Dengue fever is an important emerging public health concern, with several million viral infections occurring annually, for which no effective therapy currently exists. The NS3 protein from Dengue virus is a multifunctional protein of 69 kDa, endowed with protease, helicase, and nucleoside 5-triphosphatase (NTPase) activities. Thus, NS3 plays an important role in viral replication and represents a very interesting target for the development of specific antiviral inhibitors. We present the structure of an enzymatically active fragment of the Dengue virus NTPase/helicase catalytic domain to 2.4 Å resolution. The structure is composed of three domains, displays an asymmetric distribution of charges on its surface, and contains a tunnel large enough to accommodate single-stranded RNA. Its C-terminal domain adopts a new fold compared to the NS3 helicase of hepatitis C virus, which has interesting implications for the evolution of the Flaviviridae replication complex. A bound sulfate ion reveals residues involved in the metal-dependent NTPase catalytic mechanism. Comparison with the NS3 hepatitis C virus helicase complexed to single-stranded DNA would place the 3 singlestranded tail of a nucleic acid duplex in the tunnel that runs across the basic face of the protein. A possible model for the unwinding mechanism is proposed.

Mycobacterium heme utilization degrader (MhuD) is a heme-degrading protein from Mycobacterium tuberculosis responsible for extracting the essential nutrient iron from host-derived heme. MhuD has been previously shown to produce unique organic products compared to those of canonical heme oxygenases (HOs) as well as those of the IsdG/I heme-degrading enzymes from Staphylococcus aureus. Here, we report the X-ray crystal structure of cyanide-inhibited MhuD (MhuD–heme–CN) as well as detailed 1H nuclear magnetic resonance (NMR), UV/vis absorption, and magnetic circular dichroism (MCD) spectroscopic characterization of this species. There is no evidence for an ordered network of water molecules on the distal side of the heme substrate in the X-ray crystal structure, as was previously reported for canonical HOs. The degree of heme ruffling in the crystal structure of MhuD is greater than that observed for HO and less than that observed for IsdI. As a consequence, the Fe 3dxz-, 3dyz-, and 3dxy-based MOs are very close in energy, and the room-temperature 1H NMR spectrum of MhuD–heme–CN is consistent with population of both a 2Eg electronic state with a (dxy)2(dxz,dyz)3 electron configuration, similar to the ground state of canonical HOs, and a 2B2g state with a (dxz,dyz)4(dxy)1 electron configuration, similar to the ground state of cyanide-inhibited IsdI. Variable temperature, variable field MCD saturation magnetization data establishes that MhuD–heme–CN has a 2B2g electronic ground state with a low-lying 2Eg excited state. Our crystallographic and spectroscopic data suggest that there are both structural and electronic contributions to the α-meso regioselectivity of MhuD-catalyzed heme cleavage. The structural distortion of the heme substrate observed in the X-ray crystal structure of MhuD–heme–CN is likely to favor cleavage at the α- and γ-meso carbons, whereas the spin density distribution may favor selective oxygenation of the α-meso carbon.

Dengue virus, a member of the family Flaviviridae of positive-strand RNA viruses, has seven non-structural proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5. Except for enzymic activities contained within NS3 and NS5, the roles of the other proteins in virus replication and pathogenesis are not well defined. In this study, a physical interaction between NS4B and the helicase domain of NS3 was identified by using a yeast two-hybrid assay. This interaction was further confirmed by biochemical pull-down and immunoprecipitation assays, both with purified proteins and with dengue virus-infected cell lysates. NS4B co-localized with NS3 in the perinuclear region of infected human cells. Furthermore, NS4B dissociated NS3 from single-stranded RNA and consequently enhanced the helicase activity of NS3 in an in vitro unwinding assay. These results suggest that NS4B modulates dengue virus replication via its interaction with NS3. INTRODUCTIONDengue fever and its more severe form, dengue haemorrhagic fever, are mosquito-borne viral diseases that are caused by one of the four antigenically distinct serotypes of Dengue virus, DENV-1-DENV-4. Dengue fever affects 50-100 million people in the tropical and subtropical regions annually (Gubler, 1998(Gubler, , 2002. Contemporary demographical and lifestyle trends, such as population explosion and urbanization, have led to the spread of this disease to nonendemic regions. The pathogenesis of dengue fever remains poorly characterized and there are no antivirals or vaccines available to counter this emerging disease.Dengue virus belongs to the family Flaviviridae that consists of enveloped, positive-sense, single-stranded RNA (ssRNA) viruses, such as those that cause yellow fever, Japanese encephalitis, West Nile fever and hepatitis C. Its RNA genome is encapsulated in an icosahedral nucleocapsid (30 nm) that is enveloped in a lipid bilayer (10 nm) (Kuhn et al., 2002) consisting of the membrane and envelope proteins. The 11 kb, capped RNA genome encodes a single polyprotein that is processed co-and post-translationally by host signalases, as well as the virus-encoded serine protease, into the three structural and seven non-structural proteins (NS) in the order C (Core)-prM (pre-Membrane)-E (Envelope)-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5 (Chambers & Rice, 1987;Lindenbach & Rice, 2003).The polymerase, helicase and protease enzymic activities encoded by the dengue virus genome ensure virus replication and polyprotein processing. NS3 (618 aa) is a multifunctional protein with protease, helicase, NTPase and 59-terminal RNA triphosphatase activities (Arias et al., 1993;Benarroch et al., 2004;Falgout et al., 1991;Li et al., 1999;Zhang et al., 1992), whilst NS5 (900 aa) has RNAdependent RNA polymerase and methyltransferase activities (Ackermann & Padmanabhan, 2001;Chu & Westaway, 1987;Egloff et al., 2002;Kapoor et al., 1995;Tan et al., 1996). These two proteins form a functional complex that is vital for flavivirus replication (Brooks et al., 2002;Johansson et al., 2001;Yon et al., 2005). The role...

The highly contagious disease tuberculosis (TB) is caused by the bacterium Mycobacterium tuberculosis (Mtb), which has been evolving drug resistance at an alarming rate. Like all human pathogens, Mtb requires iron for growth and virulence. Consequently, Mtb iron transport is an emerging drug target. However, the development of anti-TB drugs aimed at these metabolic pathways have been restricted by the dearth of information on Mtb iron acquisition. In this review, we describe the multiple strategies utilized by Mtb to acquire ferric iron and heme-iron. Mtb iron uptake is a complex process, requiring biosynthesis and subsequent export of Mtb siderophores, followed by ferric iron scavenging and ferric-siderophore import into Mtb. Additionally, Mtb possesses two possible heme uptake pathways, and an Mtb-specific mechanism of heme degradation that yields iron and novel heme-degradation products. We conclude with perspectives for potential therapeutics that could directly target Mtb heme and iron uptake machineries. We also highlight how hijacking Mtb heme and iron acquisition pathways for drug import may facilitate drug transport through the notoriously impregnable Mtb cell-wall.

Heme oxygenase-1 (HO-1) is a stress response antioxidant enzyme which catalyzes the degradation of heme released during inflammation. HO-1 expression is upregulated in both experimental and human Mycobacterium tuberculosis infection, and in patients it is a biomarker of active disease. Whether the enzyme plays a protective versus pathogenic role in tuberculosis has been the subject of debate. To address this controversy, we administered tin protoporphyrin IX (SnPPIX), a well-characterized HO-1 enzymatic inhibitor, to mice during acute M. tuberculosis infection. These SnPPIX-treated animals displayed a substantial reduction in pulmonary bacterial loads comparable to that achieved following conventional antibiotic therapy. Moreover, when administered adjunctively with antimycobacterial drugs, the HO-1 inhibitor markedly enhanced and accelerated pathogen clearance. Interestingly, both the pulmonary induction of HO-1 expression and the efficacy of SnPPIX treatment in reducing bacterial burden were dependent on the presence of host T lymphocytes. Although M. tuberculosis expresses its own heme-degrading enzyme, SnPPIX failed to inhibit its enzymatic activity or significantly restrict bacterial growth in liquid culture. Together, the above findings reveal mammalian HO-1 as a potential target for host-directed monotherapy and adjunctive therapy of tuberculosis and identify the immune response as a critical regulator of this function.

Arabidopsis det2 mutants are small darkgreen dwarfs displaying pleiotropic defects in light-regulated development during multiple stages of the plant life cycle. The DET2 gene encodes a protein that shares Ϸ40% sequence identity with mammalian steroid 5␣-reductases and is implicated in the synthesis of a class of plant steroids, the brassinosteroids. Here we show that the DET2 protein, when expressed in human embryonic kidney 293 cells, catalyzes the 5␣-reduction of several animal steroid substrates and has similar kinetic properties to the mammalian steroid 5␣-reductase enzymes. Moreover, human steroid 5␣-reductases expressed in det2 mutant plants can substitute for DET2 in brassinosteroid biosynthesis. These data indicate that DET2 is an ortholog of the mammalian steroid 5␣-reductases and provide further evidence that brassinosteroids play an essential role in light-regulated plant development. The structural and functional conservation between DET2 and human steroid 5␣-reductases raise interesting issues concerning the evolutionary origin of the steroid hormone signaling system. Steroid hormones play important roles in mammalian development and homeostasis (1). While many steroids have been identified in plants (2), to date only the brassinosteroids (BRs) are widely distributed throughout the plant kingdom and elicit unique growth promoting activity when applied exogenously (3). Despite these properties, the physiological role of endogenous BRs in plant development has been unclear. We recently isolated the Arabidopsis DET2 gene by positional cloning (4) and found that DET2 encodes a 262-amino acid protein that shares significant sequence identity with mammalian steroid 5␣-reductases, enzymes that play crucial roles in steroid hormone metabolism and action by catalyzing an NADPHdependent reduction of the ⌬ 4,5 double bond in a variety of steroids (Fig. 1A) (5). Mutations in DET2, including a missense mutation at residue Glu-204, which is absolutely required for human steroid 5␣-reductase (hS5R) activity (5), lead to many defects in light-dependent Arabidopsis development that can be ameliorated by application of brassinolide, an active BR (4, 6). Based on these results, we proposed that DET2 encodes a 5␣-reductase in a BR biosynthetic pathway (Fig. 1B) and that BRs constitute an important class of plant hormones (4).Support for the postulated importance of BRs in plant development came from the identification of a second Arabidopsis gene, CPD (constitutive photomorphogenesis and dwarfism) (7). The CPD gene encodes a protein that shares sequence identity with several mammalian cytochrome P450 proteins including steroid hydroxylases. Mutations in CPD cause phenotypic defects that are similar to those of det2 mutations. Moreover, brassinolide treatment also restores a wild-type phenotype to cpd mutants. Rescue studies with intermediates in the BR biosynthetic pathway suggest that CPD may encode a steroid 23-hydroxylase (7).In mammals, steroid hormones are synthesized from cholesterol via pregnenolone thr...

We performed a mutational analysis of the NS3 helicase of dengue virus to test insights gleaned from its crystal structure and identified four residues in the full-length protein that severely impaired either its RTPase and ATPase (Arg-457-458, Arg-460, Arg-463) or helicase (Ile-365, Arg-376) activity. Alanine substitution of Lys-396, which is located at the surface of domain II, drastically reduced all three enzymatic activities. Our study points to a pocket at the surface of domain II that may be suitable for the design of allosteric inhibitors.The dengue virus NS3 protein contains protease, RNA helicase, 5Ј-nucleoside triphosphatase (NTPase), and RNA 5Ј-triphosphatase (RTPase) activities (1,2,5,15,20,21). NS3 proteins from the four dengue virus serotypes share a minimum of 67% amino acid sequence identity. The enzymatic activities of NS3 proteins from several members of the Flaviviridae have been studied, including hepatitis C virus (HCV) (9), yellow fever virus (YFV) (19), Japanese encephalitis virus (18,19), and West Nile virus (4). The unwinding of duplex RNA structures yielding individual RNA strands is thought to be required for an efficient viral genomic RNA synthesis by the NS5 RNA-dependent RNA polymerase. The essentiality of the helicase activity of NS3 in viral replication has been demonstrated through site-directed mutagenesis (8, 17); hence, it is an attractive target for the design of antiviral compounds. Two three-dimensional (3D) structures of active flavivirus helicase/ NTPase catalytic domains from dengue virus (23) and yellow fever virus (22), respectively, were recently reported. As with the HCV NS3 helicase (12), the structure can be divided into three domains. The seven sequence motifs characteristic of superfamily 2 helicases (7) are present in domains I and II, situated at the N-terminal end of the protein. The NTPase site resides between these two domains. The C-terminal domain III differs most with its HCV counterpart (22, 23) and was suggested to bind NS5 (10). A tunnel that runs across the interface between domain III and the tip of domains I and II was proposed to accommodate a single-stranded nucleic acid tail along which the enzyme could translocate, following interdomain motions triggered by NTP hydrolysis (22,23). Interestingly, recent studies on HCV helicase (14) suggest that the energy derived from nucleic acid binding may be used to unwind several base pairs at the fork essentially without ATP. However, the unidirectional translocation of the enzyme along a long stretch of DNA is derived from ATP hydrolysis.Matusan et al. (17) reported mutations in the conserved motifs of dengue virus NS3 helicase. Here, based on their evolutionary conservation and structural insights (22, 23), we targeted 14 residues within the NS3 helicase (Fig. 1) using site-directed mutagenesis. The mutant proteins were tested for their involvement in the RNA-stimulated NTPase, RTPase, and double-stranded RNA (dsRNA) unwinding activity. While the truncated carboxyl-terminal two-thirds of NS3 used to dete...

The recent increase in extensively drug-resistant bacterial pathogens and the associated increase of morbidity and mortality demonstrate the immediate need for new antibiotic backbones with novel mechanisms of action. Here, we report the development of the PepSAVI-MS pipeline for bioactive peptide discovery. This highly versatile platform employs mass spectrometry and statistics to identify bioactive peptide targets from complex biological samples. We validate the use of this platform through the successful identification of known bioactive peptides from a botanical species, Viola odorata. Using this pipeline, we have widened the known antimicrobial spectrum for V. odorata cyclotides, including antibacterial activity of cycloviolacin O2 against A. baumannii. We further demonstrate the broad applicability of the platform through the identification of novel anticancer activities for cycloviolacins by their cytotoxicity against ovarian, breast, and prostate cancer cell lines.