acidemia. In this study, 30 Turkish patients diagnosed with mut methylmalonic acidemia were screened for mutations using custom designed sequencing microarrays. The study resulted in detection of 22 different mutations, 10 of which were novel: p.Q132*, p.A137G, c.753+1T, p.T387I, p.Q514E, p.P615L, p.D625V, c.1962_1963delTC, p.L674F, and c.2115_2116insA. The most common, p.P615T, was identified in 28.0% of patients. These results suggest that microarray based sequencing is a useful tool for the detection of mutations in MUT in patients with mut methylmalonic acidemia.