مدل سازی و تعیین ساختار و ویژگی‌های آنزیم‌های سیتوکروم P450 در قارچ بیمارگر حشرات بووریا بازیانا
محورهای موضوعی : قارچ شناسی
1 - تنوع زیستی، پژوهشکده علوم محیطی، پژوهشگاه علوم و تکنولوژی پیشرفته و علوم محیطی، دانشگاه تحصیلات تکمیلی صنعتی و فناوری پیشرفته، کرمان
2 - گروه بیوتکنولوژی، پژوهشکده علوم محیطی، پژوهشگاه علوم و تکنولوژی پیشرفته و علوم محیطی، دانشگاه تحصیلات تکمیلی صنعتی و فناوری پیشرفته،
کلید واژه: ساختار سه بعدی, همولوژی, بووریا بازیانا, سیتوکرومP450, توالی حفاظت شده,
چکیده مقاله :
سابقه و هدف: آنزیم‌های سیتوکروم P450خانواده بزرگی از پروتین‌های دارای آهن هستند و نقش مهمی در متابولیسم ترکیبات مختلف دارند.قارچ بیمارگر حشرات، بووریا بازیانا نیز حاوی این آنزیم‌ها برای هدف‌گیری آلکان‌ها در حشرات است. هدف از این پژوهش، بررسی ویژگی‌ها و ساختار پروتینی هفت آنزیم سیتوکروم P450در این قارچ و پیش‌بینی ساختار سه بعدی این آنزیم‌ها با روش مدلسازی هومولوژی بود.مواد و روش‌ها: برای پیش‌بینی احتمال حضور و موقعیت سایت‌های برشی پپتیدهای سیگنال در توالی‌های اسیدآمینه از برنامه SignalP4.1 استفاده شد. برخی پارامترهای فیزیکی و شیمیایی پروتین‌های مورد نظر با برنامه ProtParam و مدل سازی در پایگاه Modebase انجام شد.یافته‌ها: در تمامی توالی‌ها یک ناحیة حفاظت‌شده با حدود 200 اسیدآمینه مرتبط با P450شناسایی شد. این ناحیة حفاظت‌شده در توالی 45-96 تا حدود 477-520 قرار داشت. پروتئین‌های CYP539B5 و CYP52G11 پایدارند. CYP584Q1 بیشترین و CYP617N1 کمترین میزان اسیدآمینه آب دوست را داشت. از بین مدل های انتخاب شده مدلی که دارای کمترین ارزش e و بالاترین پوشش بود، به عنوان بهترین مدل انتخاب شد. CYP5337A1 و CYP52G11 فاقد مارپیچ و CYP617N1 دارای دو مارپیچ بود. تعداد اسیدهای‌آمینه تخمین‌زده‌شده در مارپیچ، در تمام موارد به جز CYP584Q1، CYP5337A1 و CYP52G11 بیشتر از 18 بود. اما هیچ سیگنال پپتیدی شناسایی نشد.نتیجه‌گیری: در این تحقیق، برای اولین بار ساختار‌های سه بعدی هفت آنزیم سیتوکروم P450در قارچ بووریا بازیانا مدل سازی شد. پایداری آنزیم‌ها و مقاومت آن‌ها در مقابل عوامل نامطلوب محیطی مانند دما و اشعة ماورای بنفش می‌تواند میزان بیمارگری قارچ را علیه آفات افزایش دهد.
Background & Objectives: Cytochrome P450 enzymes are a large family of proteins containing haem and play an important role in metabolism of various compounds. Entomopathogenic fungus, Beauveria bassiana also contains these enzymes for targeting insect alkanes. The aim of this study was to investigate the properties and protein structure of seven cytochrome P 450 enzymes in this fungus and to predict the three-dimensional structure of these enzymes by homology modeling method. Materials & Methods: Predicting possible situations with intracellular helices was performed with the TMHMM program. SignalP4.1 program was used to predict the probability of the presence and location of shear sites of signal peptides in amino acid sequences. Some of the physical and chemical parameters of the proteins were performed with the ProtParam program. The modeling was conducted in the Modebase database. Results: In all sequences, a motif was identified and had about 200 amino acids that were related to P450. The motif was in the sequence of 96-45 to about 477-520. CYP539B5 and CYP52G11 proteins were stable. CYP584Q1 had the highest and CYP617N1 had the lowest hydrophilic acid of all. Among the selected models, the model with lowest e-value and the highest coverage was selected as the best model. CYP5337A1 and CYP52G11 lacked helix and CYP617N1 had two helices. Number of estimated amino acids in the helices, in all cases except CYP584Q1, CYP5337A1 and CYP52G11, was greater than 18. No signal peptide was detected. Conclusion: In this study, the modeled structures of seven cytochrome P450 enzymes were reported for the first time in the fungus B. bassiana. The enzymes' stability and their resistance to adverse environmental factors such as temperature and UV can increase the fungal pathogenicity against the pests.
generalist entomopathogenic fungus Beauveria bassiana (Bals.). ungal Genet and Biol. 2007;
44: 1231-1241.
2. aria MRde, Wraight SP. Mycoinsecticides and mycoacaricides: a comprehensive list with
worldwide coverage and international classification of formulation types. Biol Control. 2007;
43: 237-256.
3. Inglis GD, Goettel MS, Butt TM, Strasser H. Use of hyphomycetous fungi for managing insect
pests. In: Butt TM, Jackson C, Magan N, editors. ungi as biocontrol agents: progress,
problems and potential. CABI Publishing; 2001: 23-69.
4. Pedrini N, hang Sh, Juarez MP, Keyhani NO. Molecular characterization and expression
analysis of a suite of cytochrome P450 enzymes implicated in insect hydrocarbon degradation
in the entomopathogenic fungus Beauveria bassiana. Microbiology. 2010; 156: 2549-2557.
5. Stoilov I. Cytochrome P450s: coupling development and environment. Trends Genet. 2001; 17
(11): 629-632.
6. Poulos TL, Johnson E . Structures of Cytochrome P450 Enzymes. In: Montellano PROde,
editors. Cytochrome P450. Springer International Publishing Switzerland; 2015: 3-32.
7. Crooks GE, Hon G, Chandonia JM, Brenner SE. WebLogo: a sequence logo generator. Genome
Res. 2004; 14(6): 1188-1190.
8. Sonnhammer ELL, von Heijne G, Krogh A. A hidden Markov model for predicting
transmembrane helices in protein sequences. In: Glasgow J, Littlejohn T, Major , Lathrop R,
Sankoff D, Sensen C, editors. Proceeding of the Sixth International Conference on intelligent
systems for molecular biology. AAAI press, Menlo Park, CA; 1998: 175-182.
9. Krogh A, Larsson B, von Heigne G, Sonnhammer ELL. Predicting transmembrane protein
topology with a hidden Markov modeling application to complete genomes. J Mol Biol. 2001;
305: 567-580.
10. Petersen TN, Brunak S, von Heijne G, Nielsen H. SignalP 4.0: discriminating signal peptides
from transmembrane regions. Nat Methods. 2011; 8: 785-786.
11. Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, Bairoch A. Protein
identification and analysis tools on the ExPASy server. In: Walker M, editor. The proteomics
protocols Handbook. Humana press; 2005: 571-607.
12. Pieper U, Webb B M, Dong GQ, Schneidman-Duhovny D, an H, Kim SJ, Khuri N, Spill YG,
Weinkam P, Hammel M, Tainer JA, Nilges M, Sali A. ModBase, a database of annotated
comparative protein structure models and associated resources. Nucleic Acids Res. 2014; 42:
D336-46.
13. Guex N, Peitsch MC. SWISS-MODEL and the SWISS-PDBViewer: An environment for
comparative protein modeling. Electrophoresis. 1997; 18: 2714-2723.
14. Sippl MJ. Recognition of errors in three-dimensional structures of proteins. Proteins. 1993; 17:
355–362.
15. Wiederstein M, Sippl MJ. ProSA-web: interactive web service for the recognition of errors in
three-dimensional structures of proteins. Nucleic Acids Res. 2007; 35, doi:10.1093/nar/
gkm290.
16. Schuler M, Duan H, Bilgin M, Ali S. Arabidopsis cytochrome P450s through the looking
glass: a window on plant biochemistry. Phytochem Rev. 2006; 5: 205-237.
17. White SH, Ladokhin AS, Jayasinghe S, Hristova K. How membranes shape protein structure. J
Biol Chem. 2001; 276: 32395-32398.
18. hao B, Moody SC, Hider RC, Lei L, Kelly SL, Waterman MR, Lamb DC. Structural analysis
of cytochrome P450 105N1 involved in the biosynthesis of the incophore, Coelibactin. Int J
Mol Sci. 2012; 13: 8500-8513.
19. Szczesna-Skorupa E, Mallah B, Kemper B. luorescence resonance energy transfer analysis
of cytochromes P450 2C2 and 2E1 molecular interactions in living cells. J Biol Chem. 2003;
278: 31269-31276.
20. Davydov DR. Microsomal monooxygenase as a multienzyme system: the role of P450-P450
interactions. E ert Opin Drug Metab Toxicol. 2011; 7: 543-558.
21. Nagano S, Li H, Shimizu H, Nishida C, Ogura H, Ortiz de Montellano PR, Poulos TL. Crystal
structures of epothilone D-bound, epothilone B-bound, and substrate-free forms of cytochrome
P450epoK. J Biol Chem. 2003; 278: 44886-44893.
22. Watanabe Y, ukuyoshi S, Hiratsuka M, Yamaotsu N, Hirono S, Takahashi O, Oda A.
Prediction of three-dimensional structures and structural flexibilities of wild-type and mutant
cytochrome P450 1A2 using molecular dynamics simulations. J Mol Graph Model. 2016; 68:
48-56.
23. Montellano PROde. Cytochrome P450: structure, mechanism, and biochemistry. New York:
Kluwer Academic/Plenum; 2005.
24. Tanaka A, ukui S. Metabolism of n-alkanes. In: Tanaka A, ukui S, editors. The yeast. New
York: Academic Press; 1989: 261-287.
25. van Beilen JB, Li , Duetz WA, Smits THM,Witholt B. Diversity of alkane hydroxylase
systems in the environment. Oil Gas Sci Technol. 2003; 58: 427-440.
26. Yazdanpanah-Samani M, amani MR, Motallebi M, Moghaddassi Jahromi . Heterologous
expression of Chit36 from Trichoder a atroviride in prokaryotic system. J Cell Mol. Res.
2015; 28(3): 448-457. [In Persian]
_||_
generalist entomopathogenic fungus Beauveria bassiana (Bals.). ungal Genet and Biol. 2007;
44: 1231-1241.
2. aria MRde, Wraight SP. Mycoinsecticides and mycoacaricides: a comprehensive list with
worldwide coverage and international classification of formulation types. Biol Control. 2007;
43: 237-256.
3. Inglis GD, Goettel MS, Butt TM, Strasser H. Use of hyphomycetous fungi for managing insect
pests. In: Butt TM, Jackson C, Magan N, editors. ungi as biocontrol agents: progress,
problems and potential. CABI Publishing; 2001: 23-69.
4. Pedrini N, hang Sh, Juarez MP, Keyhani NO. Molecular characterization and expression
analysis of a suite of cytochrome P450 enzymes implicated in insect hydrocarbon degradation
in the entomopathogenic fungus Beauveria bassiana. Microbiology. 2010; 156: 2549-2557.
5. Stoilov I. Cytochrome P450s: coupling development and environment. Trends Genet. 2001; 17
(11): 629-632.
6. Poulos TL, Johnson E . Structures of Cytochrome P450 Enzymes. In: Montellano PROde,
editors. Cytochrome P450. Springer International Publishing Switzerland; 2015: 3-32.
7. Crooks GE, Hon G, Chandonia JM, Brenner SE. WebLogo: a sequence logo generator. Genome
Res. 2004; 14(6): 1188-1190.
8. Sonnhammer ELL, von Heijne G, Krogh A. A hidden Markov model for predicting
transmembrane helices in protein sequences. In: Glasgow J, Littlejohn T, Major , Lathrop R,
Sankoff D, Sensen C, editors. Proceeding of the Sixth International Conference on intelligent
systems for molecular biology. AAAI press, Menlo Park, CA; 1998: 175-182.
9. Krogh A, Larsson B, von Heigne G, Sonnhammer ELL. Predicting transmembrane protein
topology with a hidden Markov modeling application to complete genomes. J Mol Biol. 2001;
305: 567-580.
10. Petersen TN, Brunak S, von Heijne G, Nielsen H. SignalP 4.0: discriminating signal peptides
from transmembrane regions. Nat Methods. 2011; 8: 785-786.
11. Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, Bairoch A. Protein
identification and analysis tools on the ExPASy server. In: Walker M, editor. The proteomics
protocols Handbook. Humana press; 2005: 571-607.
12. Pieper U, Webb B M, Dong GQ, Schneidman-Duhovny D, an H, Kim SJ, Khuri N, Spill YG,
Weinkam P, Hammel M, Tainer JA, Nilges M, Sali A. ModBase, a database of annotated
comparative protein structure models and associated resources. Nucleic Acids Res. 2014; 42:
D336-46.
13. Guex N, Peitsch MC. SWISS-MODEL and the SWISS-PDBViewer: An environment for
comparative protein modeling. Electrophoresis. 1997; 18: 2714-2723.
14. Sippl MJ. Recognition of errors in three-dimensional structures of proteins. Proteins. 1993; 17:
355–362.
15. Wiederstein M, Sippl MJ. ProSA-web: interactive web service for the recognition of errors in
three-dimensional structures of proteins. Nucleic Acids Res. 2007; 35, doi:10.1093/nar/
gkm290.
16. Schuler M, Duan H, Bilgin M, Ali S. Arabidopsis cytochrome P450s through the looking
glass: a window on plant biochemistry. Phytochem Rev. 2006; 5: 205-237.
17. White SH, Ladokhin AS, Jayasinghe S, Hristova K. How membranes shape protein structure. J
Biol Chem. 2001; 276: 32395-32398.
18. hao B, Moody SC, Hider RC, Lei L, Kelly SL, Waterman MR, Lamb DC. Structural analysis
of cytochrome P450 105N1 involved in the biosynthesis of the incophore, Coelibactin. Int J
Mol Sci. 2012; 13: 8500-8513.
19. Szczesna-Skorupa E, Mallah B, Kemper B. luorescence resonance energy transfer analysis
of cytochromes P450 2C2 and 2E1 molecular interactions in living cells. J Biol Chem. 2003;
278: 31269-31276.
20. Davydov DR. Microsomal monooxygenase as a multienzyme system: the role of P450-P450
interactions. E ert Opin Drug Metab Toxicol. 2011; 7: 543-558.
21. Nagano S, Li H, Shimizu H, Nishida C, Ogura H, Ortiz de Montellano PR, Poulos TL. Crystal
structures of epothilone D-bound, epothilone B-bound, and substrate-free forms of cytochrome
P450epoK. J Biol Chem. 2003; 278: 44886-44893.
22. Watanabe Y, ukuyoshi S, Hiratsuka M, Yamaotsu N, Hirono S, Takahashi O, Oda A.
Prediction of three-dimensional structures and structural flexibilities of wild-type and mutant
cytochrome P450 1A2 using molecular dynamics simulations. J Mol Graph Model. 2016; 68:
48-56.
23. Montellano PROde. Cytochrome P450: structure, mechanism, and biochemistry. New York:
Kluwer Academic/Plenum; 2005.
24. Tanaka A, ukui S. Metabolism of n-alkanes. In: Tanaka A, ukui S, editors. The yeast. New
York: Academic Press; 1989: 261-287.
25. van Beilen JB, Li , Duetz WA, Smits THM,Witholt B. Diversity of alkane hydroxylase
systems in the environment. Oil Gas Sci Technol. 2003; 58: 427-440.
26. Yazdanpanah-Samani M, amani MR, Motallebi M, Moghaddassi Jahromi . Heterologous
expression of Chit36 from Trichoder a atroviride in prokaryotic system. J Cell Mol. Res.
2015; 28(3): 448-457. [In Persian]