Mycobacterium tuberculosis (Mtb) acquires non-heme iron through salicylate-derived siderophores termed mycobactins whereas heme iron is obtained through a cascade of heme uptake proteins. Three proteins are proposed to mediate Mtb heme iron uptake, a secreted heme transporter (Rv0203), and MmpL3 and MmpL11, which are potential transmembrane heme transfer proteins. Furthermore, MhuD, a cytoplasmic heme-degrading enzyme, has been identified. Rv0203, MmpL3 and MmpL11 are mycobacteria-specific proteins, making them excellent drug targets. Importantly, MmpL3, a necessary protein, has also been implicated in trehalose monomycolate export. Recent drug-discovery efforts revealed that MmpL3 is the target of several compounds with antimycobacterial activity. Inhibition of the Mtb heme uptake pathway has yet to be explored. We propose that inhibitor design could focus on heme analogs, with the goal of blocking specific steps of this pathway. In addition, heme uptake could be hijacked as a method of importing drugs into the mycobacterial cytosol.

Iron is an essential nutrient for the survival of organisms. Bacterial pathogens possess specialized pathways to acquire heme from their human hosts. In this review, we present recent structural and biochemical data that provide mechanistic insights into several bacterial heme uptake pathways, encompassing the sequestration of heme from human hemoproteins to secreted or membrane-associated bacterial proteins, the transport of heme across bacterial membranes, and the degradation of heme within the bacterial cytosol to liberate iron. The pathways for heme transport into the bacterial cytosol are divergent, harboring non-homologous protein sequences, novel structures, varying numbers of proteins, and different mechanisms. Congruously, the breakdown of heme within the bacterial cytosol by sequence-divergent proteins releases iron and distinct degradation products.

Abstract Background Bacterial Disulfide bond forming (Dsb) proteins facilitate proper folding and disulfide bond formation of periplasmic and secreted proteins. Previously, we have shown that Mycobacterium tuberculosis Mt-DsbE and Mt-DsbF aid in vitro oxidative folding of proteins. The M. tuberculosis proteome contains another predicted membrane-tethered Dsb protein, Mt-DsbA, which is encoded by an essential gene. Results Herein, we present structural and biochemical analyses of Mt-DsbA. The X-ray crystal structure of Mt-DsbA reveals a two-domain structure, comprising a canonical thioredoxin domain with the conserved CXXC active site cysteines in their reduced form, and an inserted α-helical domain containing a structural disulfide bond. The overall fold of Mt-DsbA resembles that of other DsbA-like proteins and not Mt-DsbE or Mt-DsbF. Biochemical characterization demonstrates that, unlike Mt-DsbE and Mt-DsbF, Mt-DsbA is unable to oxidatively fold reduced, denatured hirudin. Moreover, on the substrates tested in this study, Mt-DsbA has disulfide bond isomerase activity contrary to Mt-DsbE and Mt-DsbF. Conclusion These results suggest that Mt-DsbA acts upon a distinct subset of substrates as compared to Mt-DsbE and Mt-DsbF. One could speculate that Mt-DsbE and Mt-DsbF are functionally redundant whereas Mt-DsbA is not, offering an explanation for the essentiality of Mt-DsbA in M. tuberculosis.

Replicative DNA polymerases are highly efficient enzymes that maintain stringent geometric control over shape and orientation of the template and incoming nucleoside triphosphate. In a surprising twist to this paradigm, a naturally occurring bacterial DNA polymerase I member isolated from Geobacillus stearothermophilus (Bst) exhibits an innate ability to reverse transcribe RNA and other synthetic congeners (XNAs) into DNA. This observation raises the interesting question of how a replicative DNA polymerase is able to recognize templates of diverse chemical composition. Here, we present crystal structures of natural Bst DNA polymerase that capture the post-translocated product of DNA synthesis on templates composed entirely of 2'-deoxy-2'-fluoro-β-d-arabino nucleic acid (FANA) and α-l-threofuranosyl nucleic acid (TNA). Analysis of the enzyme active site reveals the importance of structural plasticity as a possible mechanism for XNA-dependent DNA synthesis and provides insights into the construction of variants with improved activity.

Mycobacterium tuberculosis, the pathogen that causes tuberculosis, has evolved sophisticated mechanisms for evading assault by the human host. This review focuses on M. tuberculosis regulatory metalloproteins that are sensitive to exogenous stresses attributed to changes in the levels of gaseous molecules (i.e., molecular oxygen, carbon monoxide and nitric oxide) to elicit an intracellular response. In particular, we highlight recent developments on the subfamily of Whi proteins, redox sensing WhiB-like proteins that contain iron-sulfur clusters, sigma factors and their cognate anti-sigma factors of which some are zinc-regulated, and the dormancy survival regulon DosS/DosT-DosR heme sensory system. Mounting experimental evidence suggests that these systems contribute to a highly complex and interrelated regulatory network that controls M. tuberculosis biology. This review concludes with a discussion of strategies that M. tuberculosis has developed to maintain redox homeostasis, including mechanisms to regulate endogenous nitric oxide and carbon monoxide levels.

Darwinian evolution experiments carried out on xeno-nucleic acid (XNA) polymers require engineered polymerases that can faithfully and efficiently copy genetic information back and forth between DNA and XNA. However, current XNA polymerases function with inferior activity relative to their natural counterparts. Here, we report five X-ray crystal structures that illustrate the pathway by which α-(L)-threofuranosyl nucleic acid (TNA) triphosphates are selected and extended in a template-dependent manner using a laboratory-evolved polymerase known as Kod-RI. Structural comparison of the apo, binary, open and closed ternary, and translocated product detail an ensemble of interactions and conformational changes required to promote TNA synthesis. Close inspection of the active site in the closed ternary structure reveals a sub-optimal binding geometry that explains the slow rate of catalysis. This key piece of information, which is missing for all naturally occurring archaeal DNA polymerases, provides a framework for engineering new TNA polymerase variants.

Amyloid oligomers play a central role in Alzheimer's and other amyloid diseases, and yet the structures of these heterogeneous and unstable species are not well understood. To better understand the structures of oligomers formed by amyloid-β peptide (Aβ), we have incorporated a key amyloidogenic region of Aβ into a macrocyclic peptide that stabilizes oligomers and facilitates structural elucidation by X-ray crystallography. This paper reports the crystallographic structures of oligomers and oligomer assemblies formed by a macrocycle containing the Aβ(15-23) nonapeptide. The macrocycle forms hydrogen-bonded β-sheets that assemble into cruciform tetramers consisting of eight β-strands in a two-layered assembly. Three of the cruciform tetramers assemble into a triangular dodecamer. These oligomers further assemble in the lattice to form hexagonal pores. Molecular modeling studies suggest that the natural Aβ peptide can form similar oligomers and oligomer assemblies. The crystallographic and molecular modeling studies suggest the potential for interaction of the oligomers with cell membranes and provide insights into the role of oligomers in amyloid diseases.