Crystal structure and mechanism of tryptophan 2,3-dioxygenase, a heme enzyme involved in tryptophan catabolism and in quinolinate biosynthesis

The structure of tryptophan 2,3-dioxygenase (TDO) from Ralstonia metallidurans was determined at 2.4 A. TDO catalyzes the irreversible oxidation of l-tryptophan to N-formyl kynurenine, which is the initial step in tryptophan catabolism. TDO is a heme-containing enzyme and is highly specific for its substrate l-tryptophan. The structure is a tetramer with a heme cofactor bound at each active site. The monomeric fold, as well as the heme binding site, is similar to that of the large domain of indoleamine 2,3-dioxygenase, an enzyme that catalyzes the same reaction except with a broader substrate tolerance. Modeling of the putative (S)-tryptophan hydroperoxide intermediate into the active site, as well as substrate analogue and mutagenesis studies, are consistent with a Criegee mechanism for the reaction.     

Viral paratransgenesis in the malaria vector Anopheles gambiae

Paratransgenesis, the genetic manipulation of insect symbiotic microorganisms, is being considered as a potential method to control vector-borne diseases such as malaria. The feasibility of paratransgenic malaria control has been hampered by the lack of candidate symbiotic microorganisms for the major vector Anopheles gambiae. In other systems, densonucleosis viruses (DNVs) are attractive agents for viral paratransgenesis because they infect important vector insects, can be genetically manipulated and are transmitted to subsequent generations. However, An. gambiae has been shown to be refractory to DNV dissemination. We discovered, cloned and characterized the first known DNV (AgDNV) capable of infection and dissemination in An. gambiae. We developed a flexible AgDNV-based expression vector to express any gene of interest in An. gambiae using a two-plasmid helper-transducer system. To demonstrate proof-of-concept of the viral paratransgenesis strategy, we used this system to transduce expression of an exogenous gene (enhanced green fluorescent protein; EGFP) in An. gambiae mosquitoes. Wild-type and EGFP-transducing AgDNV virions were highly infectious to An. gambiae larvae, disseminated to and expressed EGFP in epidemiologically relevant adult tissues such as midgut, fat body and ovaries and were transmitted to subsequent mosquito generations. These proof-of-principle data suggest that AgDNV could be used as part of a paratransgenic malaria control strategy by transduction of anti-Plasmodium peptides or insect-specific toxins in Anopheles mosquitoes. AgDNV will also be extremely valuable as an effective and easy-to-use laboratory tool for transient gene expression or RNAi in An. gambiae.     

Histidine 55 of tryptophan 2,3-dioxygenase is not an active site base but regulates catalysis by controlling substrate binding

Tryptophan 2,3-dioxygenase (TDO) from Xanthomonas campestris is a highly specific heme-containing enzyme from a small family of homologous enzymes, which includes indoleamine 2,3-dioxygenase (IDO). The structure of wild type (WT TDO) in the catalytically active, ferrous (Fe (2+)) form and in complex with its substrate l-tryptophan ( l-Trp) was recently reported [Forouhar et al. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 473-478] and revealed that histidine 55 hydrogen bonds to l-Trp, precisely positioning it in the active site and implicating it as a possible active site base. In this study the substitution of the active site residue histidine 55 by alanine and serine (H55A and H55S) provides insight into the molecular mechanism used by the enzyme to control substrate binding. We report the crystal structure of the H55A and H55S mutant forms at 2.15 and 1.90 A resolution, respectively, in binary complexes with l-Trp. These structural data, in conjunction with potentiometric and kinetic studies on both mutants, reveal that histidine 55 is not essential for turnover but greatly disfavors the mechanistically unproductive binding of l-Trp to the oxidized enzyme allowing control of catalysis. This is demonstrated by the difference in the K d values for l-Trp binding to the two oxidation states of wild-type TDO (3.8 mM oxidized, 4.1 microM reduced), H55A TDO (11.8 microM oxidized, 3.7 microM reduced), and H55S TDO (18.4 microM oxidized, 5.3 microM reduced).     

The second enzyme in pyrrolnitrin biosynthetic pathway is related to the heme-dependent dioxygenase superfamily

Pyrrolnitrin is a commonly used and clinically effective treatment for fungal infections and provides the structural basis for the more widely used fludioxinil. The pyrrolnitrin biosynthetic pathway consists of four chemical steps, the second of which is the rearrangement of 7-chloro-tryptophan by the enzyme PrnB, a reaction that is so far unprecedented in biochemistry. When expressed in Pseudomonas fluorescens, PrnB is red in color due to the fact that it contains 1 mol of heme b per mole of protein. The crystal structure unexpectedly establishes PrnB as a member of the heme-dependent dioxygenase superfamily with significant structural but not sequence homology to the two-domain indoleamine 2,3-dioxygenase enzyme (IDO). The heme-binding domain is also structurally similar to that of tryptophan 2,3-dioxygenase (TDO). Here we report the binary complex structures of PrnB with d- and l-tryptophan and d- and l-7-chloro-tryptophan. The structures identify a common hydrophobic pocket for the indole ring but exhibit unusual heme ligation and substrate binding when compared with that observed in the TDO crystal structures. Our solution studies support the heme ligation observed in the crystal structures. Purification of the hexahistidine-tagged PrnB yields homogeneous protein that only displays in vitro activity with 7-chloro-l-tryptophan after reactivation with crude extract from the host strain, suggesting that an as yet unknown cofactor is required for activity. Mutation of the proximal heme ligand results, not surprisingly, in inactive enzyme. Redox titrations show that PrnB displays a significantly different reduction potential to that of IDO or TDO, indicating possible differences in the PrnB catalytic cycle. This is confirmed by the absence of tryptophan dioxygenase activity in PrnB, although a stable oxyferrous adduct (which is the first intermediate in the TDO/IDO catalytic cycle) can be generated. We propose that PrnB shares a key catalytic step with TDO and IDO, generation of a tryptophan hydroperoxide intermediate, although this species suffers a different fate in PrnB, leading to the eventual formation of the product, monodechloroaminopyrrolnitrin.     

Identification and biochemical characterization of the Anopheles gambiae 3-hydroxykynurenine transaminase

Spontaneous oxidation of 3-hydroxykynureine (3-HK), a metabolic intermediate of the tryptophan degradation pathway, elicits a remarkable oxidative stress response in animal tissues. In the yellow fever mosquito Aedes aegypti the excess of this toxic metabolic intermediate is efficiently removed by a specific 3-HK transaminase, which converts 3-HK into the more stable compound xanthurenic acid. In anopheline mosquitoes transmitting malaria, xanthurenic acid plays an important role in Plasmodium gametocyte maturation and fertility. Using the sequence information provided by the Anopheles gambiae genome and available ESTs, we adopted a PCR-based approach to isolate a 3-HK transaminase coding sequence from the main human malaria vector A. gambiae. Tissue and developmental expression analysis revealed an almost ubiquitary profile, which is in agreement with the physiological role of the enzyme in mosquito development and 3-HK detoxification. A high yield procedure for the expression and purification of a fully active recombinant version of the protein has been developed. Recombinant A. gambiae 3-HK transaminase is a dimeric pyridoxal 5'-phosphate dependent enzyme, showing an optimum pH of 7.8 and a comparable catalytic efficiency for both 3-HK and its immediate catabolic precursor kynurenine. This study may be useful for the identification of 3-HK transaminase inhibitors of potential interest as malaria transmission-blocking drugs or effective insecticides.     

Characterization of the 3-HKT gene in important malaria vectors in India, viz: Anopheles culicifacies and Anopheles stephensi (Diptera: Culicidae)

The 3-hydroxykynurenine transaminase (3-HKT) gene plays a vital role in the development of malaria parasites by participating in the synthesis of xanthurenic acid, which is involved in the exflagellation of microgametocytes in the midgut of malaria vector species. The 3-HKT enzyme is involved in the tryptophan metabolism of Anophelines. The gene had been studied in the important global malaria vector, Anopheles gambiae. In this report, we have conducted a preliminary investigation to characterize this gene in the two important vector species of malaria in India, Anopheles culicifacies and Anopheles stephensi. The analysis of the genetic structure of this gene in these species revealed high homology with the An. gambiae gene. However, four non-synonymous mutations in An. stephensi and seven in An. culicifacies sequences were noted in the exons 1 and 2 of the gene; the implication of these mutations on enzyme structure remains to be explored.     

Thioredoxin reductase from the malaria mosquito Anopheles gambiae.

The mosquito, Anopheles gambiae, is an important vector of Plasmodium falciparum malaria. Full genome analysis revealed that, as in Drosophila melanogaster, the enzyme glutathione reductase is absent in A. gambiae and functionally substituted by the thioredoxin system. The key enzyme of this system is thioredoxin reductase-1, a homodimeric FAD-containing protein of 55.3 kDa per subunit, which catalyses the reaction NADPH + H+ + thioredoxin disulfide-->NADP+ + thioredoxin dithiol. The A. gambiae trxr gene is located on chromosome X as a single copy; it represents three splice variants coding for two cytosolic and one mitochondrial variant. The predominant isoform, A. gambiae thioredoxin reductase-1, was recombinantly expressed in Escherichia coli and functionally compared with the wild-type enzyme isolated in a final yield of 1.4 U.ml(-1) of packed insect cells. In redox titrations, the substrate A. gambiae thioredoxin-1 (Km=8.5 microm, kcat=15.4 s(-1) at pH 7.4 and 25 degrees C) was unable to oxidize NADPH-reduced A. gambiae thioredoxin reductase-1 to the fully oxidized state. This indicates that, in contrast to other disulfide reductases, A. gambiae thioredoxin reductase-1 oscillates during catalysis between the four-electron reduced state and a two-electron reduced state. The thioredoxin reductases of the malaria system were compared. A. gambiae thioredoxin reductase-1 shares 52% and 45% sequence identity with its orthologues from humans and P. falciparum, respectively. A major difference among the three enzymes is the structure of the C-terminal redox centre, reflected in the varying resistance of catalytic intermediates to autoxidation. The relevant sequences of this centre are Thr-Cys-Cys-SerOH in A. gambiae thioredoxin reductase, Gly-Cys-selenocysteine-GlyOH in human thioredoxin reductase, and Cys-X-X-X-X-Cys-GlyOH in the P. falciparum enzyme. These differences offer an interesting approach to the design of species-specific inhibitors. Notably, A. gambiae thioredoxin reductase-1 is not a selenoenzyme but instead contains a highly unusual redox-active Cys-Cys sequence.     

Towards a species-selective acetylcholinesterase inhibitor to control the mosquito vector of malaria, Anopheles gambiae

Anopheles gambiae is the major mosquito vector of malaria in sub-Saharan Africa. At present, insecticide-treated nets (ITNs) impregnated with pyrethroid insecticides are widely used in malaria-endemic regions to reduce infection; however the emergence of pyrethroid-resistant mosquitoes has significantly reduced the effectiveness of the pyrethroid ITNs. An acetylcholinesterase (AChE) inhibitor that is potent for An. gambiae but weakly potent for the human enzyme could potentially be safely deployed on a new class of ITNs. In this paper we provide a preliminary pharmacological characterization of An. gambiae AChE, discuss structural features of An. gambiae and human AChE that could lead to selective inhibition, and describe compounds with 130-fold selectivity for inhibition of An. gambiae AChE relative to human AChE.     

Mapping the ranges and relative abundance of the two principal African malaria vectors,Anopheles gambiae sensu stricto and An. arabiensis,using climate data.

Members of the Anopheles gambiae complex are major malaria vectors in Africa. We tested the hypothesis that the range and relative abundance of the two major vectors in the complex, An. gambiae sensu stricto and An. arabiensis, could be defined by climate. Climate was characterized at mosquito survey sites by extracting data for each location from climate surfaces using a Geographical Information System. Annual precipitation, together with annual and wet season temperature, defined the ranges of both vectors and were used to map suitable climate zones. Using data from West Africa, we found that where the species were sympatric, An. gambiae s.s. predominated in saturated environments, and An. arabiensis was more common in sites subject to desiccation (r2 = 0.875, p < 0.001). We used the nonlinear equation that best described this relationship to map habitat suitability across Africa. This simple model predicted accurately the relative abundance of both vectors in Tanzania (rs = 0.745, p = 0.002), where species composition is highly variable. The combined maps of species'' range and relative abundance showed very good agreement with published maps. This technique represents a new approach to mapping the distribution of malaria vectors over large areas and may facilitate species-specific vector control activities.     

Synthetic propeptide inhibits mosquito midgut chitinase and blocks sporogonic development of malaria parasite

Incessant transmission of the parasite by mosquitoes makes most attempts to control malaria fail. Blocking of parasite transmission by mosquitoes therefore is a rational strategy to combat the disease. Upon ingestion of blood meal mosquitoes secrete chitinase into the midgut. This mosquito chitinase is a zymogen which is activated by the removal of a propeptide from the N-terminal. Since the midgut peritrophic matrix acts as a physical barrier, the activated chitinase is likely to contribute to the further development of the malaria parasite in the mosquito. Earlier it has been shown that inhibiting chitinase activity in the mosquito midgut blocked sporogonic development of the malaria parasite. Since synthetic propeptides of several zymogens have been found to be potent inhibitors of their respective enzymes, we tested propeptide of mosquito midgut chitinase as an inhibitor and found that the propeptide almost completely inhibited the recombinant or purified native Anopheles gambiae chitinase. We also examined the effect of the inhibitory peptide on malaria parasite development. The result showed that the synthetic propeptide blocked the development of human malaria parasite Plasmodium falciparum in the African malaria vector An. gambiae and avian malaria parasite Plasmodium gallinaceum in Aedes aegypti mosquitoes. This study implies that the expression of inhibitory mosquito midgut chitinase propeptide in response to blood meal may alter the mosquito's vectorial capacity. This may lead to developing novel strategies for controlling the spread of malaria.     

Immune response of Anopheles gambiae to the early sporogonic stages of the human malaria parasite Plasmodium falciparum

Deciphering molecular interactions between the malaria parasite and its mosquito vector is an emerging area of research that will be greatly facilitated by the recent sequencing of the genomes of Anopheles gambiae mosquito and of various Plasmodium species. So far, most such studies have focused on Plasmodium berghei, a parasite species that infects rodents and is more amenable to studies. Here, we analysed the expression pattern of nine An.gambiae genes involved in immune surveillance during development of the human malaria parasite P.falciparum in mosquitoes fed on parasite-containing blood from patients in Cameroon. We found that P.falciparum ingestion triggers a midgut-associated, as well as a systemic, response in the mosquito, with three genes, NOS, defensin and GNBP, being regulated by ingestion of gametocytes, the infectious stage of the parasite. Surprisingly, we found a different pattern of expression of these genes in the An.gambiae-P.berghei model. Therefore, differences in mosquito reaction against various Plasmodium species may exist, which stresses the need to validate the main conclusions suggested by the P.berghei-An.gambiae model in the P.falciparum-An.gambiae system.     

Change in composition of the Anopheles gambiae complex and its possible implications for the transmission of malaria and lymphatic filariasis in north-eastern Tanzania

ABSTRACT: BACKGROUND: A dramatic decline in the incidence of malaria due to Plasmodium falciparum infection in coastal East Africa has recently been reported to be paralleled (or even preceded) by an equally dramatic decline in malaria vector density, despite absence of organized vector control. As part of investigations into possible causes for the change in vector population density, the present study analysed the Anopheles gambiae s.l. sibling species composition in north-eastern Tanzania. METHODS: The study was in two parts. The first compared current species complex composition in freshly caught An. gambiae s.l. complex from three villages to the composition reported from previous studies carried out 2-4 decades ago in the same villages. The second took advantage of a sample of archived dried An. gambiae s.l. complex specimens collected regularly from a fourth study village since 2005. Both fresh and archived dried specimens were identified to sibling species of the An. gambiae s.l. complex by PCR. The same specimens were moreover examined for Plasmodium falciparum and Wuchereria bancrofti infection by PCR. RESULTS: As in earlier studies, An. gambiae s.s., Anopheles merus and Anopheles arabiensis were identified as sibling species found in the area. However, both study parts indicated a marked change in sibling species composition over time. From being by far the most abundant in the past An. gambiae s.s. was now the most rare, whereas An. arabiensis had changed from being the most rare to the most common. P. falciparum infection was rarely detected in the examined specimens (and only in An. arabiensis) whereas W. bancrofti infection was prevalent and detected in all three sibling species. CONCLUSION: The study indicates that a major shift in An. gambiae s.l. sibling species composition has taken place in the study area in recent years. Combined with the earlier reported decline in overall malaria vector density, the study suggests that this decline has been most marked for An. gambiae s.s., and least for An. arabiensis, leading to current predominance of the latter. Due to differences in biology and vectorial capacity of the An. gambiae s.l. complex the change in sibling species composition will have important implications for the epidemiology and control of malaria and lymphatic filariasis in the study area.     

Controlling malaria transmission with genetically-engineered, Plasmodium-resistant mosquitoes: milestones in a model system

We are developing transgenic mosquitoes resistant to malaria parasites to test the hypothesis that genetically-engineered mosquitoes can be used to block the transmission of the parasites. We are developing and testing many of the necessary methodologies with the avian malaria parasite, Plasmodium gallinaceum, and its laboratory vector, Aedes aegypti, in anticipation of engaging the technical challenges presented by the malaria parasite, P. falciparum, and its major African vector, Anopheles gambiae. Transformation technology will be used to insert into the mosquito a synthetic gene for resistance to P. gallinaceum. The resistance gene will consist of a promoter of a mosquito gene controlling the expression of an effector protein that interferes with parasite development and/or infectivity. Mosquito genes whose promoter sequences are capable of sex- and tissue-specific expression of exogenous coding sequences have been identified, and stable transformation of the mosquito has been developed. We now are developing the expressed effector portion of the synthetic gene that will interfere with the transmission of the parasites. Mouse monoclonal antibodies that recognize the circumsporozoite protein of P. gallinaceum block sporozoite invasion of mosquito salivary glands, as well as abrogate the infectivity of sporozoites to a vertebrate host, the chicken, Gallus gallus, and block sporozoite invasion and development in susceptible cell lines in vitro. Using the genes encoding these antibodies, we propose to clone and express single-chain antibody constructs (scFv) that will serve as the effector portion of the gene that interferes with transmission of P. gallinaceum sporozoites.     

The role of serine 167 in human indoleamine 2,3-dioxygenase: a comparison with tryptophan 2,3-dioxygenase

The initial step in the l-kynurenine pathway is oxidation of l-tryptophan to N-formylkynurenine and is catalyzed by one of two heme enzymes, tryptophan 2,3-dioxygenase (TDO) or indoleamine 2,3-dioxygenase (IDO). Here, we address the role of the conserved active site Ser167 residue in human IDO (S167A and S167H variants), which is replaced with a histidine in other mammalian and bacterial TDO enzymes. Our kinetic and spectroscopic data for S167A indicate that this residue is not essential for O 2 or substrate binding, and we propose that hydrogen bond stabilization of the catalytic ferrous-oxy complex involves active site water molecules in IDO. The data for S167H show that the ferrous-oxy complex is dramatically destabilized in this variant, which is similar to the behavior observed in human TDO [Basran et al. (2008) Biochemistry 47, 4752-4760], and that this destabilization essentially destroys catalytic activity. New kinetic data for the wild-type enzyme also identify the ternary [enzyme-O 2-substrate] complex. The data reveal significant differences between the IDO and TDO enzymes, and the implications of these results are discussed in terms of our current understanding of IDO and TDO catalysis.     

Linking individual phenotype to density-dependent population growth: the influence of body size on the population dynamics of malaria vectors

Understanding the endogenous factors that drive the population dynamics of malaria mosquitoes will facilitate more accurate predictions about vector control effectiveness and our ability to destabilize the growth of either low- or high-density insect populations. We assessed whether variation in phenotypic traits predict the dynamics of Anopheles gambiae sensu lato mosquitoes, the most important vectors of human malaria. Anopheles gambiae dynamics were monitored over a six-month period of seasonal growth and decline. The population exhibited density-dependent feedback, with the carrying capacity being modified by rainfall (97% wAIC(c) support). The individual phenotypic expression of the maternal (p = 0.0001) and current (p = 0.040) body size positively influenced population growth. Our field-based evidence uniquely demonstrates that individual fitness can have population-level impacts and, furthermore, can mitigate the impact of exogenous drivers (e.g. rainfall) in species whose reproduction depends upon it. Once frontline interventions have suppressed mosquito densities, attempts to eliminate malaria with supplementary vector control tools may be attenuated by increased population growth and individual fitness.