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Cobalamin is a complex organometallic cofactor that is processed and targeted via a network of chaperones to its dependent enzymes. AdoCbl (5′-deoxyadenosylcobalamin) is synthesized from cob(II)alamin in a reductive adenosylation reaction catalyzed by adenosyltransferase (ATR), which also serves as an escort, delivering AdoCbl to methylmalonyl-CoA mutase (MCM). The mechanism by which ATR signals that its cofactor cargo is ready (AdoCbl) or not [cob(II)alamin] for transfer to MCM, is not known. In this study, we have obtained crystallographic snapshots that reveal ligand-induced ordering of the N terminus of Mycobacterium tuberculosis ATR, which organizes a dynamic cobalamin binding site and exerts exquisite control over coordination geometry, reactivity, and solvent accessibility. Cob(II)alamin binds with its dimethylbenzimidazole tail splayed into a side pocket and its corrin ring buried. The cosubstrate, ATP, enforces a four-coordinate cob(II)alamin geometry, facilitating the unfavorable reduction to cob(I)alamin. The binding mode for AdoCbl is notably different from that of cob(II)alamin, with the dimethylbenzimidazole tail tucked under the corrin ring, displacing the N terminus of ATR, which is disordered. In this solvent-exposed conformation, AdoCbl undergoes facile transfer to MCM. The importance of the tail in cofactor handover from ATR to MCM is revealed by the failure of 5′-deoxyadenosylcobinamide, lacking the tail, to transfer. In the absence of MCM, ATR induces a sacrificial cobalt–carbon bond homolysis reaction in an unusual reversal of the heterolytic chemistry that was deployed to make the same bond. The data support an important role for the dimethylbenzimidazole tail in moving the cobalamin cofactor between active sites.

Itaconate is an immunometabolite with both anti-inflammatory and bactericidal effects. Its coenzyme A (CoA) derivative, itaconyl-CoA, inhibits B12-dependent methylmalonyl-CoA mutase (MCM) by an unknown mechanism. We demonstrate that itaconyl-CoA is a suicide inactivator of human and Mycobacterium tuberculosis MCM, which forms a markedly air-stable biradical adduct with the 5′-deoxyadenosyl moiety of the B12 coenzyme. Termination of the catalytic cycle in this way impairs communication between MCM and its auxiliary repair proteins. Crystallography and spectroscopy of the inhibited enzyme are consistent with a metal-centered cobalt radical ~6 angstroms away from the tertiary carbon-centered radical and suggest a means of controlling radical trajectories during MCM catalysis. Mycobacterial MCM thus joins enzymes in the glyoxylate shunt and the methylcitrate cycle as targets of itaconate in pathogen propionate metabolism.

Coenzyme B 12 (or 5′-deoxyadenosylcobalamin [AdoCbl]) is a cofactor for methylmalonyl-CoA mutase (MCM), which is important for propionate metabolism in Mycobacterium tuberculosis and for anaplerosis in humans. AdoCbl is synthesized by adenosyltransferase (ATR), which doubles as an escort, delivering the cofactor to MCM. The mechanism by which this large cofactor is translocated from ATR to MCM is not known. Our crystal structures of M. tuberculosis ATR reveal that mobile loops dynamically create a customized pocket to control cofactor coordination and conformation, regulating its reactivity and transportability. These changes also control solvent exposure and signaling to MCM when ATR is ready to transfer cargo, in a process where the cofactor tail plays an important role in the handover between proteins. Cobalamin is a complex organometallic cofactor that is processed and targeted via a network of chaperones to its dependent enzymes. AdoCbl (5′-deoxyadenosylcobalamin) is synthesized from cob(II)alamin in a reductive adenosylation reaction catalyzed by adenosyltransferase (ATR), which also serves as an escort, delivering AdoCbl to methylmalonyl-CoA mutase (MCM). The mechanism by which ATR signals that its cofactor cargo is ready (AdoCbl) or not [cob(II)alamin] for transfer to MCM, is not known. In this study, we have obtained crystallographic snapshots that reveal ligand-induced ordering of the N terminus of Mycobacterium tuberculosis ATR, which organizes a dynamic cobalamin binding site and exerts exquisite control over coordination geometry, reactivity, and solvent accessibility. Cob(II)alamin binds with its dimethylbenzimidazole tail splayed into a side pocket and its corrin ring buried. The cosubstrate, ATP, enforces a four-coordinate cob(II)alamin geometry, facilitating the unfavorable reduction to cob(I)alamin. The binding mode for AdoCbl is notably different from that of cob(II)alamin, with the dimethylbenzimidazole tail tucked under the corrin ring, displacing the N terminus of ATR, which is disordered. In this solvent-exposed conformation, AdoCbl undergoes facile transfer to MCM. The importance of the tail in cofactor handover from ATR to MCM is revealed by the failure of 5′-deoxyadenosylcobinamide, lacking the tail, to transfer. In the absence of MCM, ATR induces a sacrificial cobalt–carbon bond homolysis reaction in an unusual reversal of the heterolytic chemistry that was deployed to make the same bond. The data support an important role for the dimethylbenzimidazole tail in moving the cobalamin cofactor between active sites.

The human brain possesses three predominate phospholipids, phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS), which account for approximately 35–40%, 35–40%, and 20% of the brain’s phospholipids, respectively. Mitochondrial membranes are relatively diverse, containing the aforementioned PC, PE, and PS, as well as phosphatidylinositol (PI) and phosphatidic acid (PA); however, cardiolipin (CL) and phosphatidylglycerol (PG) are exclusively present in mitochondrial membranes. These phospholipid interactions play an essential role in mitochondrial fusion and fission dynamics, leading to the maintenance of mitochondrial structural and signaling pathways. The essential nature of these phospholipids is demonstrated through the inability of mitochondria to tolerate alteration in these specific phospholipids, with changes leading to mitochondrial damage resulting in neural degeneration. This review will emphasize how the structure of phospholipids relates to their physiologic function, how their metabolism facilitates signaling, and the role of organ- and mitochondria-specific phospholipid compositions. Finally, we will discuss the effects of global ischemia and reperfusion on organ- and mitochondria-specific phospholipids alongside the novel therapeutics that may protect against injury.

Peripheral neuropathy can be a rare symptom of erythrocytosis through an ischemia-related mechanism. Vitamin B12 deficiency is another cause of peripheral neuropathy through the impaired maintenance of peripheral myelin sheaths. Patients with myeloproliferative disorders causing erythrocytosis can present clinically with functional, symptomatic vitamin B12 deficiencies despite normal serum levels. We present a case of peripheral neuropathy occurring secondary to both erythrocytosis and a functional B12 deficiency. The patient's pattern of peripheral neuropathy could not be explained by erythrocytosis alone, supporting a joint etiology involving a functional B12 deficiency. The patient was treated with therapeutic phlebotomy and vitamin B12 injections to address both underlying causes of his symptoms. He was discharged to follow up with an outpatient hematologist and reported mild symptom improvement at the two-week and one-month follow-up appointments.

The performance management process is seen as a closed loop control system which deploys policy and strategy, and obtains feedback from various levels in order to manage the performance of the business. The performance measurement system is the information system which is at the heart of the performance management process and it is of critical importance to the effective and efficient functioning of the performance management system. Research identifies two critical elements with respect to the content and structure of the performance measurement system. These are: integrity and deployment. The viable systems model (VSM) provides a framework for assessing the integrity of the performance measurement system. The reference model developed for integrated performance measurement systems provides a framework against which performance measurement systems can be designed and audited.

The performance management process is seen as a closed loop control system which deploys policy and strategy, and obtains feedback from various levels in order to manage the performance of the business.

WalKR (YycFG) is the only essential two-component regulator in the human pathogen Staphylococcus aureus . WalKR regulates peptidoglycan synthesis, but this function alone does not explain its essentiality. Here, to further understand WalKR function, we investigate a suppressor mutant that arose when WalKR activity was impaired; a histidine to tyrosine substitution (H271Y) in the cytoplasmic Per-Arnt-Sim (PAS CYT ) domain of the histidine kinase WalK. Introducing the WalK H271Y mutation into wild-type S. aureus activates the WalKR regulon. Structural analyses of the WalK PAS CYT domain reveal a metal-binding site, in which a zinc ion (Zn 2+ ) is tetrahedrally-coordinated by four amino acids including H271. The WalK H271Y mutation abrogates metal binding, increasing WalK kinase activity and WalR phosphorylation. Thus, Zn 2+ -binding negatively regulates WalKR. Promoter-reporter experiments using S. aureus confirm Zn 2+ sensing by this system. Identification of a metal ligand recognized by the WalKR system broadens our understanding of this critical S. aureus regulon. WalKR is an essential two-component regulator that controls peptidoglycan synthesis in the human pathogen Staphylococcus aureus . Here, the authors provide biochemical, structural, and functional evidence supporting that the binding of a zinc ion inhibits autophosphorylation and thus alters WalKR regulatory activity.

Among the 16 two-component systems in the opportunistic human pathogen Staphylococcus aureus , only WalKR is essential. Like the orthologous systems in other Bacillota, S. aureus WalKR controls autolysins involved in peptidoglycan remodeling and is therefore intimately involved in cell division. However, despite the importance of WalKR in S. aureus , the basis for its essentiality is not understood and the regulon is poorly defined. Here, we defined a consensus WalR DNA-binding motif and the direct WalKR regulon by using functional genomics, including chromatin immunoprecipitation sequencing, with a panel of isogenic walKR mutants that had a spectrum of altered activities. Consistent with prior findings, the direct regulon includes multiple autolysin genes. However, this work also revealed that WalR directly regulates at least five essential genes involved in lipoteichoic acid synthesis ( ltaS ): translation ( rplK ), DNA compaction ( hup ), initiation of DNA replication ( dnaA , hup ) and purine nucleotide metabolism ( prs ). Thus, WalKR in S. aureus serves as a polyfunctional regulator that contributes to fundamental control over critical cell processes by coordinately linking cell wall homeostasis with purine biosynthesis, protein biosynthesis, and DNA replication. Our findings further address the essentiality of this locus and highlight the importance of WalKR as a bona fide target for novel anti-staphylococcal therapeutics. The opportunistic human pathogen Staphylococcus aureus uses an array of protein sensing systems called two-component systems (TCS) to sense environmental signals and adapt its physiology in response by regulating different genes. This sensory network is key to S. aureus versatility and success as a pathogen. Here, we reveal for the first time the full extent of the regulatory network of WalKR, the only staphylococcal TCS that is indispensable for survival under laboratory conditions. We found that WalKR is a master regulator of cell growth, coordinating the expression of genes from multiple, fundamental S. aureus cellular processes, including those involved in maintaining cell wall metabolism, protein biosynthesis, nucleotide metabolism, and the initiation of DNA replication.

Phenotypically agnostic screens for positive selection in pathogen populations provide a means of pinpointing genes and regulatory regions involved in adaptation to the local environment or host population. We screened a large ( n  = 2506) collection of Vietnamese Mycobacterium tuberculosis ( Mtb ) isolates, finding targets of selection to be lineage-specific, and encompass diverse functions, including dormancy ( Rv0080 ), zinc homeostasis ( zur ), and virulence (ESX-1 structure). Extending our screen to the wider Mtb complex (MTBC) phylogeny demonstrated Rv0080 to display an extraordinarily dynamic evolutionary history, acquiring premature stop codons or putative functional mutations on branches upstream of 8 of the 10 human-adapted lineages, and undergoing positive selection in the remaining 2. Lineage 1, which is one of two such lineages retaining the ancestral Rv0080 sequence, displays a rate of selection for this gene (dN/dS=9.37) exceeding any other in the Mtb genome, save a transcription factor linked to its expression ( Rv0042c ; dN/dS=11.02). Deletion of Rv0080 ’s M. smegmatis orthologue confers a survival advantage in hypoxic conditions, as does the evolution of nonsense or missense mutations on an ancestral Rv0080 background. We show the dormancy survival regulon experienced recurrent episodes of reductive evolution across the MTBC phylogeny, illuminating a novel mechanism via which it adapted to human populations. Mycobacterium tuberculosis owes its success partly to its ability to enter a ‘dormant’, non-replicative state, reactivating years or even decades after initial infection. In this work, authors find that a key alteration in a gene involved in this dormancy response has evolved, or is evolving, in parallel in human-adapted lineages across the globe.