Mycobacterium tuberculosis PanD Structure-Function Analysis and Identification of a Potent Pyrazinoic Acid-Derived Enzyme Inhibitor.

A common strategy employed in antibacterial drug discovery is the targeting of biosynthetic processes that are essential and specific for the pathogen. Specificity in particular avoids undesirable interactions with potential enzymatic counterparts in the human host, and it ensures on-target toxicity. Synthesis of pantothenate (Vitamine B5), which is a precursor of the acyl carrier coenzyme A, is an example of such a pathway. In Mycobacterium tuberculosis (Mtb), which is the causative agent of tuberculosis (TB), pantothenate is formed by pantothenate synthase, utilizing D-pantoate and ß-Ala as substrates. ß-Ala is mainly formed by the decarboxylation of l-aspartate, generated by the decarboxylase PanD, which is a homo-oliogomer in solution. Pyrazinoic acid (POA), which is the bioactive form of the TB prodrug pyrazinamide, binds and inhibits PanD activity weakly. Here, we generated a library of recombinant Mtb PanD mutants based on structural information and PZA/POA resistance mutants. Alterations in oligomer formation, enzyme activity, and/or POA binding were observed in respective mutants, providing insights into essential amino acids for Mtb PanD's proper structural assembly, decarboxylation activity and drug interaction. This information provided the platform for the design of novel POA analogues with modifications at position 3 of the pyrazine ring. Analogue 2, which incorporates a bulky naphthamido group at this position, displayed a 1000-fold increase in enzyme inhibition, compared to POA, along with moderately improved antimycobacterial activity. The data demonstrate that an improved understanding of mechanistic and enzymatic features of key metabolic enzymes can stimulate design of more-potent PanD inhibitors.

Features and Functional Importance of Key Residues of the Mycobacterium tuberculosis Cytochrome bd Oxidase.

Cytochrome bd (cyt-bd) oxygen reductases have a high affinity to oxygen and use the two electrons provided by ubiquinol or menaquinol, like in mycobacteria, to reduce oxygen to water. Although they do not pump protons from the cytoplasmic to the periplasmic side, they generate a proton motive force due to the release of protons after quinol oxidation. Here, we show that the mycobacterial cyt-bd has a number of specific features, including a 17-residue stretch (307SGVTLQGIRDLQQEYQQ323) near the Q-loop of the Mycobacterium tuberculosis subunit CydA and a 412QLVRLTVKA420 region on the periplasmic side. Site directed mutagenesis and whole-bacteria assays demonstrated that these mycobacteria-specific stretches are essential for the oxidase's function. Single amino acid substitutions around the 307SGVTLQGIRDLQQEYQQ323 stretch revealed the importance of the aromatic residue Y330 in oxygen consumption and consequently in ATP synthesis. A moderate reduction and no effect was observed for mutants F325 and Y321, respectively, while the double mutant CydAY321/F325 drastically reduced enzyme activity. In addition, single mutants of the mycobacterial cyt-bd were generated to probe the role of proposed critical residues for proton shuffling. Further data demonstrate that amino acids W64 and F18 in the CydB subunit might be important as any slight destabilization of the hydrophobic environment near them makes the enzyme inactive. Finally, the potential of the mycobacterial cyt-bd as a drug target is discussed.

Structure and flexibility of non-structural proteins 3 and -5 of Dengue- and Zika viruses in solution.

Dengue- (DENV) and Zika viruses (ZIKV) rely on their non-structural protein 5 (NS5) including a methyl-transferase (MTase) and a RNA-dependent RNA polymerase (RdRp) for capping and synthesis of the viral RNA, and the non-structural protein 3 (NS3) with its protease and helicase domain for polyprotein possessing, unwinding dsRNA proceeding replication, and NTPase/RTPase activities. Accumulation of data for DENV- and ZIKV NS3 and NS5 in solution during recent years provides information about their overall shape, substrate-induced alterations, oligomeric forms and flexibility, with the latter being essential for domain-domain crosstalk. The importance and differences of the linker regions that connect the two domains of NS3 or NS5 are highlighted in particular with respect to the different DENV serotypes (DENV-1 to -4) as well as to the sequence diversities between the DENV and ZIKV proteins. Novel mutants of the French Polynesia ZIKV NS3 linker presented, identify critical residues in protein stability and enzymatic activity.

Substrate-induced structural alterations of Mycobacterial mycothione reductase and critical residues involved.

Redox homeostasis is a prerequisite for survival of the pathogen Mycobacterium tuberculosis (Mtb) which employs the low molecular weight thiol mycothiol (MSH). The Mycobacterial NADPH-dependent mycothione reductase (MtMtr), composed of an NADPH-, FAD-, and a dimerization-domain connected by linkers, regulates the balance of oxidized-reduced MSH. Here, we demonstrate by small-angle X-ray scattering, that NADPH-binding alters the oligomeric state equilibrium of the protein with no significant overall structural change after MSH-binding. Mutation of critical residues in the linker regions of MtMtr eliminate partially or totally the NADPH-induced oligomerization effect with simultaneous effect on enzyme activity. The data provide insight into the MtMtr linker regions involved in the novel oligomerization equilibrium of the Mycobacterial enzyme.

AhpC of the mycobacterial antioxidant defense system and its interaction with its reducing partner Thioredoxin-C.

Despite the highly oxidative environment of the phagosomal lumen, the need for maintaining redox homeostasis is a critical aspect of mycobacterial biology. The pathogens are equipped with the sophisticated thioredoxin- (Trx) and peroxiredoxin system, including TrxC and the alkyl hydroperoxide reductase subunit C (AhpC), whereby TrxC is one of the reducing partners of AhpC. Here we visualize the redox modulated dodecamer ring formation of AhpC from Mycobacterium bovis (BCG strain; MbAhpC) using electron microscopy and present novel insights into the unique N-terminal epitope (40 residues) of mycobacterial AhpC. Truncations and amino acid substitutions of residues in the unique N-terminus of MbAhpC provide insights into their structural and enzymatic roles, and into the evolutionary divergence of mycobacterial AhpC versus that of other bacteria. These structural details shed light on the epitopes and residues of TrxC which contributes to its interaction with AhpC. Since human cells lack AhpC, the unique N-terminal epitope of mycobacterial AhpC as well as the MbAhpC-TrxC interface represent an ideal drug target.

Structural features of NS3 of Dengue virus serotypes 2 and 4 in solution and insight into RNA binding and the inhibitory role of quercetin.

Dengue virus (DENV), which has four serotypes (DENV-1 to DENV-4), is the causative agent of the viral infection dengue. DENV nonstructural protein 3 (NS3) comprises a serine protease domain and an RNA helicase domain which has nucleotide triphosphatase activities that are essential for RNA replication and viral assembly. Here, solution X-ray scattering was used to provide insight into the overall structure and flexibility of the entire NS3 and its recombinant helicase and protease domains for Dengue virus serotypes 2 and 4 in solution. The DENV-2 and DENV-4 NS3 forms are elongated and flexible in solution. The importance of the linker residues in flexibility and domain-domain arrangement was shown by the compactness of the individual protease and helicase domains. Swapping of the 174PPAVP179 linker stretch of the related Hepatitis C virus (HCV) NS3 into DENV-2 NS3 did not alter the elongated shape of the engineered mutant. Conformational alterations owing to RNA binding are described in the protease domain, which undergoes substantial conformational alterations that are required for the optimal catalysis of bound RNA. Finally, the effects of ATPase inhibitors on the enzymatically active DENV-2 and DENV-4 NS3 and the individual helicases are presented, and insight into the allosteric effect of the inhibitor quercetin is provided.

The uniqueness of subunit α of mycobacterial F-ATP synthases: An evolutionary variant for niche adaptation.

The F1F0 -ATP (F-ATP) synthase is essential for growth of Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). In addition to their synthase function most F-ATP synthases possess an ATP-hydrolase activity, which is coupled to proton-pumping activity. However, the mycobacterial enzyme lacks this reverse activity, but the reason for this deficiency is unclear. Here, we report that a Mycobacterium-specific, 36-amino acid long C-terminal domain in the nucleotide-binding subunit α (Mtα) of F-ATP synthase suppresses its ATPase activity and determined the mechanism of suppression. First, we employed vesicles to show that in intact membrane-embedded mycobacterial F-ATP synthases deletion of the C-terminal domain enabled ATPase and proton-pumping activity. We then generated a heterologous F-ATP synthase model system, which demonstrated that transfer of the mycobacterial C-terminal domain to a standard F-ATP synthase α subunit suppresses ATPase activity. Single-molecule rotation assays indicated that the introduction of this Mycobacterium-specific domain decreased the angular velocity of the power-stroke after ATP binding. Solution X-ray scattering data and NMR results revealed the solution shape of Mtα and the 3D structure of the subunit α C-terminal peptide 521PDEHVEALDEDKLAKEAVKV540 of M. tubercolosis (Mtα(521-540)), respectively. Together with cross-linking studies, the solution structural data lead to a model, in which Mtα(521-540) comes in close proximity with subunit γ residues 104-109, whose interaction may influence the rotation of the camshaft-like subunit γ. Finally, we propose that the unique segment Mtα(514-549), which is accessible at the C terminus of mycobacterial subunit α, is a promising drug epitope.

Structural features of Zika virus non-structural proteins 3 and -5 and its individual domains in solution as well as insights into NS3 inhibition.

Zika virus (ZIKV) has emerged as a pathogen of major health concern. The virus relies on its non-structural protein 5 (NS5) including a methyl-transferase (MTase) and a RNA-dependent RNA polymerase (RdRp) for capping and synthesis of the viral RNA and the nonstructural protein 3 (NS3) with its protease and helicase domain for polyprotein possessing, unwinding dsRNA proceeding replication, and NTPase/RTPase activities. In this study we present for the first time insights into the overall structure of the entire French Polynesia ZIKV NS3 in solution. The protein is elongated and flexible in solution. Solution studies of the individual protease- and helicase domains show the compactness of the two monomeric enzymes as well as the contribution of the 10-residues linker region to the flexibility of the entire NS3. We show also the solution X-ray scattering data of the French Polynesia ZIKV NS5, which is dimeric in solution and switches to oligomers in a concentration-dependent manner. The solution shapes of the MTase and RdRp domains are described. The dimer arrangement of ZIKV NS5 is discussed in terms of its importance for MTase-RdRp communication and concerted interaction with its flexible and monomeric counterpart NS3 during viral replication and capping. The comparison of ZIKV NS3 and -NS5 solution data with the related DENV nonstructural proteins shed light into the similarities and diversities of these classes of enzymes. Finally, the effect of ATPase inhibitors to the enzymatic active ZIKV NS3 and the individual helicase are provided.

Identification of the critical linker residues conferring differences in the compactness of NS5 from Dengue virus serotype 4 and NS5 from Dengue virus serotypes 1-3.

Dengue virus (DENV) nonstructural protein 5 (NS5) consists of a methyltransferase (MTase) domain and an RNA-dependent RNA polymerase (RdRp) domain. The cross-talk between these domains occurs via a ten-residue linker. Recent solution studies of DENV NS5 from all four serotypes (DENV-1 to DENV-4) showed that NS5 adopts multiple conformations owing to its flexible linker and that DENV-4 NS5 is more compact and less flexible compared with NS5 from DENV-1 to DENV-3 [Saw et al. (2015), Acta Cryst. D71, 2309-2327]. Here, using a variety of single, double, triple and quadruple mutants of DENV-4 NS5 combined with solution X-ray scattering studies, insight into the critical residues responsible for the differential flexibility of DENV-4 NS5 is presented. The DENV-4 NS5 mutants K271T and S266N/T267A as well as the deletion mutant ΔS266T267 showed enlarged dimensions and flexibility similar to those of DENV-3 NS5. The data indicate that the residues Lys271, Ser266 and Thr267 are important for the compactness of DENV-4 NS5 and therefore may be critical for the regulation of virus replication. Furthermore, quantitative characterization of the flexibility of these DENV-4 NS5 linker mutants using the ensemble-optimization method revealed that these mutants possess a similar conformational distribution to DENV-3 NS5, confirming that these residues in the linker region cause the higher compactness of DENV-4 NS5.

Structural insight and flexible features of NS5 proteins from all four serotypes of Dengue virus in solution.

Infection by the four serotypes of Dengue virus (DENV-1 to DENV-4) causes an important arthropod-borne viral disease in humans. The multifunctional DENV nonstructural protein 5 (NS5) is essential for capping and replication of the viral RNA and harbours a methyltransferase (MTase) domain and an RNA-dependent RNA polymerase (RdRp) domain. In this study, insights into the overall structure and flexibility of the entire NS5 of all four Dengue virus serotypes in solution are presented for the first time. The solution models derived revealed an arrangement of the full-length NS5 (NS5FL) proteins with the MTase domain positioned at the top of the RdRP domain. The DENV-1 to DENV-4 NS5 forms are elongated and flexible in solution, with DENV-4 NS5 being more compact relative to NS5 from DENV-1, DENV-2 and DENV-3. Solution studies of the individual MTase and RdRp domains show the compactness of the RdRp domain as well as the contribution of the MTase domain and the ten-residue linker region to the flexibility of the entire NS5. Swapping the ten-residue linker between DENV-4 NS5FL and DENV-3 NS5FL demonstrated its importance in MTase-RdRp communication and in concerted interaction with viral and host proteins, as probed by amide hydrogen/deuterium mass spectrometry. Conformational alterations owing to RNA binding are presented.

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. The EcAhpF structures (2.0 and 2.65 Šresolution) reveal an open and elongated conformation, while that of EcAhpC (3.3 Šresolution) forms a decameric ring. Solution X-ray scattering analysis of EcAhpF unravels the flexibility of its N-terminal domain, and its binding to EcAhpC was demonstrated by isothermal titration calorimetry. These studies suggest a novel overall mechanistic model of AhpR as a hydroperoxide scavenger, in which the dimeric, extended AhpF prefers complex formation with the AhpC ring to accelerate the catalytic activity and thus to increase the chance of rescuing the cell from ROS.