Proline Dehydrogenase and Pyrroline 5 Carboxylate Dehydrogenase from Mycobacterium tuberculosis: Evidence for Substrate Channeling.

In Mycobacterium tuberculosis, proline dehydrogenase (PruB) and ∆1-pyrroline-5-carboxylate (P5C) dehydrogenase (PruA) are monofunctional enzymes that catalyze proline oxidation to glutamate via the intermediates P5C and L-glutamate-γ-semialdehyde. Both enzymes are essential for the replication of pathogenic M. tuberculosis. Highly active enzymes were expressed and purified using a Mycobacterium smegmatis expression system. The purified enzymes were characterized using natural substrates and chemically synthesized analogs. The structural requirements of the quinone electron acceptor were examined. PruB displayed activity with all tested lipoquinone analogs (naphthoquinone or benzoquinone). In PruB assays utilizing analogs of the native naphthoquinone [MK-9 (II-H2)] specificity constants Kcat/Km were an order of magnitude greater for the menaquinone analogs than the benzoquinone analogs. In addition, mycobacterial PruA was enzymatically characterized for the first time using exogenous chemically synthesized P5C. A Km value of 120 ± 0.015 µM was determined for P5C, while the Km value for NAD+ was shown to be 33 ± 4.3 µM. Furthermore, proline competitively inhibited PruA activity and coupled enzyme assays, suggesting that the recombinant purified monofunctional PruB and PruA enzymes of M. tuberculosis channel substrate likely increase metabolic flux and protect the bacterium from methylglyoxal toxicity.

Innovations in targeting RNA by fragment-based ligand discovery.

A subset of functional regions within large RNAs fold into complex structures able to bind small-molecule ligands with high affinity and specificity. Fragment-based ligand discovery (FBLD) offers notable opportunities for discovery and design of potent small molecules that bind pockets in RNA. Here we share an integrated analysis of recent innovations in FBLD, emphasizing opportunities resulting from fragment elaboration via both linking and growing. Analysis of elaborated fragments emphasizes that high-quality interactions form with complex tertiary structures in RNA. FBLD-inspired small molecules have been shown to modulate RNA functions by competitively inhibiting protein binding and by selectively stabilizing dynamic RNA states. FBLD is creating a foundation to interrogate the relatively unknown structural space for RNA ligands and for discovery of RNA-targeted therapeutics.

Vanadium Chloro-Substituted Schiff Base Catecholate Complexes are Reducible, Lipophilic, Water Stable, and Have Anticancer Activities.

A hydrophobic Schiff base catecholate vanadium complex was recently discovered to have anticancer properties superior to cisplatin and suited for intratumoral administration. This [VO(HSHED)(DTB)] complex, where HSHED is N-(salicylideneaminato)-N'-(2-hydroxyethyl)-1,2-ethanediamine and the non-innocent catecholato ligand is di-t-butylcatecholato (DTB), has higher stability compared to simpler catecholato complexes. Three new chloro-substituted Schiff base complexes of vanadium(V) with substituted catecholates as co-ligands were synthesized for comparison with their non-chlorinated Schiff base vanadium complexes, and their properties were characterized. Up to four geometric isomers for each complex were identified in organic solvents using 51V and 1H NMR spectroscopies. Spectroscopy was used to characterize the structure of the major isomer in solution and to demonstrate that the observed isomers are exchanged in solution. All three chloro-substituted Schiff base vanadium(V) complexes with substituted catecholates were also characterized by UV-vis spectroscopy, mass spectrometry, and electrochemistry. Upon testing in human glioblastoma multiforme (T98g) cells as an in vitro model of brain gliomas, the most sterically hindered, hydrophobic, and stable compound [t1/2 (298 K) = 15 min in cell medium] was better than the two other complexes (IC50 = 4.1 ± 0.5 µM DTB, 34 ± 7 µM 3-MeCat, and 19 ± 2 µM Cat). Furthermore, upon aging, the complexes formed less toxic decomposition products (IC50 = 9 ± 1 µM DTB, 18 ± 3 µM 3-MeCat, and 8.1 ± 0.6 µM Cat). The vanadium complexes with the chloro-substituted Schiff base were more hydrophobic, more hydrolytically stable, more easily reduced compared to their corresponding parent counterparts, and the most sterically hindered complex of this series is only the second non-innocent vanadium Schiff base complex with a potent in vitro anticancer activity that is an order of magnitude more potent than cisplatin under the same conditions.

Chemistry of Lipoquinones: Properties, Synthesis, and Membrane Location of Ubiquinones, Plastoquinones, and Menaquinones.

Lipoquinones are the topic of this review and are a class of hydrophobic lipid molecules with key biological functions that are linked to their structure, properties, and location within a biological membrane. Ubiquinones, plastoquinones, and menaquinones vary regarding their quinone headgroup, isoprenoid sidechain, properties, and biological functions, including the shuttling of electrons between membrane-bound protein complexes within the electron transport chain. Lipoquinones are highly hydrophobic molecules that are soluble in organic solvents and insoluble in aqueous solution, causing obstacles in water-based assays that measure their chemical properties, enzyme activities and effects on cell growth. Little is known about the location and ultimately movement of lipoquinones in the membrane, and these properties are topics described in this review. Computational studies are particularly abundant in the recent years in this area, and there is far less experimental evidence to verify the often conflicting interpretations and conclusions that result from computational studies of very different membrane model systems. Some recent experimental studies have described using truncated lipoquinone derivatives, such as ubiquinone-2 (UQ-2) and menaquinone-2 (MK-2), to investigate their conformation, their location in the membrane, and their biological function. Truncated lipoquinone derivatives are soluble in water-based assays, and hence can serve as excellent analogs for study even though they are more mobile in the membrane than the longer chain counterparts. In this review, we will discuss the properties, location in the membrane, and syntheses of three main classes of lipoquinones including truncated derivatives. Our goal is to highlight the importance of bridging the gap between experimental and computational methods and to incorporate properties-focused considerations when proposing future studies relating to the function of lipoquinones in membranes.

Electron Transport Lipids Fold Within Membrane-Like Interfaces.

Lipoquinones, such as ubiquinones (UQ) and menaquinones (MK), function as essential lipid components of the electron transport system (ETS) by shuttling electrons and protons to facilitate the production of ATP in eukaryotes and prokaryotes. Lipoquinone function in membrane systems has been widely studied, but the exact location and conformation within membranes remains controversial. Lipoquinones, such as Coenzyme Q (UQ-10), are generally depicted simply as "Q" in life science diagrams or in extended conformations in primary literature even though specific conformations are important for function in the ETS. In this study, our goal was to determine the location, orientation, and conformation of UQ-2, a truncated analog of UQ-10, in model membrane systems and to compare our results to previously studied MK-2. Herein, we first carried out a six-step synthesis to yield UQ-2 and then demonstrated that UQ-2 adopts a folded conformation in organic solvents using 1H-1H 2D NOESY and ROESY NMR spectroscopic studies. Similarly, using 1H-1H 2D NOESY NMR spectroscopic studies, UQ-2 was found to adopt a folded, U-shaped conformation within the interface of an AOT reverse micelle model membrane system. UQ-2 was located slightly closer to the surfactant-water interface compared to the more hydrophobic MK-2. In addition, Langmuir monolayer studies determined UQ-2 resided within the monolayer water-phospholipid interface causing expansion, whereas MK-2 was more likely to be compressed out and reside within the phospholipid tails. All together these results support the model that lipoquinones fold regardless of the headgroup structure but that the polarity of the headgroup influences lipoquinone location within the membrane interface. These results have implications regarding the redox activity near the interface as quinone vs. quinol forms may facilitate locomotion of lipoquinones within the membrane. The location, orientation, and conformation of lipoquinones are critical for their function in generating cellular energy within membrane ETS, and the studies described herein shed light on the behavior of lipoquinones within membrane-like environments.

Interactions of Truncated Menaquinones in Lipid Monolayers and Bilayers.

Menaquinones (MK) are hydrophobic molecules that consist of a naphthoquinone headgroup and a repeating isoprenyl side chain and are cofactors used in bacterial electron transport systems to generate cellular energy. We have previously demonstrated that the folded conformation of truncated MK homologues, MK-1 and MK-2, in both solution and reverse micelle microemulsions depended on environment. There is little information on how MKs associate with phospholipids in a model membrane system and how MKs affect phospholipid organization. In this manuscript, we used a combination of Langmuir monolayer studies and molecular dynamics (MD) simulations to probe these questions on truncated MK homologues, MK-1 through MK-4 within a model membrane. We observed that truncated MKs reside farther away from the interfacial water than ubiquinones are are located closer to the phospholipid tails. We also observed that phospholipid packing does not change at physiological pressure in the presence of truncated MKs, though a difference in phospholipid packing has been observed in the presence of ubiquinones. We found through MD simulations that for truncated MKs, the folded conformation varied, but MKs location and association with the bilayer remained unchanged at physiological conditions regardless of side chain length. Combined, this manuscript provides fundamental information, both experimental and computational, on the location, association, and conformation of truncated MK homologues in model membrane environments relevant to bacterial energy production.

Mycobacterium tuberculosis Survival in J774A.1 Cells Is Dependent on MenJ Moonlighting Activity, Not Its Enzymatic Activity.

MenJ, a flavoprotein oxidoreductase, is responsible for the saturation of the ß-isoprene unit of mycobacterial menaquinone, resulting in the conversion of menaquinone with nine isoprene units (MK-9) to menaquinone with nine isoprene units where the double bond in the second unit is reduced [MK-9(II-H2)]. The hydrogenation of MK-9 increases the efficiency of the mycobacterial electron transport system, whereas the deletion of MenJ results in decreased survival of the bacteria inside J774A.1 macrophage-like cells but is not required for growth in culture. Thus, it was suggested that MenJ may represent a contextual drug target in M. tuberculosis, that is, a drug target that is valid only in the context of an infected macrophage. However, it was unclear if the conversion of MK-9 to MK-9(II-H2) or the MenJ protein itself was responsible for bacterial survival. In order to resolve this issue, a plasmid expressing folded, full-length, inactive MenJ was engineered. Primary sequence analysis data revealed that MenJ shares conserved FAD binding, NADH binding, and catalytic and C-terminal motifs with archaeal geranylgeranyl reductases. A MenJ mutant deficient in any one of these motifs is devoid of reductase activity. Therefore, point mutations of highly conserved amino acids in the conserved motifs were generated and the recombinant proteins were monitored for conformational changes by circular dichroism and oxidoreductase activity. The mutational analysis indicates that amino acids tryptophan 215 (W215) and cysteine 46 (C46) of M. tuberculosis MenJ, conserved in known archaeal geranylgeranyl reductases and putative menaquinone saturases, are essential to the hydrogenation of MK-9. The mutation of either C46 to serine (C46S) or W215 to leucine (W215L) in MenJ completely abolishes the catalytic activity in vitro, and menJ knockout strains of M. tuberculosis expressing either the C46S or W215L mutant protein are unable to convert MK-9 to MK-9(II-H2) but survive inside the J774A.1 cells. Thus, surprisingly, the survival of M. tuberculosis in J774A.1 cells is dependent on the expression of MenJ rather than its oxidoreductase activity, the conversion of MK-9 to MK-9(II-H2) as previously hypothesized. Overall, the current data suggest that MenJ is a moonlighting protein.

A Short-Lived but Highly Cytotoxic Vanadium(V) Complex as a Potential Drug Lead for Brain Cancer Treatment by Intratumoral Injections.

The chemistry and short lifetimes of metal-based anti-cancer drugs can be turned into an advantage for direct injections into tumors, which then allow the use of highly cytotoxic drugs. The release of their less toxic decomposition products into the blood will lead to decreased toxicity and can even have beneficial effects. We present a ternary VV complex, 1 ([VOL1 L2 ], where L1 is N-(salicylideneaminato)-N'-(2-hydroxyethyl)ethane-1,2-diamine and L2 is 3,5-di-tert-butylcatechol), which enters cells intact to induce high cytotoxicity in a range of human cancer cells, including T98g (glioma multiforme), while its decomposition products in cell culture medium were ≈8-fold less toxic. 1 was 12-fold more toxic than cisplatin in T98g cells and 6-fold more toxic in T98g cells than in a non-cancer human cell line, HFF-1. Its high toxicity in T98g cells was retained in the presence of physiological concentrations of the two main metal-binding serum proteins, albumin and transferrin. These properties favor further development of 1 for brain cancer treatment by intratumoral injections.

Hydrophobicity may enhance membrane affinity and anti-cancer effects of Schiff base vanadium(v) catecholate complexes.

Anti-cancer activities of vanadium compounds have generated recent interest because of a combination of desirable properties for chemotherapy, i.e., strong cytotoxicities, anti-metastatic activities and relatively low systemic toxicities. Certain hydrophobic vanadium(v) Schiff base/catecholate compounds, which as shown herein, have increased stability in aqueous media and affinity for membrane interfaces. Depending on their hydrophobicity, they may be able to enter cells intact. In this manuscript, two hydrophobic V(v) catecholate substituted analogues, [VO(Hshed)(cat)] and [VO(Hshed)(dtb)], (Hshed = N-(salicylideneaminato)-N'-(2-hydroxyethyl)-1,2-ethanediamine, cat = pyrocatechol, and dtb = 3,5-di(tert-butyl)catechol and the vanadium(v) precursor [V(O)2(Hshed)]) were synthesized for their ability to interact with membranes and their anti-cancer effects. Using 51V and 1H NMR spectroscopy, the presence and location of the free ligand, H2shed, and the three V(v) complexes were examined in a model membrane microemulsion system. The stability of the three complexes was measured in aqueous solution, cell media and an inhomogeneous microemulsion system. Our results demonstrated that free ligand H2shed and the intact V(v) complexes associated with the interface but that the V-complexes hydrolyzed to some extent because oxovanadates were observed by 51V NMR spectroscopy and decreasing complex by absorption spectroscopy in cell media. When determining the effects of V(v) catecholate complexes on bone cancer cells, the strongest effects were observed with the more stable hydrophobic complex [VO(Hshed)(dtb)] that was able to best associate and penetrate the model membrane system intact. These studies are consistent with the membrane permeability studies being a good predictor for in vitro cytotoxicity assays because [VO(Hshed)(dtb)] can pass through the cellular membrane intact, which may enhance its anti-cancer activities.

Investigating Substrate Analogues for Mycobacterial MenJ: Truncated and Partially Saturated Menaquinones.

Menaquinones (MKs) are essential for electron transport in prokaryotes, and importantly, partially saturated MKs represent a novel virulence factor. However, little is known regarding how the degree of saturation in the isoprenyl side chain influences conformation or quinone redox potential. MenJ is an enzyme that selectively reduces the second isoprene unit on MK-9 and is contextually essential for the survival of Mycobacterium tuberculosis in J774A.1 macrophage-like cells, suggesting that MenJ may be a conditional drug target for pathogenic mycobacteria. Therefore, fundamental information about the properties of this system is important, and we synthesized the simplest MKs, unsaturated MK-1 and the saturated analogue, MK-1(H2). Using two-dimensional nuclear magnetic resonance spectroscopy, we established that MK-1 and MK-1(H2) adopted similar folded-extended conformations (i.e., the isoprenyl side chain folds upward) in each solvent examined but the folded-extended conformations differed slightly between organic solvents. Saturation of the isoprenyl side chain slightly altered the MK-1 analogue conformation in each solvent. We used molecular mechanics to illustrate the MK-1 analogue conformations. The measured quinone redox potentials of MK-1 and MK-1(H2) differed between organic solvents (presumably due to differences in dielectric constants), and remarkably, an ∼20 mV semiquinone redox potential difference was observed between MK-1 and MK-1(H2) in pyridine, acetonitrile, and dimethyl sulfoxide, demonstrating that the degree of saturation in the isoprenyl side chain of MK-1 influences the quinone redox potential. Finally, MK-1 and MK-1(H2) interacted with Langmuir phospholipid monolayers and Aerosol-OT reverse micelle (RM) model membrane interfaces, where MK-1 adopted a slightly different folded conformation within the RM model membrane interface.

Mycobacterial MenJ: An Oxidoreductase Involved in Menaquinone Biosynthesis.

MenJ, annotated as an oxidoreductase, was recently demonstrated to catalyze the reduction (saturation) of a single double bond in the isoprenyl side-chain of mycobacterial menaquinone. This modification was shown to be essential for bacterial survival in J774A.1 macrophage-like cells, suggesting that MenJ may be a conditional drug target in Mycobacterium tuberculosis and other pathogenic mycobacteria. Recombinant protein was expressed in a heterologous host, and the activity was characterized. Although highly regiospecific in vivo, the activity is not absolutely regiospecific in vitro; in addition, the enzyme is not specific for naphthoquinones vs benzoquinones. Coenzyme Q-1 (a benzoquinone, UQ-1) was used as the lipoquinone substrate, and NADH oxidation was followed spectrophotometrically as the activity readout. NADPH could not be substituted for NADH in the reaction mixture. The enzyme contains a FAD binding site that was 72% occupied in the purified recombinant protein. Enzyme activity was maximal at 37 °C and pH 7.0; addition of divalent cations, EDTA, and reducing agents such as dithiothreitol to the reaction mixture had no effect on activity. The addition of detergents did not stimulate activity, and addition of saturating levels of FAD had relatively little effect on the observed kinetic parameters. These properties allowed the development of a facile assay needed to study this potential drug target, which is also amenable to high throughput screening. The Km values for UQ-1 using recombinant MenJ from Mycobacterium smegmatis or M. tuberculosis without saturating concentrations of FAD were found to be 52 ± 9.6 and 44 ± 4.8 µM, respectively, while the KmNADH values were determined to be 59 ± 14 and 64 ± 15 µM. The Km for MK-1, the menaquinone analogue of UQ-1, using recombinant MenJ from M. tuberculosis without saturating concentrations of FAD but in the presence of 0.5% Tween 80 was shown to be 30 ± 2.9 µM. Thus, this is the first report of a kinetic characterization of a member of the geranylgeranyl reductase family of enzymes.

Synthesis and Characterization of Partially and Fully Saturated Menaquinone Derivatives.

Menaquinones (MKs) contain both a redox active quinone moiety and a hydrophobic repeating isoprenyl side chain of varying lengths and degrees of saturation. This characteristic structure allows MKs to play a key role in the respiratory electron transport system of some prokaryotes by shuttling electrons and protons between membrane-bound protein complexes, which act as electron acceptors and donors. Hydrophobic MK molecules with partially and fully saturated isoprenyl side chains are found in a wide range of eubacteria and archaea, and the structural variations of the MK analogues are evolutionarily conserved but poorly understood. For example, Mycobacterium tuberculosis, the causative agent of tuberculosis, uses predominantly MK-9(II-H2) (saturated at the second isoprene unit) as its electron carrier and depends on the synthesis of MK-9(II-H2) for survival in host macrophages. Thus, MKs with partially saturated isoprenyl side chains may represent a novel virulence factor. Naturally occurring longer MKs are very hydrophobic, whereas MK analogues that have a truncated (i.e., one to three isoprenes) isoprenyl side chain are less hydrophobic. This improves their solubility in aqueous solutions, allowing rigorous study of their structure and biological activity. We present the synthesis and characterization of two partially saturated MK analogues, MK-2(II-H2) and MK-3(II-H2), and two novel fully saturated MK derivatives, MK-2(I,II-H4) and MK-3(I,II,III-H6).

A Synthetic Isoprenoid Lipoquinone, Menaquinone-2, Adopts a Folded Conformation in Solution and at a Model Membrane Interface.

Menaquinones (naphthoquinones, MK) are isoprenoids that play key roles in the respiratory electron transport system of some prokaryotes by shuttling electrons between membrane-bound protein complexes acting as electron acceptors and donors. Menaquinone-2 (MK-2), a truncated MK, was synthesized, and the studies presented herein characterize the conformational and chemical properties of the hydrophobic MK-2 molecule. Using 2D NMR spectroscopy, we established for the first time that MK-2 has a folded conformation defined by the isoprenyl side-chain folding back over the napthoquinone in a U-shape, which depends on the specific environmental conditions found in different solvents. We used molecular mechanics to illustrate conformations found by the NMR experiments. The measured redox potentials of MK-2 differed in three organic solvents, where MK-2 was most easily reduced in DMSO, which may suggest a combination of solvent effect (presumably in part because of differences in dielectric constants) and/or conformational differences of MK-2 in different organic solvents. Furthermore, MK-2 was found to associate with the interface of model membranes represented by Langmuir phospholipid monolayers and Aerosol-OT (AOT) reverse micelles. MK-2 adopts a slightly different U-shaped conformation within reverse micelles compared to within solution, which is in sharp contrast to the extended conformations illustrated in literature for MKs.