Structure and subunit arrangement of Mycobacterial FF ATP synthase and novel features of the unique mycobacterial subunit δ.

In contrast to other prokaryotes, the Mycobacterial FF ATP synthase (α:β:γ:δ:ε:a:b:b':c) is essential for growth. The mycobacterial enzyme is also unique as a result of its 111 amino acids extended δ subunit, whose gene is fused to the peripheral stalk subunit b. Recently, the crystallographic structures of the mycobacterial α:β:γ:ε-domain and c subunit ring were resolved.

The NMR solution structure of Mycobacterium tuberculosis F-ATP synthase subunit ε provides new insight into energy coupling inside the rotary engine.

Unlabelled: Mycobacterium tuberculosis (Mt) F F ATP synthase (α :β :γ:δ:ε:a:b:b':c ) is essential for the viability of growing and nongrowing persister cells of the pathogen. Here, we present the first NMR solution structure of Mtε, revealing an N-terminal β-barrel domain (NTD) and a C-terminal domain (CTD) composed of a helix-loop-helix with helix 1 and -2 being shorter compared to their counterparts in other bacteria. The C-terminal amino acids are oriented toward the NTD, forming a domain-domain interface between the NTD and CTD.

Atomic structure and enzymatic insights into the vancomycin-resistant Enterococcus faecalis (V583) alkylhydroperoxide reductase subunit C.

The Enterococcus faecalis alkyl hydroperoxide reductase complex (AhpR) with its subunits AhpC (EfAhpC) and AhpF (EfAhpF) are of paramount importance to restore redox homeostasis. Recently, the novel phenomenon of swapping of the catalytic domains of EfAhpF was uncovered. Here, we visualized its counterpart EfAhpC (187 residues) from the vancomycin-resistant E.

Pyrazinoic Acid Inhibits Mycobacterial Coenzyme A Biosynthesis by Binding to Aspartate Decarboxylase PanD.

Previously, we showed that a major in vitro and in vivo mechanism of resistance to pyrazinoic acid (POA), the bioactive component of the critical tuberculosis (TB) prodrug pyrazinamide (PZA), involves missense mutations in the aspartate decarboxylase PanD, an enzyme required for coenzyme A biosynthesis. What is the mechanism of action of POA? Upon demonstrating that treatment of M. bovis BCG with POA resulted in a depletion of intracellular coenzyme A and confirming that this POA-mediated depletion is prevented by either missense mutations in PanD or exogenous supplementation of pantothenate, we hypothesized that POA binds to PanD and that this binding blocks the biosynthetic pathway.

Crystallographic and solution structure of the N-terminal domain of the Rel protein from Mycobacterium tuberculosis.

Modulation of intracellular guanosine 3',5'-bispyrophosphate ((p)ppGpp) level, the effector of the stringent response, is crucial for survival as well as optimal growth of prokaryotes and, thus, for bacterial pathogenesis and dormancy. In Mycobacterium tuberculosis (Mtb), (p)ppGpp synthesis and degradation are carried out by the bifunctional enzyme MtRel, which consists of 738 residues, including an N-terminal hydrolase- and synthetase-domain (N-terminal domain or NTD) and a C-terminus with a ribosome-binding site. Here, we present the first crystallographic structure of the enzymatically active MtRel NTD determined at 3.

Structural and mechanistic insights into Mycothiol Disulphide Reductase and the Mycoredoxin-1-alkylhydroperoxide reductase E assembly of Mycobacterium tuberculosis.

Mycobacteria employ a versatile machinery of the mycothiol-dependent system, containing the proteins mycothiol disulfide reductase (Mtr), the oxido-reductase Mycoredoxin-1 (Mrx-1) and the alkyl-hydroperoxide subunit E (AhpE). The mycothiol-dependent protein ensemble regulates the balance of oxidized-reduced mycothiol, to ensure a reductive intracellular environment for optimal functioning of its proteins even upon exposure to oxidative stress. Here, we determined the first low-resolution solution structure of Mycobacterium tuberculosis Mtr (MtMtr) derived from small-angle X-ray scattering data, which provides insight into its dimeric state.

Redox chemistry of Mycobacterium tuberculosis alkylhydroperoxide reductase E (AhpE): Structural and mechanistic insight into a mycoredoxin-1 independent reductive pathway of AhpE via mycothiol.

Mycobacterium tuberculosis (Mtb) has the ability to persist within the human host for a long time in a dormant stage and re-merges when the immune system is compromised. The pathogenic bacterium employs an elaborate antioxidant defence machinery composed of the mycothiol- and thioredoxin system in addition to a superoxide dismutase, a catalase, and peroxiredoxins (Prxs). Among the family of Peroxiredoxins, Mtb expresses a 1-cysteine peroxiredoxin, known as alkylhydroperoxide reductase E (MtAhpE), and defined as a potential tuberculosis drug target.

Low resolution solution structure of an enzymatic active AhpC10:AhpF2 ensemble of the Escherichia coli Alkyl hydroperoxide Reductase.

The ability of bacteria to combat oxidative stress is imperative for their survival. The Alkyl hydroperoxide Reductase (AhpR) system, composed of the AhpC and AhpF proteins, is one of the dominant antioxidant defense systems required for scavenging hydrogen peroxide and organic peroxide. Therefore, it is necessary to understand the mechanism of the AhpR ensemble formation.

NMR studies reveal a novel grab and release mechanism for efficient catalysis of the bacterial 2-Cys peroxiredoxin machinery.

In bacteria, an ensemble of alkyl hydroperoxide reductase subunits C (AhpC) and F (AhpF) is responsible for scavenging H2O2. AhpC donates electrons for the reduction of H2O2, which are provided after NADH oxidation by AhpF. The latter contains an N-terminal domain (NTD), catalyzing the electron transfer from NADH via a FAD of the C-terminal domain (CTD) into AhpC.

Crystallographic and solution studies of NAD(+)- and NADH-bound alkylhydroperoxide reductase subunit F (AhpF) from Escherichia coli provide insight into sequential enzymatic steps.

Redox homeostasis is significant for the survival of pro- and eukaryotic cells and is crucial for defense against reactive oxygen species like superoxide and hydrogen peroxide. In Escherichia coli, the reduction of peroxides occurs via the redox active disulfide center of the alkyl hydroperoxide reductase C subunit (AhpC), whose reduced state becomes restored by AhpF. The 57kDa EcAhpF contains an N-terminal domain (NTD), which catalyzes the electron transfer from NADH via an FAD of the C-terminal domain into EcAhpC.

Structure, mechanism and ensemble formation of the alkylhydroperoxide reductase subunits AhpC and AhpF from Escherichia coli.

Hydroperoxides are reactive oxygen species (ROS) that are toxic to all cells and must be converted into the corresponding alcohols to alleviate oxidative stress. In Escherichia coli, the enzyme primarily responsible for this reaction is alkylhydroperoxide reductase (AhpR). Here, the crystal structures of both of the subunits of EcAhpR, EcAhpF (57 kDa) and EcAhpC (21 kDa), have been solved.

Key roles of the Escherichia coli AhpC C-terminus in assembly and catalysis of alkylhydroperoxide reductase, an enzyme essential for the alleviation of oxidative stress.

2-Cys peroxiredoxins (Prxs) are a large family of peroxidases, responsible for antioxidant function and regulation in cell signaling, apoptosis and differentiation. The Escherichia coli alkylhydroperoxide reductase (AhpR) is a prototype of the Prxs-family, and is composed of an NADH-dependent AhpF reductase (57 kDa) and AhpC (21 kDa), catalyzing the reduction of H2O2. We show that the E.