Present status and future prospects to safeguard Nepali citrus industry against Chinese citrus fly (Bactrocera minax Enderlein)

Unlike other Tephrid flies, the Chinese citrus fly (Bactrocera minax Enderlein) is univoltine and oligophagous species strictly restricted to citrus fruits. It has been a serious threat to the citrus industry in China, Bhutan, India and Nepal causing up to 100% of fruit drop before the harvest. Citrus groves, especially tight-skinned cultivars, sweet orange (Citrus sinensis L. Osbeck) in mid-hill districts like Ramechhap, Sindhuli, Dolakha, Kavre, Syangja, Gulmi, etc. have been threatened while in some pockets, lemon, acid lime, and mandarin have vanished due to the Chinese citrus fly (CCF). The driver behind the spread of this invasive pest seems to be poor research works on the phenology of the pest, ill-equipped management practices, flying nature of adult fly and easy movement of infested fruits. Therefore, with reviewing published data, this study aimed to figure out the most appropriate management technology for curbing the CCF and make comprehensive material for safeguarding the citrus industry in the future. Since Area-Wide Integrated Pest Management (AW-IPM) or Area-Wide Control Program (AWCP) was found to be an effective tool to control the CCF, individual practices are crucial to incorporate. Monitoring the pest with the lure of protein hydrolase (PH) and subsequently killing adults with attractive protein baits of 25% hydrolyzed protein + insecticide as lethal dinner is mentioned exceptionally better. In AWCP domestic practice: orchard sanitation is not so effective if the orchards are sloppy while shallow tillage adds less to the natural enemy mechanism of CCF pupae in the soil. Equally, we conclude that Sterile Insect Technology (SIT) is not so economical and the boons of natural enemies, parasitoid and entomo-pathogens against CCF, is yet to be exploited

The Mechanism of Substrate Inhibition in Human Indoleamine 2,3-Dioxygenase

Indoleamine 2,3-dioxygenase catalyzes the O(2)-dependent oxidation of L-tryptophan (L-Trp) to N-formylkynurenine (NFK) as part of the kynurenine pathway. Inhibition of enzyme activity at high L-Trp concentrations was first noted more than 30 years ago, but the mechanism of inhibition has not been established. Using a combination of kinetic and reduction potential measurements, we present evidence showing that inhibition of enzyme activity in human indoleamine 2,3-dioxygenase (hIDO) and a number of site-directed variants during turnover with L-tryptophan (L-Trp) can be accounted for by the sequential, ordered binding of O(2) and L-Trp. Analysis of the data shows that at low concentrations of L-Trp, O(2) binds first followed by the binding of L-Trp; at higher concentrations of L-Trp, the order of binding is reversed. In addition, we show that the heme reduction potential (E(m)(0)) has a regulatory role in controlling the overall rate of catalysis (and hence the extent of inhibition) because there is a quantifiable correlation between E(m)(0) (that increases in the presence of L-Trp) and the rate constant for O(2) binding. This means that the initial formation of ferric superoxide (Fe(3+)-O(2)(•-)) from Fe(2+)-O(2) becomes thermodynamically less favorable as substrate binds, and we propose that it is the slowing down of this oxidation step at higher concentrations of substrate that is the origin of the inhibition. In contrast, we show that regeneration of the ferrous enzyme (and formation of NFK) in the final step of the mechanism, which formally requires reduction of the heme, is facilitated by the higher reduction potential in the substrate-bound enzyme and the two constants (k(cat) and E(m)(0)) are shown also to be correlated. Thus, the overall catalytic activity is balanced between the equal and opposite dependencies of the initial and final steps of the mechanism on the heme reduction potential. This tuning of the reduction potential provides a simple mechanism for regulation of the reactivity, which may be used more widely across this family of enzymes

The Mechanism of Formation of N-Formylkynurenine by Heme Dioxygenases

[Image: see text] Heme dioxygenases catalyze the oxidation of l-tryptophan to N-formylkynurenine (NFK), the first and rate-limiting step in tryptophan catabolism. Although recent progress has been made on early stages in the mechanism, there is currently no experimental data on the mechanism of product (NFK) formation. In this work, we have used mass spectrometry to examine product formation in a number of dioxygenases. In addition to NFK formation (m/z = 237), the data identify a species (m/z = 221) that is consistent with insertion of a single atom of oxygen into the substrate during O(2)-driven turnover. The fragmentation pattern for this m/z = 221 species is consistent with a cyclic amino acetal structure; independent chemical synthesis of the 3a-hydroxypyrroloindole-2-carboxylic acid compound is in agreement with this assignment. Labeling experiments with (18)O(2) confirm the origin of the oxygen atom as arising from O(2)-dependent turnover. These data suggest that the dioxygenases use a ring-opening mechanism during NFK formation, rather than Criegee or dioxetane mechanisms as previously proposed

Human heme dioxygenases

The L-kynurenine pathway, which leads to the formation of NAD, is the major catabolic route of L-tryptophan metabolism in biology. The initial step in this pathway is oxidation of L-tryptophan to N-formyl-kynurenine. In all biological systems examined to date, this is catalysed by one of two heme enzymes, indoleamine 2,3-dioxygenase (IDO) or tryptophan 2,3-dioxygenase (TDO). In this thesis the reaction mechanism, the reactive catalytic intermediates involved in this reaction and the nature of substrate (L-tryptophan and dioxygen)-protein interactions, if any, present within the active site of rhIDO have been examined.;In Chapter 2, we addressed the role of S I67 in rhIDO (S167A and S167H), which is replaced with a histidine residue in TDO enzymes. Kinetic and spectroscopic data for S I67A indicate that this residue is not essential for O2 or substrate binding. The data for S167H show that the ferrous-oxy complex is dramatically destabilised, which is similar to the behaviour observed in rhTDO. The implications of these results are discussed in terms of our current understanding of IDO and TDO catalysis.;In Chapter 3, it was shown that 1-methyL-tryptophan is a substrate for rhIDO and S167A. However, no activity was observed for rhTDO. Substitution of an active site histidine residue in rhTDO (H76S) allows accommodation of the additional methyl group and 1-methyL-tryptophan turnover to occur. These observations suggest that deprotonation of the indole N 1 is not essential for catalysis, and an alternative reaction mechanism is presented. Additional experiments using EPR and 1H ENDOR spectroscopy were used to examine the surrounding environment of the heme iron. The results reveal important information on the surrounding environment of the heme-bound dioxygen and the interactions present in the ternary complex. The mechanistic implications of such interactions are discussed in this work.;In Chapter 5, we undertook site-directed mutagenesis of several active site residues and the role of each residue on dioxygen, substrate binding and in catalysis was examined. We found the conserved residue R231 plays a key role in substrate binding and is likely to do so in all heme dioxygenase enzymes. The F227A variant was found to be catalytically competent for L-tryptophan turnover and suggests that this residue is not involved in substrate recognition like previously proposed.;In Chapter 6, we have shown that rhTDO and rhIDO can utilise hydrogen peroxide as an alternative oxygen source to dioxygen. For rhTDO, approximately two equivalents of H2O2 were consumed in the production of one molecule of N-formyl-kynurenine, suggesting that an alternative mechanistic pathway is used with hydrogen peroxide

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 O2 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-O2-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. © 2008 American Chemical Society

The Mechanism of Substrate Inhibition in Human Indoleamine 2,3-Dioxygenase

Indoleamine 2,3-dioxygenase catalyzes the O₂-dependent oxidation of l-tryptophan (l-Trp) to <i>N</i>-formylkynurenine (NFK) as part of the kynurenine pathway. Inhibition of enzyme activity at high l-Trp concentrations was first noted more than 30 years ago, but the mechanism of inhibition has not been established. Using a combination of kinetic and reduction potential measurements, we present evidence showing that inhibition of enzyme activity in human indoleamine 2,3-dioxygenase (hIDO) and a number of site-directed variants during turnover with l-tryptophan (l-Trp) can be accounted for by the sequential, ordered binding of O₂ and l-Trp. Analysis of the data shows that at low concentrations of l-Trp, O₂ binds first followed by the binding of l-Trp; at higher concentrations of l-Trp, the order of binding is reversed. In addition, we show that the heme reduction potential (<i>E</i>_m⁰) has a regulatory role in controlling the overall rate of catalysis (and hence the extent of inhibition) because there is a quantifiable correlation between <i>E</i>_m⁰ (that increases in the presence of l-Trp) and the rate constant for O₂ binding. This means that the initial formation of ferric superoxide (Fe³⁺–O₂^•–) from Fe²⁺-O₂ becomes thermodynamically less favorable as substrate binds, and we propose that it is the slowing down of this oxidation step at higher concentrations of substrate that is the origin of the inhibition. In contrast, we show that regeneration of the ferrous enzyme (and formation of NFK) in the final step of the mechanism, which formally requires reduction of the heme, is facilitated by the higher reduction potential in the substrate-bound enzyme and the two constants (<i>k</i>_cat and <i>E</i>_m⁰) are shown also to be correlated. Thus, the overall catalytic activity is balanced between the equal and opposite dependencies of the initial and final steps of the mechanism on the heme reduction potential. This tuning of the reduction potential provides a simple mechanism for regulation of the reactivity, which may be used more widely across this family of enzymes