Progress 05/15/20 to 06/20/23
Outputs
Target Audience:Our primary target audience are researchers working on organisms from the Arthropoda phylum with a special emphasis on researchers working on herbivory mites. Moreover, as significant fraction of our targets is involved in pesticide/acaricide resistance or metabolism we target as well specialist involved in pest management. In addition, we have presented our results to a broad audience of scientists working in the field of biological and biomedical sciences. Changes/Problems:The biggest change is related to move of the Chruszcz group to Michigan State University. Unfortunately, the move affected our productivity at the end of 2022 and the beginning of 2023. At this point, we finalize the transfer of the grant from the University of South Carolina to Michigan State University. The process of the grant transfer is taking significantly more time than we anticipated. What opportunities for training and professional development has the project provided?Since May 2023, there have been two graduate and one undergraduate students who have worked on this project in the Chruszcz group. The students worked on production of recombinant versions of the target enzymes, and were involved in characterization of these proteins. We were mainly focused on characterization of enzymatic activity and structure of the target proteins, which subsequently was used to identify physiological substrates of the enzyme and their biological roles. Therefore, the students had an opportunity to become familiar with various biochemical and biophysical methods. The methods/techniques used included DNA manipulation, bacterial protein expression protein purification, spectroscopic methods for testing enzyme activity, mass spectrometry, X-ray crystallography, etc. Currently, there is one graduate studnet from the Makris group who works on the project. The student's work focuses on production and charaterization of cytochromes P450. How have the results been disseminated to communities of interest?There were two conference abstract that we have submitted during the last two months. These abstrat are related to presentations that will take place during 2023 Meeting of Phytochemical Society of North America (July 16-20, East Lansing, MI) 1. "Intradiol ring-cleavage dioxygenases and their role in the xenobiotic detoxification and host adaptation in Tetranychidae mites." Brendan Abiskaroon, Ricardo Hernandez, Naoki Takeda, Kristi Hemstreet, Vishvendra Chouhan, Vojislava Grbic, Takeshi Suzuki, Maksymilian Chruszcz 2. "TuGSTmu12, but not its close homologue TuGSTmu06, is required for detoxification of the glucosinolate-myrosinase defenses and Tetranychus urticae adaptation to Arabidopsis." Kriti Khatri, Ricardo Hernandez, Brendan Abiskaroon, Sameer Dixit, Vinayak Singh, Vladimir Zhurov, Emilie Widemann, Chetan Sharma, Anna Micaela Camini, Golnaz Salehipourshirazi, Brendan Walshe-Roussel, Kristie Bruinsma, Zuzana Rosenbergova Luis Fernando Saraiva Macedo Timmers, Martin Rebros, Miodrag Grbic, Vojislava Grbic, Maksymilian Chruszcz What do you plan to do during the next reporting period to accomplish the goals?This is our final report for the USC. However, at MSU we hope to continue our work on the project.
Impacts
What was accomplished under these goals?
During the last two months since the last report, we were continuing work on TSSM enzymes. We focus on cytochrome P450 family of proteins, as well as on intradiol ring-cleavage dioxygenases (ID-RCDs), glutathione S-transferases (GSTs) and UDP-glycosyl transferases (UGTs). As mentioned in previous repost we have over 30 of these proteins produced in recombinant form, and we identify their physiological substrates and functions.
Publications
|
Progress 05/15/22 to 05/14/23
Outputs
Target Audience:Our primary target audience are researchers working on organisms from the Arthropoda phylum with a special emphasis on researchers working on herbivory mites. Moreover, as significant fraction of our targets is involved in pesticide/acaricide resistance or metabolism we target as well specialist involved in pest management. In addition, we have presented our results to a broad audience of scientists working in the field of biological and biomedical sciences. Changes/Problems:The biggest change is related to move of the Chruszcz group to Michigan State University. Unfortunately, the move affected our productivity at the end of 2022 and the beginning of 2023. At this point, we finalize the transfer of the grant from the University of South Carolina to Michigan State University. What opportunities for training and professional development has the project provided?Since May 15th 2022, there have been several graduate and undergraduate students who have worked on this project in the Chruszcz group. The students worked on production of recombinant versions of the target enzymes, and were involved in characterization of these proteins. We were mainly focused on characterization of enzymatic activity and structure of the target proteins, which subsequently was used to identify physiological substrates of the enzyme and their biological roles. Therefore, the students had an opportunity to become familiar with various biochemical and biophysical methods. The methods/techniques used included DNA manipulation, bacterial protein expression protein purification, spectroscopic methods for testing enzyme activity, mass spectrometry, X-ray crystallography, etc. Student from the Chruszcz group working on this project during the past funding period: Brendan Abiskaroon (4.5 calendar months) - currently 1st year graduate student at the Department of Biochemistry and Molecular Biology (MSU) Ricardo Hernandez Arriaza (3 calendar months) - currently 3rd year Chemistry & Biochemistry (USC) graduate student Kriti Khatri (3 calendar months) - currently 4th year Chemistry & Biochemistry graduate student. Kriti Katri successfully defended her dissertation and will graduate this May. Andrea O'Malley (3 calendar months) - currently 4th year Chemistry & Biochemistry (USC) graduate student. Andrea O'Malley successfully defended her dissertation and will graduate this May. Christina Plantz (2.5 calendar months) - currently 1st year graduate student at the Department of Biochemistry and Molecular Biology (MSU) Hayley Cash (4.5 months) - undergraduate student (USC) and Biochemistry and Molecular Biology major and violin performance certification minor Vishvendra Chouhan (2.5 months) - undergraduate student (MSU) and Biochemistry major Robert Pokora (4.5 months)- undergraduate student (USC) and Biochemistry and Molecular Biology major The results originated from this grant are also used in teaching of undergraduate and graduate students. For example, Dr. Chruszcz was teaching CHEM 753 (Protein Chemistry and Enzymology - graduate level class) in Fall 2022. This course has parts dedicated to enzymes; therefore, experimental data generated for proteins focused on in this proposal were used in this class. Dr. Makris teaches BCH 451 (~80 students each semester), which introduces students from diverse majors to protein structure and enzyme mechanisms. Students from the Makris group working on this project during the past funding period: Hannah Gering (12 calendar months) - currently 3rd year graduate student How have the results been disseminated to communities of interest?The Chruszcz group gave an oral presentation titled "Structural, Functional and Biochemical Studies to Elucidate the Molecular Basis of Xenobiotic Metabolism" at the 12th Spider Mite Genome Meeting (Logroño, La Rioja, Spain; October 17-20, 2022). Dr. Chruszcz presented seminar titled "Mites affecting humans - from a source of allergens to agricultural pests) at North Carolina State University (Raleigh, NC; April 6th, 2023). Ricardo Hernandez Arriaza from the Chruszcz group, who was applying for postdoctoral positions, has presented his work on TuUGTs at Stanford University and Massachusetts Institute of Technology. Here we would like to mention, that he has secured the postdoctoral research associate position at Stanford University, where he will continue his academic career starting from January 2024. The Chruszcz group plans to participate in the 62nd annual meeting of the Phytochemical Society of North America at Michigan State University (East Lansing) and we will present at least to posters. Hannah Gering from the Makris laboratory presented work on CYPs at the International Conference on Cytochrome P450 (ICPP450) in Washington DC (July 17-22, 2022) In summary, during the last funding period there were four oral presentations that were delivered for researchers from different universities. What do you plan to do during the next reporting period to accomplish the goals?We can produce approximately 30 recombinant enzymes originating from TSSM. We will continue to work on enzymes that we started to characterize during the previous reporting periods. A special focus will be placed on cytochrome P450 family of proteins. In the fourth year we hope to add additional 5-10 recombinant proteins to our list. In addition, we will continue our work on identification of physiological substrates for CYPs, ID-RCDs, GSTs and UGTs. In parallel to the work on recombinant proteins we will perform gene silencing experiments, and perform in situ hybridization for these proteins. At his point we work both on Arabidopsis and tomato model systems, and focus on these plants defenses against TSSM.
Impacts
What was accomplished under these goals?
We continued the characterization of proteins from our original target list. In addition, we have expanded the list of targets and we also worked on several proteins from the glutathione S-transferase (GST) and UDP-glycosyltransferase (UGT) families. The addition of these targets was prompted by our and literature data showing involvement of the proteins in adaptation to a new host or their involvement during host shift. TuCAS (β-cyanoalanine synthase) We have performed several experiments in order to evaluate the role of TuCAS in protection of T. urticae against indole glucosinolates. However, no major conclusions on the role of this enzyme in this process can be drawn. However, the experiments with indole glucosinolates implicated GSTs that play a role in detoxification of these compounds. This will be discussed in the GST-related section. Intradiol ring-cleavage dioxygenases (ID-RCDs) Two new ID-RCDs, Tetur20g01790 and Tetur20g01790_KSM were produced in recombinant form. The enzymes are available in significant quantities, and are currently being characterized (Fig.1). Tetur20g01790_KSM is originated from T. kanzawai and was identified, last year, as required for mite adaptation to tea plants. Interestingly, these two enzymes only differ in two amino acids: Q127A and T203A (Tetur20g01790 compared to Tetur20g01790_KSM).Enzymatic assays have been performed using the epicatechins mentioned above and Tetur20g01790, as well as Tetur07g05930 (required for Arabidopsis adaptation). Our results show that both enzymes can cleave these four epicatechins, showing the highest activity against (-)-epicatechin. On the other hand, mass-spectrometry studies to identify the product of the reaction suggests that these two enzymes do not cleave between the same diol groups, in the case of (-)-epicatechin gallate. Ongoing experiments include the determination of the kinetic parameters for the two enzymes and the four epicatechins, along with Tetur20g01790_KSM. TuGSTs Analysis of the role of mu class GSTs in T. urticae adaptation to Arabidopsis GSTmu enzymes are characteristic for vertebrates and are not found in insects. However, this subclass of GST enzymes is present in T. urticae and contains 12 members. We focused on study of two members of the family, TuGSTmu12 and TuGSTmu06 as they: have high sequence similarity TuGSTmu12 is constitutively upregulated in mites adapted to Arabidopsis, while TuGSTmu06 is responsive to general xenobiotic challenge and is regulated upon transfer of any mite from bean to Arabidopsis. provide an experimental framework to address the specificity of detoxification mechanisms in T. urticae associated with mite adaptation to new host. We have used an RNAi approach to show that TuGSTmu12 is required for mite adaptation to Arabidopsis and for the mitigation of the negative effects of indole glucosinolates (I3M) on mite fitness. Consistent with the accumulation of I3M-GSH in mites upon exposure to I3M, we identified the ability of TuGSTmu12 but not TuGSTmu06 to use indole-isothiocyanates (indole-ITC) as a substrate in an in vitro reaction that consisted of an I3M, myrosinases TGG1, glutathione, and TuGSTmu12 or TuGSTmu06. As indole-ITC is toxic compound that deters herbivory of wide range of herbivores, its conjugation with GSH by TuGSTmu12 establishes a detoxification pathway that counteracts indole-ITC toxicity. Characterization of TuGSTmu12 in mite adaptation to Arabidopsis adds to our previous findings that TuCAS is required for the detoxification of I3M-derived cyanide (Fig. 4). Our data demonstrate: specificity of detoxification machinery in Arabidopsis-adapted mites complexity of plant I3M defense compounds complexity of mite counter-responses that enable them to adapt to Arabidopsis. TuUGTs Structural, functional, and computational studies performed on Tetur22g00270 (UGT202A2) have elucidated the molecular basis of this enzyme promiscuity. Low-regioselectivity and an open-ended catalytic site are two of the major attributes contributing to this promiscuity (Fig. 7). Additionally, we have developed a pipeline to test the activity of any TuUGT against a library of hundreds or thousands of compounds. Figure 8 shows the results obtained from testing the activity of UGT202A2 against a library of natural compounds (around 400 chemicals) from Michigan State University (MSU). This pipeline will be used to find new and novel natural substrates for this family of enzymes. Production of recombinant CYP392s in an E. coli expression system Thus far, we have been able to successfully overexpress and purify two CYPs, CYP392A13v2 and CYP392A16. Overexpression of CYP392A13v2 yields high levels of well-behaved, pure enzyme that is suitable for spectroscopic characterization, crystallography trials and activity assays. Optimization was required for isolation of CYP392A16, which involved multiple different truncations of the membrane-bound N-terminus. We were able to obtain low yields of active enzyme that are suitable for activity assays. We have adopted the same N-terminal modification to two other target CYPs, CYP392D8 and CYP392E4, which were unable to express in their previous forms. Expression trials of these target CYPs with this new strategy are currently underway. Characterization of CYP392A13v2 The activity of CYP392A13v2 was evaluated using a 7-ethoxycoumarin O-deethylation assay, which is a substrate that is commonly used to monitor the monooxygenase activity of CYPs. Purified recombinant rCPR was used as a surrogate redox partner. The O-deethylation product, 7-hydroxycoumarin, has fluorescent properties which is used to monitor product formation and can be used in a high-throughput manner. The KM and kcat for the reaction of CYP392A13v2 with 7-ethoxycoumarin was determined to be 143 ± 26 µM and 0.17 ± 0.02 min-1, respectively, as shown in figure 9. These results demonstrate that the surrogate redox partner, rCPR, is effective in supporting catalysis of CYP392A13v2. One of the first steps of oxygen activation in CYPs that is critical in catalysis is the generation of the oxy-complex. This intermediate is formed when oxygen binds to the ferrous heme after the first electron transfer step and also serves as the first branchpoint from productive catalysis to "autooxidation," or decay of the oxy complex in the absence of a second electron to enable further oxygen activation. This intermediate has been studied extensively in both human and bacterial CYPs, and the relative stabilities change drastically between enzymes. The stability of the oxy complex is typically altered by the presence of substrate, which is thought to stabilize this intermediate to allow further electron transfer to occur. We have previously reported that CYP392A13v2 binds the acaricide abamectin with a KD of ~2 µM. Thus, we analyzed the formation and decay of the oxy-intermediate in the presence and absence of abamectin as a pseudo-substrate using stopped-flow spectrophotometry. As depicted in figure 2a, the substrate-free oxy complex (red trace) displays absorption characteristics that are in line with other reported CYP oxy complexes, with a Soret maximum at 422 nm and a merged charge-transfer band at 556 nm. The oxy complex decays to the low spin species (blue trace) in a biphasic manner with a rate constant of ~4 s-1. In contrast, the oxy complex of abamectin-bound CYP392A13v2 (Fig. 10B) decays an order of magnitude slower at around 0.1 s-1, highlighting the role of a bound molecule in the modulation of CYP-mediated oxygen activation. This study is the first reported characterization of an oxygenated intermediate in this family of CYPs.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
1. Kader S., Arriaza R.H., Khatri K., OMalley A., Grbic V., Grbic M., Chruszcz M. (2023) Current status of structural studies of proteins originating from Arachnida. Systematic & Applied Acarology 28, 298-308. DOI: 10.11158/saa.28.2.12
- Type:
Journal Articles
Status:
Under Review
Year Published:
2023
Citation:
2. Kettlin Ruffatto, Camila Rockenbach da Silva, Eduardo Vieira de Souza, Ricardo Hernandez Arriaza, Maksymilian Chruszcz, Raul Antonio Sperotto, Luis Fernando Saraiva Macedo Timmers (2023) Identification of natural compounds able to inhibit Tetranychus urticae-specific proteins aiming to develop new bioacaricides. Journal of Pest Science - revisions submitted
- Type:
Journal Articles
Status:
Other
Year Published:
2023
Citation:
3. Arriaza, R.H.; Daneshian, L.; Kluza, A; Abiskaroon, B; Patel, M; Snoeck, S.; Grbic, V.; Dermauw, W.; Borowski, T.; Chruszcz, M. (2023) Structural and functional studies reveal molecular basis of substrate promiscuity of a glycosyltransferase originating from a major agricultural pest. final stage of preparation.
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Progress 05/15/21 to 05/14/22
Outputs
Target Audience:Our primary target audience are researchers working on organisms from the Arthropoda phylum with a special emphasis on researchers working on herbivory mites. Moreover, as significant fraction of our targets is involved in pesticide/acaricide resistance or metabolism we target as well specialist involved in pest management. In addition, we have presented our results to a broad audience of scientists working in the field of biological and biomedical sciences. Changes/Problems:As almost all restrictions related to the COVID-19 pandemic were lifted, we currently are nearly on track with completing our goals that were set before the pandemic. We still experience some delays related to difficulties in production of some of CYPS. However, at the same time a new biological data allowed us to expand a list of our targets, and we have made a significant progress on GSTs and UGTs. During the last funding period we were also working with both plant model systems (Arabidopsis and tomato), which were proposed to be studied in our grant application. What opportunities for training and professional development has the project provided?Since May 15th 2021, there have been several graduate and undergraduate students who have worked on this project in the Chruszcz group. The students worked on production of recombinant versions of the target enzymes, and were involved in characterization of these proteins. We were mainly focused on characterization of enzymatic activity and structure of the target proteins, which subsequently was used to identify physiological substrates of the enzyme and their biological roles. Therefore, the students had an opportunity to become familiar with various biochemical and biophysical methods. The methods/techniques used included DNA manipulation, bacterial protein expression protein purification, spectroscopic methods for testing enzyme activity, mass spectrometry, X-ray crystallography, etc. Student from the Chruszcz group working on this project during the past funding period: Ricardo Hernandez Arriaza (9 calendar months) - currently 2nd year Chemistry and Biochemistry graduate student Kriti Khatri (3 calendar months) - currently 3rd year Chemistry & Biochemistry graduate student Andrea O'Malley (3 calendar months) - currently 3rd year Chemistry & Biochemistry graduate student Hayley Cash (4.5 months) - undergraduate student and Biochemistry and Molecular Biology major and violin performance certification minor Shannon Henry (4.5 months) - undergraduate student and Biochemistry and Molecular Biology major Arina Lomoff (9 months) - undergraduate student and Biochemistry and Molecular Biology major Robert Pokora (4.5 months)- undergraduate student and Biochemistry and Molecular Biology major Isabella Renggli (4.5 calendar months) - undergraduate student who graduated in December 2021; - Biochemistry and Molecular Biology major The results originated from this grant are also used in teaching of undergraduate and graduate students. For example, Dr. Chruszcz was teaching CHEM 753 (Protein Chemistry and Enzymology - graduate level class) and CHEM 555/ BIOL 545 (Biochemistry I) courses in Fall 2021 and Spring 2022, respectively. Both courses have parts dedicated to enzymes; therefore, experimental data generated for proteins focused on in this proposal were used in both these classes. Students from the Makris group working on this project during the past funding period: Suman Das (12 calendar months) - currently 5th graduate student who is planning to defend his dissertation in May 2022 Hannah Gering (9 calendar months) - currently 2nd year graduate student How have the results been disseminated to communities of interest?We have published three manuscripts and we have one manuscript in the final stages of preparation. The results from our work were also disseminated in a form of seminars and oral presentations. The Chruszcz group had four poster presentations. One presented during the Discover UofSC event (April 22, 2022; Columbia SC), and three during the 12th Annual Southeast Enzyme Conference (April 23, 2022; Atlanta, GA). Presentations given (or planned this summer): By Dr. Makris: Pacifichem Biochemistry Club for undergraduate students in the Department of Molecular and Structural Biochemistry at NCSU University of Texas San Antonio, Department of Chemistry International Conference on Porphyrins and Phthalocyanines International Conference on Cytochrome P450 By Suman Das (who is currently applying for industrial positions): Pfizer Regeneron In summary, during the last funding period there were seven oral presentations that were delivered for researchers from three different universities and three different commercial companies. What do you plan to do during the next reporting period to accomplish the goals?We can produce approximately 20 recombinant enzymes originating from TSSM. We will continue to work on enzymes that we started to characterize during the first and second reporting periods. A special focus will be placed on cytochrome P450 family of proteins. In the third year we hope to add additional 8-10 recombinant proteins to our list. In addition, we will work on identification of physiological substrates for CYPs, ID-RCDs, GSTs and UGTs. In parallel to the work on recombinant proteins we will perform gene silencing experiments, and perform in situ hybridization for these proteins. At his point we are ready to work both on Arabidopsis and tomato model systems, and focus on these plants defenses against TSSM.
Impacts
What was accomplished under these goals?
We continued the characterization of proteins from our original target list. In addition, we have expanded the list of targets and we also worked on several proteins from the glutathione S-transferase (GST) and UDP-glycosyltransferase (UGT) families. The addition of these targets was prompted by our reported in literature data showing involvement of the proteins in adaptation to a new host or their involvement during host shift. TuCAS (β-cyanoalanine synthase) We have now published (Plant Physiology) a paper describing the role of TuCAS in mite adaptation to indole glucosinolates. We showed that TuCAS is required for mite adaptation to indole glucosinolates through its β-cyanoalanine synthase activity. Consistent with β-cyanoalanine synthase's role in detoxification of hydrogen cyanide, we demonstrated that, upon mite herbivory, Arabidopsis plants generate hydrogen cyanide through the indole glucosinolate pathway. Follow up studies aimed at characterizing the source of cyanide in Arabidopsis, as well as alternative Arabidopsis metabolites as substrates to TuCAS, are underway. In addition, our manuscript describing biochemical and structural characterization of TuCAS was published in Insect Biochemistry and Molecular Biology (most of the findings reported in this paper were reported last year). Intradiol ring-cleavage dioxygenases (ID-RCDs) Two ID-RCDs (Tetur07g02040 and Tetur07g05930) are available as recombinant proteins, and are being characterized. We still work on production of Tetur06g00450, as the expression of this ID_RCD is poor. Meanwhile, two other ID-RDCs - Tetur20g01730 and Tetur01g00490 - were identified as REQUIRED for mite adaptation to tea (T. kanzawai) and Arabidopsis (T. urticae), respectively. A blend of tea catechins were identified as natural substrates for Tetur20g01730. Comparative analysis of enzymatic properties of these ID-RCDs using candidate substrates and plant extracts will be used to characterize their substrate specificity. Identification of plant defense substrates will be attempted. CYPs CYP392A13v2, CYP392E2, CYP392E4, associated with mite adaptation to Arabidopsis, are available as recombinant proteins that have a TEV-cleavable C-terminal hexahistidine tag to facilitate purification. A synthetic gene for the sole cytochrome P450 reductase (Tetur18g03390) in TSSM also became available. RNAi induced by the chimeric dsRNA targeting CYP392A13v2, CYP392E2, CYP392E4 showed their requirement for mite adaptation status to Arabidopsis. Comparative analysis of enzymatic properties of these P450s relative to other P450s available using candidate substrates and plant extracts will be used to characterize their substrate specificity. Identification of plant defense substrates will be attempted. Analysis of substrate binding to A13v2 CYPs are believed to play an essential role in detoxifying xenobiotics and plant secondary metabolites in TSSM. Avermectin B1 is a globally popular acaricide to fight against two-spotted spider mite (TSSM) infestation. Riga, M. et al. have shown that CYP392A16, a generalist cytochrome P450 found in TSSM, can catalyze the hydroxylation of avermectin B1. However, they did not characterize the implications of avermectin B1 binding to CYP392A16. Herein we have determined the binding affinity of Avermectin with A13v2. Some cytochrome P450s undergo a spectroscopic shift of the soret maxima upon substrate binding. Generally, the soret peak shifts from ~418 nm to ~390 nm. This optical change in the soret maxima is caused by the change in the redox state of iron (III). The aqua-ligated octahedral iron (III) heme b complex usually has a very low redox potential (~300 mV; low-spin or spin-paired Fe3+ form). The substrate molecules often replace the distal water molecule from the active site. This makes the redox potential of the CYP more positive (~225 mV; high-spin or spin-free Fe3+ form), and iron (III) takes up a penta-coordinated geometry. Avermectin can induce the same optical change on A13v2 . Though only ~15% of the low-spin iron (III) was converted into the high-spin form, the optical change was enough to be detected on the UV-Vis spectra of the binding titration. The absorbance changes at 390 nm (high-spin form) and 418 nm (low-spin form) were added and plotted against the total Avermectin concentration. The data points were fitted with a Morrison quadratic equation, and the dissociation constant (KD) of Avermectin for A13v2 was very low, only 1.5±0.4 µM . Hexythiazox is a miticide and is often used to eradicate TSSM infestation. In order to determine the KD of Hexythiazox inhibition to A13v2, a binding titration assay was carried out. But Hexythiazox did not impart any optical change upon binding to A13v2. No detectable change in absorbance was observed at ~390 nm, and therefore KD of Hexythiazox inhibition could not be determined. Avermectin B1 (molecular weight=873.1 g/mol) has a very bulky structure compared to Hexythiazox (molecular weight=352.9 g/mol), and probably the former molecule was able to make strong interactions in the active site, ultimately displacing the active-site water ligand from the low-spin resting state of A13v2. Looking at the low percentage of high-spin conversion (~15%) when fully saturated with Avermectin, it is likely that CYP392A13v2 has a low propensity for the high-spin state in general. Tetur06g04520 (CYP392A16) We have chosen CYP392A16, a P450 that has been shown to be associated with abamectin resistance in T. urticae1, to pilot expression trials for our remaining target CYPs. After unsuccessful attempts at expression and purification of our original N-terminal truncated constructs with all target CYPs but CYP392A13v2, we have designed two new CYP392A16 constructs. These designs, shown below, are based on modifications of other membrane-associated plant or mammalian CYPs that have proved successful in producing soluble, active P450s. TuGSTs In parallel with work on the original proposal target, we have worked on other T. urticae proteins that belong to glutathione S-transferase family. Genome analysis of T. urticae revealed the presence of a set of 32 genes that code for secreted proteins belonging to the GST family of enzymes. A number of recombinant GSTs are now available, including tetur05g05300 TuGSTm12, tetur12g03900 TuGSTo02, tetur01g02510 TuGSTd05, tetur05g05260 TuGSTm09, tetur01g02230 TuGSTd01 - associated with mite adaptation to Arabidopsis, tetur05g05220 TuGSTm06, tetur26g02802 TuGSTd10, tetur29g00220 TuGSTd14 - xenobiotically-induced GSTs. Such a panel of GSTs should enable us to compare their enzymatic properties and substrate specificities, testing the hypothesis that host-associated GSTs may have narrower substrate ranges relative to xenobiotically-induced ones. In addition, the identification of plant defense substrates will be attempted. Silencing of some of the GSTs indicate that they are required for mite adaptation to Arabidopsis. TuUGTs A number of recombinant UGTs are now available, including: Tetur02g09850 (UGT204B2), Tetur04g02350 (UGT203A2), Tetur05g05050 (UGT201B9), Tetur22g00270 (UGT202A2), Tetur22g00440 (UGT202A15), Tetur07g06390 (UGT201B11). Tetur07g06390 (D4) and tetur08g00190 (D5) are associated with mite adaptation to Arabidopsis and are required for the adaptation of Col-a mites to Arabidopsis. We have crystallized several of UGTs both alone, as well as in complexes with substrates. In addition, we have started their enzymatic characterization. Similarly, as in the case of GSTs and ID-RCDs, we plan to perform identification of TuUGTs natural substrates using Arabidopsis or tomato plant extracts.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
1. Leily Daneshian, Isabella Renggli, Ryan Hanaway, Lesa R. Offermann, Caleb R. Schlachter, Ricardo A. Hernandez, Shannon Henry, Rahul Prakash, Nicky Wybouw, Wannes Dermauw, Linda S. Shimizu, Thomas Van Leeuwen, Thomas Makris, Vojislava Grbic, Miodrag Grbic, Maksymilian Chruszcz (2022) Structural and Functional Characterization of ?-Cyanoalanine Synthase from Tetranychus urticae. Insect Biochemistry and Molecular Biology 142, 103722. DOI: 10.1016/j.ibmb.2022.103722
- Type:
Journal Articles
Status:
Accepted
Year Published:
2022
Citation:
2. Sameer Dixit, Emilie Widemann, Nicolas Bensoussan, Golnaz Salehipourshirazi, Kristie Bruinsma, Maja Milojevic, Akanchha Shukla, Luis C Romero, Vladimir Zhurov, Mark A Bernards, Maksymilian Chruszcz, Miodrag Grbi?, Vojislava Grbi? (2022) ?-Cyanoalanine Synthase Protects Mites against Arabidopsis Defenses.
Plant Physiology kiac147. DOI: 10.1093/plphys/kiac147.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2022
Citation:
3. Christine Njiru, Wenxin Xue, Sander De Rouck, Juan Alba, Merijn Kant, Maksymillian Chrusz, Bartel Vanholme, Wannes Dermauw, Nicky Wybouw, Thomas Van Leeuwen (2022) Intradiol ring cleavage dioxygenases from herbivorous spider mites, a new detoxification enzyme family in animals. BMC Biology
- Type:
Journal Articles
Status:
Other
Year Published:
2022
Citation:
4. Kettlin Ruffatto, Camila Rockenbach da Silva, Eduardo Vieira de Souza, Ricardo Hernandez Arriaza, Maksymilian Chruszcz, Raul Antonio Sperotto, Luis Fernando Saraiva Macedo Timmers (2022) Identification of natural compounds able to inhibit Tetranychus urticae-specific proteins aiming to develop new bioacaricides. - final stages of preparation
|
Progress 05/15/20 to 05/14/21
Outputs
Target Audience:Our primary target audience are researchers working on organisms from the Arthropoda phylum with a special emphasis on researchers working on herbivory mites. Moreover, as significant fraction of our targets is involved in pesticide/acaricide resistance or metabolism we target as well specialist involved in pest management. Changes/Problems:There were several developments that had some impact on our productivity. First of all, Dr. Makris group has moved from the UofSC to the NC State University, and as a result of the delay in grant transfer, only had less than 6 months to spend the first installment of the grant. Many of our costs are in the summer (e.g. salaries for myself, lab members, and travel registrations for conferences). The timing of the award meant that no graduate students were directly supported by the grant for salary, but through NCSU startup. Research expenditures were impacted by the move and gradual phasing of experimental work due to COVID. For example, some gene synthesis has taken 6 months (from order to arrival). Therefore, I expect to use the full year's funding allocation in the upcoming years as my group expands. Moreover, some of our activities were affected by COVID-19 pandemic. The impact of the pandemic was mainly related to limited access to laboratories and facilities. This was especially affecting the groups from Western University. As already mentioned, the pandemic significantly affected production of synthetic DNA. Usually, production of synthetic DNA coding for protein of interest takes 2-3 weeks. However, during the last year the commercial companies that we were using for DNA synthesis were operating significantly slower and the delivery time was usually 2-3 months, instead of weeks. As we had to wait with recombinant protein production, work on some proteins from our list is somewhat delayed. However, we expect to be on the originally planned track within the next half a year. What opportunities for training and professional development has the project provided?Since May 15th 2020, there have been several graduate and undergraduate students who have worked on this project in the Chruszcz group. The students worked on production of recombinant versions of the target enzymes, and were involved in characterization of these proteins. We were mainly focused on characterization of enzymatic activity and structure of the target proteins, which subsequently was used to identify physiological substrates of the enzyme and their biological roles. Therefore, the students had an opportunity to become familiar with various biochemical and biophysical methods. The methods/techniques used included DNA manipulation, bacterial protein expression protein purification, spectroscopic methods for testing enzyme activity, mass spectrometry, X-ray crystallography, etc. Student from the Chruszcz group working on this project during the past funding period: Leily Daneshian - graduate student who finished in May 2021 Ricardo Hernandez Arriaza (9 calendar months) - currently 1st year Chemistry and Biochemistry graduate student Kriti Khatri (3 calendar months) - currently 2nd year Chemistry & Biochemistry graduate student Andrea O'Malley (3 calendar months) - currently 2nd year Chemistry & Biochemistry graduate student Isabella Renggli (4.5 calendar months) - undergraduate student who graduated in December 2020; - Biochemistry and Molecular Biology major Shannon Henry (4.5 months) - undergraduate student and Biochemistry and Molecular Biology major The results originated from this grant are also used in teaching of undergraduate and graduate students. For example, Dr. Chruszcz was teaching CHEM 753 (Protein Chemistry and Enzymology - graduate level class) and CHEM 759 (Macromolecular Crystallography - graduate level class) courses in Fall 2020 and Spring 2021, respectively. Both courses have parts dedicated to enzymes, therefore experimental data generated for proteins being in focus of this proposal were used in both these classes. In the Makris laboratory, two graduate students were involved in the project in the past funding period: Suman Das (12 calendar months) - Biochemistry PhD graduate student (currently 4th year) Hannah Gering (6 calendar months) - Biochemistry PhD graduate student (currently 1st year) The Makris laboratory has remained committed to the training of undergraduate students. Our normal level of involvement has obviously been limited due to restrictions of undergraduates in research labs due to COVID and our move to NCSU. However, two undergraduate students have just joined and will participate in the project for the next funding period. CYPs In order to identify natural substrates of the selected TSSM proteins, we have performed bioinformatics analyses of the 17 CYPs identified in the original proposal and analyzed substrate recognition sites (SRSs) to ascertain whether certain CYPs may have different substrate-scopes and metabolic profiles. We ordered synthetic genes optimized for E. coli expression for the following CYPs where the N-terminal transmembrane domain was truncated to enhance expression and solubilization (CYP392D8, CYP392A16, CYP392A1, CYP392A13v2, CYP392E2, CYP392E4), and a TEV-cleavable C-terminal hexahistidine tag was added to facilitate purification. A synthetic gene for the sole cytochrome P450 reductase (Tetur18g03390) in TSSM was also ordered. Adopting a similar subcloning approach for all of the aforementioned genes, we have subcloned these targets into 3 promoter systems (T5, T7 and a double taq promoter). Sequence verification and expression screening are in progress. How have the results been disseminated to communities of interest?We have published one manuscript and we have two other manuscripts in the final stages of preparation. Moreover, one PhD dissertation was finalized. The dissertation is not yet available for the public, as we have asked to put an embargo on its release. This is related to the fact that the dissertation contains a significant amount of data that is currently used to prepare the publications. The results from our work were also disseminated in a form of seminars and oral presentations. All these presentations were delivered remotely. The Chruszcz group gave two seminars at the UofSC, and one oral presentation during the Discover UofSC event. Moreover, the work performed by our group was presented during seminars in the following institutions: Imanis Life Sciences (December 2020) Terrray Therapeutics (December 2020) Stanford University (December 2020) Rutgers University (February 2021) BP (April 2021) The listed presentations allowed Leily Daneshian to secure three job offers. Dr. Daneshian has decided to pursue her academic career as a postdoctoral research associate at the Department of Biology at Stanford University. Unfortunately, engagement in conferences and seminars has been limited in the current funding period due to travel restrictions and cancellations of numerous planned engagements (e.g. Pacifichem, Eurobic, ICCP450). In lieu, Dr. Makris has engaged in several local presentations to the NCSU Departments of Molecular and Structural Biochemistry and Chemistry in several graduate recruiting seminars. In addition, the PI met with three undergraduates from the CH203 course for undergraduate (freshmen) in the Department of Chemistry. In summary, during the last funding period there were seven oral presentations that were delivered for researchers from three different universities and three different commercial companies. Intradiol ring-cleavage dioxygenases (ID-RCDs) Two ID-RCDs (Tetur06g00450 and Tetur07g06560) are on our target list. Tetur06g00450 is associated with host shift, while Tetur07g06560 is reported to be important for host adaptation (both data are for Arabidopsis). We have produced three different recombinant forms of Tetur06g00450 that have a polyhistidine tag located on N- or C-terminal end, or without any additional tags. In addition, to increase protein solubility and yield of the production we have added N-terminal maltose-binding protein to the construct with C-terminal polyhistidine tag. Currently we work on optimization of this protein expression and purification. We are already able to produce Tetur07g06560 in significant quantities. The protein is active and we were able to determine its basic kinetic parameters. In addition, we have determined optimal pH conditions for activity of this ID-RCD (Fig. 5). Interestingly, this ID-RCD displays the highest activity in a lower pH range in comparison with the previously characterized by us Tetur07g02040 (Schlachter et al. 2019). What do you plan to do during the next reporting period to accomplish the goals?We will continue to work on enzymes that we started to characterize during the first reporting period, and in addition we will focus more on cytochrome P450 family of proteins. We will characterize the six CYPs and CPR which we have cloned during the past funding period. Our first stage of the project will be to screen the ability of the CYPs to metabolize acaricides (e.g. avermectin) or plant metabolites as indicated by the metabolomics approaches performed by the Grbic groups. The binding of these metabolites will first be performed by simple optical spectroscopic while the metabolism will be followed both by O2 consumption and downstream LC-MS to look for oxygen-integration into the products. We will also attempt to solve the structures of one member of each subfamily (392A, 392D, and 392E). In addition, we will work on identification of physiological substrates for ID-RCDs. In parallel to the work on recombinant proteins we will perform gene silencing experiments, develop antibodies for some of the protein target, and perform in situ hybridization for these proteins. We will continue studies of pH in TSSM gut.
Impacts
What was accomplished under these goals?
We have started characterization of several proteins from our target list. Currently we work on: Tetur10g01570 (TuCAS), Terur06g00450 (ID-RCD), Tetur07g06560 (ID-RCD), Tetur03g05070 (CYP392D8), Tetur06g04520 (CYP392A16), Tetur07g06410 (CYP392A1), Tetur03g00020 (CYP392A13v2), Tetur06g02400 (CYP392E2), Tetur27g02598 (CYP392E4). A more detailed description of the research progress on particular group of proteins is provided below. TuCAS Among genes correlated with mite adaptation to indole glucosinolates we identified TuCAS, a gene encoding an enzyme with cysteine and β-cyanoalanine synthase activities (Wybouw et al., 2014). We showed that TuCAS is required for mite adaptation to indole glucosinolates through its β-cyanoalanine synthase activity. Consistent with β-cyanoalanine synthase's role in detoxification of hydrogen cyanide, we demonstrated that upon mite herbivory, Arabidopsis plants generate hydrogen cyanide through the indole glucosinolate pathway. TuCAS is constitutively expressed in adult spider mites (Fig. 1A-C). Using dsRNA-TuCAS, we silenced TuCAS expression in Arabidopsis-adapted mites (Fig. 1D). Reduced expression of TuCAS resulted in a significant reduction of mite fecundity when fed on the Col-0 wild-type leaves. When mites treated with dsRNA-TuCAS fed on cyp79b2 cyp79b3 leaves, there was a modest but significant decrease of their fecundity (14% reduction), while reduced TuCAS expression did not affect mite fitness when they infested bean leaves (Fig. 1E). Thus, we established the requirement of the high expression of TuCAS to counteract Trp-derived Arabidopsis defenses. The TuCAS is a bifunctional enzyme that catalyzes cysteine synthesis and synthesises of β-cyanoalanine from cyanide (HCN) and cysteine. We showed that cysteine synthase activity of TuCAS is not required for mite adaptation to Arabidopsis, but that TuCAS β-cyanoalanine synthase activity is required for cyanide resistance of Col-a mites (data not shown). While overexpression of TuCAS confers mite tolerance to cyanide, the existence and the source of defensive cyanide against mite herbivory in Arabidopsis are not known. We were able to detect cyanide, whose levels increase upon mite feeding, in Arabidopsis leaves (Fig. 2A). As seen in Figure 2B and C, mite-induced cyanide is formed through indole glucosinolate pathway. The significant decrease in fecundity of Arabidopsis-adapted mites treated with dsRNA-TuCAS upon feeding on I3M-supplemented cyp79b2 cyp79b3 leaves, Fig. 2C, demonstrates that TuCAS protects Arabidopsis-adapted mites against I3M-dependent cyanide defenses. In addition, the structure and enzymatic properties of TuCAS were studied. The crystal structures of TuCAS with pyridoxal phosphate (PLP) linked to lysine residues were determined. These structures reveal an extensive TuCAS homology with the β-substituted alanine synthase family, and this enzyme utilizes a similar chemical mechanism involving a stable α-aminoacrylate intermediate. It was demonstrated that TuCAS is more efficient in the synthesis of β-cyanoalanine, which is a product of the reaction between cysteine and cyanide, than in the synthesis of cysteine that uses O-acetyl-L-serine and sulfide. We have also found that TuCAS can detoxify cyanide using O-acetyl-L-serine as the substrate, and this not described previously reaction leads to the formation of β-cyanoalanine (Fig. 3). Moreover, we have shown that the spider mite enzyme catalyzes the reaction between the TuCAS-bound α-aminoacrylate intermediate and aromatic compounds with the thiol group. In addition, we have tested several TuCAS inhibitors (Table 1), and some of them may be used to develop a new generation of T. urticae targeting acaricides. In order to provide insights into physiological function of TuCAs, we have also performed a detailed characterization of enzyme activity with a special emphasis on pH conditions that are optimal for the enzyme activity (Fig. 4). The information on pH-dependent activity profiles of detoxification enzymes will be used in combination with data on pH values in various parts of T. urticae's digestive system. TuCAS is the first enzyme of this type that has its structure determined (Fig. 5) and originates from a eukaryotic organism that is not a plant. TuCAS is very similar to the homolog from Tetranychus evansi (93% sequence identity), as well as other arthropod and insect CASs that originate from various agricultural pests, like Pieris rapae (67% sequence identity) Intradiol ring-cleavage dioxygenases (ID-RCDs) Two ID-RCDs (Tetur06g00450 and Tetur07g06560) are on our target list. Tetur06g00450 is associated with host shift, while Tetur07g06560 is reported to be important for host adaptation (both data are for Arabidopsis). We have produced three different recombinant forms of Tetur06g00450 that have a polyhistidine tag located on N- or C-terminal end, or without any additional tags. In addition, to increase protein solubility and yield of the production we have added N-terminal maltose-binding protein to the construct with C-terminal polyhistidine tag. Currently we work on optimization of this protein expression and purification. We are already able to produce Tetur07g06560 in significant quantities. The protein is active and we were able to determine its basic kinetic parameters. In addition, we have determined optimal pH conditions for activity of this ID-RCD (Fig. 5). Interestingly, this ID-RCD displays the highest activity in a lower pH range in comparison with the previously characterized by us Tetur07g02040 (Schlachter et al. 2019). CYPs In order to identify natural substrates of the selected TSSM proteins, we have performed bioinformatics analyses of the 17 CYPs identified in the original proposal and analyzed substrate recognition sites (SRSs) to ascertain whether certain CYPs may have different substrate-scopes and metabolic profiles. We ordered synthetic genes optimized for E. coli expression for the following CYPs where the N-terminal transmembrane domain was truncated to enhance expression and solubilization (CYP392D8, CYP392A16, CYP392A1, CYP392A13v2, CYP392E2, CYP392E4), and a TEV-cleavable C-terminal hexahistidine tag was added to facilitate purification. A synthetic gene for the sole cytochrome P450 reductase (Tetur18g03390) in TSSM was also ordered. Adopting a similar subcloning approach for all of the aforementioned genes, we have subcloned these targets into 3 promoter systems (T5, T7 and a double taq promoter). Sequence verification and expression screening are in progress. TuGSTd01 In parallel with work on the original proposal target, we have worked on another T. urticae protein that belongs to glutathione S-transferase family. Genome analyses of T. urticae revealed the presence of a set of 32 genes that code for secreted proteins belonging to the GST family of enzymes. To better understand the role of these proteins in T. urticae, we have functionally characterized TuGSTd01. Several reports associated increased GSTs activity with T. urticae resistance to some acaricides. However, we found that TuGSTd01 is not able to detoxify abamectin; in fact, the acaricide inhibits the enzyme with Ki = 101 µM (Fig. 7). Therefore, we suggest that the increased GST activity observed in abamectin-resistant T. urticae field populations is a part of the compensatory feedback loop.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
1. Leily Daneshian, Caleb Schlachter, Lu�s Fernando Saraiva Macedo Timmers, Taylor Radford, Brenda Kapingidza, Travis Dias, Jana Liese, Raul Antonio Sperotto, Vojislava Grbic, Miodrag Grbic, Maksymilian Chruszcz (2021) Delta class glutathione S-transferase (TuGSTd01) from the two-spotted spider mite Tetranychus urticae is inhibited by abamectin. Pesticide Biochemistry and Physiology, 176, 104873 (DOI: 10.1016/j.pestbp.2021.104873)
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2021
Citation:
2. Sameer Dixit, Golnaz Salehipourshirazi, Kristie Bruinsma, Maja Milojevic, Luis C. Romero, Nicolas Bensoussan, Emilie Widemann, Vladimir Zhurov, Maksymilian Chruszcz, Miodrag Grbi?, Vojislava Grbi? (2021) TuCAS enables Tetranychus urticae to counteract indole glucosinolate dependent cyanide Arabidopsis defenses. final stages of preparation will be submitted in July
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2021
Citation:
3. Leily Daneshian, Isabella Renggli, Ryan Hanaway, Lesa R. Offermann, Caleb R. Schlachter, Ricardo A. Hernandez, Shannon Henry, Rahul Prakash, Nicky Wybouw, Wannes Dermauw, Linda S. Shimizu, Thomas Van Leeuwen, Thomas Makris, Vojislava Grbic, Miodrag Grbic, Maksymilian Chruszcz (2021) Structural and Functional Characterization of ?-Cyanoalanine Synthase from Tetranychus urticae. final stages of preparation; the manuscript will be submitted in July to Insect Biochemistry and Molecular Biology
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