Heterologous expression, biochemical characterization and prospects for insecticide biosensing potential of carboxylesterase Ha006a from Helicoverpa armigera.

Enzymes have attracted considerable scientific attention for their crucial role in detoxifying a wide range of harmful compounds. In today's global context, the extensive use of insecticides has emerged as a significant threat to the environment, sparking substantial concern. Insects, including economically important pests like Helicoverpa armigera, have developed resistance to conventional pest control methods through enzymes like carboxyl/cholinesterases. This study specifically focuses on a notable carboxyl/cholinesterase enzyme from Helicoverpa armigera (Ha006a), with the goal of harnessing its potential to combat environmental toxins. A total of six insecticides belonging to two different classes displayed varying inhibitory responses towards Ha006a, thereby rendering it effective in detoxifying a broader spectrum of insecticides. The significance of this research lies in discovering the bioremediation property of Ha006a, as it hydrolyzes synthetic pyrethroids (fenvalerate, λ-cyhalothrin and deltamethrin) and sequesters organophosphate (paraoxon ethyl, profenofos, and chlorpyrifos) insecticides. Additionally, the interaction studies between organophosphate insecticides and Ha006a helped in the fabrication of a novel electroanalytical sensor using a modified carbon paste electrode (MCPE). This sensor boasts impressive sensitivity, with detection limits of 0.019 µM, 0.15 µM, and 0.025 µM for paraoxon ethyl, profenofos, and chlorpyrifos, respectively. This study provides a comprehensive biochemical and biophysical characterization of the purified esterase Ha006a, showcasing its potential to remediate different classes of insecticides.

Characterization of AICAR transformylase/IMP cyclohydrolase (ATIC) bifunctional enzyme from Candidatus Liberibacer asiaticus.

The bifunctional enzyme, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/inosine monophosphate (IMP) cyclohydrolase (ATIC) is involved in catalyzing penultimate and final steps of purine de novo biosynthetic pathway crucial for the survival of organisms. The present study reports the characterization of ATIC from Candidatus Liberibacer asiaticus (CLasATIC) along with the identification of potential inhibitor molecules and evaluation of cell proliferative activity. CLasATIC showed both the AICAR Transformylase (AICAR TFase) activity for substrates, 10-f-THF (Km, 146.6 µM and Vmax, 0.95 µmol/min/mg) and AICAR (Km, 34.81 µM and Vmax, 0.56 µmol/min/mg) and IMP cyclohydrolase (IMPCHase) activitiy (Km, 1.81 µM and Vmax, 2.87 µmol/min/mg). The optimum pH and temperature were also identified for the enzyme activity. In-silico study has been conducted to identify potential inhibitor molecules through virtual screening and MD simulations. Out of many compounds, HNBSA, diosbulbin A and lepidine D emerged as lead compounds, exhibiting higher binding energy and stability for CLasATIC than AICAR. ITC study reports higher binding affinities for HNBSA and diosbulbin A (Kd, 12.3 µM and 34.2 µM, respectively) compared to AICAR (Kd, 83.4 µM). Likewise, DSC studies showed enhanced thermal stability for CLasATIC in the presence of inhibitors. CD and Fluorescence studies revealed significant conformational changes in CLasATIC upon binding of the inhibitors. CLasATIC demonstrated potent cell proliferative, wound healing and ROS scavenging properties evaluated by cell-based bioassays using CHO cells. This study highlights CLasATIC as a promising drug target with potential inhibitors for managing CLas and its unique cell protective, wound-healing properties for future biotechnological applications.

Comparative Analysis of Inhibitor Binding to Peroxiredoxins from Candidatus Liberibacter asiaticus and Its Host Citrus sinensis.

The peroxiredoxins (Prxs), potential drug targets, constitute an important class of antioxidant enzymes present in both pathogen and their host. The comparative binding potential of inhibitors to Prxs from pathogen and host could be an important step in drug development against pathogens. Huanglongbing (HLB) is a most devastating disease of citrus caused by Candidatus Liberibacter asiaticus (CLa). In this study, the binding of conoidin-A (conoidin) and celastrol inhibitor molecules to peroxiredoxin of bacterioferritin comigratory protein family from CLa (CLaBCP) and its host plant peroxiredoxin from Citrus sinensis (CsPrx) was assessed. The CLaBCP has a lower specific activity than CsPrx and is efficiently inhibited by conoidin and celastrol molecules. The biophysical studies showed conformational changes and significant thermal stability of CLaBCP in the presence of inhibitor molecules as compared to CsPrx. The surface plasmon resonance (SPR) studies revealed that the conoidin and celastrol inhibitor molecules have a strong binding affinity (KD) with CLaBCP at 33.0 µM, and 18.5 µM as compared to CsPrx at 52.0 µM and 61.6 µM, respectively. The docked complexes of inhibitor molecules showed more structural stability of CLaBCP as compared to CsPrx during the run of molecular dynamics-based simulations for 100 ns. The present study suggests that the conoidin and celastrol molecules can be exploited as potential inhibitor molecules against the CLa to manage the HLB disease.

Identification and evaluation of potential inhibitor molecules against TcyA from Candidatus Liberibacter asiaticus.

Of the two putative amino acid binding periplasmic receptors of ABC transporter family in Candidatus Liberibacter asiaticus (CLas), cystine binding receptor (CLasTcyA) has been shown to mainly express in phloem of citrus plant and is a target for inhibitor development. The crystal structure of CLasTcyA in complex with substrates has been reported earlier. The present work reports the identification and evaluation of potential candidates for their inhibitory potential against CLasTcyA. Among many compounds, selected through virtual screening, and MD simulation, pimozide, clidinium, sulfasalazine and folic acid showed significantly higher affinities and stability in complex with CLasTcyA. The SPR studies with CLasTcyA revealed significantly higher binding affinities for pimozide and clidinium (Kd, 2.73 nM and 70 nM, respectively) as compared to cystine (Kd, 1.26 µM). The higher binding affinities could be attributed to significantly increased number of interactions in the binding pocket as evident from the crystal structures of CLasTcyA in complex with pimozide and clidinium as compared to cystine. The CLasTcyA possess relatively large binding pocket where bulkier inhibitors fit quite well. In planta studies, carried out to assess the effect of inhibitors on HLB infected Mosambi plants, showed significant reduction in CLas titre in plants treated with inhibitors as compared to control plants. The results showed that pimozide exhibited higher efficiency as compared to clidinium in reducing CLas titre in treated plants. Our results showed that the inhibitor development against critical proteins like CLasTcyA can be an important strategy in management of HLB.

Mutation studies and structure-based identification of potential inhibitor molecules against periplasmic amino acid binding protein of Candidatus Liberibacter asiaticus (CLasTcyA).

Earlier reported crystal structure of CLasTcyA revealed unique features like relatively a larger substrate binding pocket, an extended C-terminal loop restricted by a disulfide bond and involvement of residues from hinge region in substrate binding. In present study, CLasTcyA mutants were created to evaluate the importance of these unique features through biophysical characterization. The Val58 in CLasTcyA was replaced by Trp, conserved in most cystine binding proteins, to reduce the size of the binding pocket. All other mutations were created in CLasTcyAV58W mutant as the presence of Trp could be used for intrinsic fluorescence studies. The CLasTcyAV58W showed a noticeable increase in binding affinity and thermal stability as compared to the native form. The mutation of two cysteines in triple mutant CLasTcyAV58W/C212S/C239S, removal of C-terminal extended loop in truncated CLasTcyAV58W/C212S and mutation of His95 from hinge region in the double mutant CLasTcyAV58W/H95A showed a marked decrease in stability-indicating the importance of the unique features in structure of CLasTcyA. The bioinformatics-based virtual screening was employed to screen the potential inhibitor molecules for detailed future studies. The results clearly establish the importance of unique features in structure-function relationship of CLasTcyA.