Structure-guided engineering of a Thermobifida fusca cutinase for enhanced hydrolysis on natural polyester substrate

Abstract

Cutinases could degrade insoluble polyester, including natural cutin and synthetic plastic. However, their turnover efficiency for polyester remains too low for industrial application. Herein, we report the 1.54-Å resolution X-ray crystal structure of a cutinase from Thermobifida fusca and modeling structure in complex with a cutin mimic oligo-polyester C₂₄H₄₂O₈. These efforts subsequently guided our design of cutinase variants with less bulky residues in the vicinity of the substrate binding site. The L90A and I213A variants exhibit increased hydrolysis activity (5- and 2.4-fold, respectively) toward cutin and also showed enhanced cotton scouring efficiency compared with the wild-type enzyme.

Keywords

Cutinase / Polyester cutin / Enzyme engineering / Cotton scouring / Loop flexibility

References

[1] Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung LW, Kapral GJ, Grosse-Kunstleve RW, McCoy AJ, Moriarty NW, Oeffner R, Read RJ, Richardson DC, Richardson JS, Terwilliger TC, Zwart PH. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr, 2010, 66(Pt 2): 213-221.

[2] Araujo R, Silva C, O’Neill A, Micaelo N, Guebitz G, Soares CM, Casal M, Cavaco-Paulo A. Tailoring cutinase activity towards polyethylene terephthalate and polyamide 6,6 fibers. J Biotechnol, 2007, 128(4): 849-857.

[3] Austin HP, Allen MD, Donohoe BS, Rorrer NA, Kearns FL, Silveira RL, Pollard BC, Dominick G, Duman R, El Omari K, Mykhaylyk V, Wagner A, Michener WE, Amore A, Skaf MS, Crowley MF, Thorne AW, Johnson CW, Woodcock HL, McGeehan JE, Beckham GT. Characterization and engineering of a plastic-degrading aromatic polyesterase. Proc Natl Acad Sci USA, 2018, 115(19): E4350-E4357.

[4] Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS, Read RJ, Rice LM, Simonson T, Warren GL. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr, 1998, 54(Pt 5): 905-921.

[5] Chen S, Tong X, Woodard RW, Du G, Wu J, Chen J. Identification and characterization of bacterial cutinase. J Biol Chem, 2008, 283(38): 25854-25862.

[6] Chen S, Su L, Billig S, Zimmermann W, Chen J, Wu J. Biochemical characterization of the cutinases from Thermobifida fusca. J Mol Catal B Enzym, 2010, 63(3–4): 121-127.

[7] Chen VB, Arendall WB 3rd, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, Murray LW, Richardson JS, Richardson DC. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr, 2010, 66(Pt 1): 12-21.

[8] Delano WL (2012) The PyMOL molecular graphics system, Version 1.50

[9] Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of coot. Acta Crystallogr D Biol Crystallogr, 2010, 66: 16.

[10] Eric Barth KK, Leimkuhler Benedict, Skeel Robert D. Algorithms for constrained molecular dynamics. J Comput Chem, 1995, 16(10): 18.

[11] Fecker T, Galaz-Davison P, Engelberger F, Narui Y, Sotomayor M, Parra LP, Ramirez-Sarmiento CA. Active site flexibility as a hallmark for efficient PET degradation by I. sakaiensis PETase. Biophys J, 2018, 114(6): 1302-1312.

[12] Friesner RA, Banks JL, Murphy RB, Halgren TA, Klicic JJ, Mainz DT, Repasky MP, Knoll EH, Shelley M, Perry JK, Shaw DE, Francis P, Shenkin PS. Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. J Med Chem, 2004, 47(7): 1739-1749.

[13] Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, Scalmani G, Barone V, Petersson GA, Nakatsuji H, Li X, Caricato M, Marenich A, Bloino J, Janesko BG, Gomperts R, Mennucci B, Hratchian HP, Ortiz JV, Izmaylov AF, Sonnenberg JL, Williams-Young D, Ding F, Lipparini F, Egidi F, Goings J, Peng B, Petrone A, Henderson T, Ranasinghe D, Zakrzewski VG, Gao J, Rega N, Zheng G, Liang W, Hada M, Ehara M, Toyota K, Fukuda R, Hasegawa J, Ishida M, Nakajima T, Honda Y, Kitao O, Nakai H, Vreven T, Throssell K, Montgomery JA Jr, Peralta JE, Ogliaro F, Bearpark M, Heyd JJ, Brothers E, Kudin KN, Staroverov VN, Keith T, Kobayashi R, Normand J, Raghavachari K, Rendell A, Burant JC, Iyengar SS, Tomasi J, Cossi M, Millam JM, Klene M, Adamo C, Cammi R, Ochterski JW, Martin RL, Morokuma K, Farkas O, Foresman JB, Fox DJ. Revision A.02, 2016, Wallingford CT: Gaussian Inc.

[14] Genheden S, Ryde U. The MM/PBSA and MM/GBSA methods to estimate ligand-binding affinities. Expert Opin Drug Discov, 2015, 10(5): 449-461.

[15] Greenwood JR, Calkins D, Sullivan AP, Shelley JC. Towards the comprehensive, rapid, and accurate prediction of the favorable tautomeric states of drug-like molecules in aqueous solution. J Comput Aided Mol Des, 2010, 24(6–7): 591-604.

[16] Han X, Liu W, Huang JW, Ma J, Zheng Y, Ko TP, Xu L, Cheng YS, Chen CC, Guo RT. Structural insight into catalytic mechanism of PET hydrolase. Nat Commun, 2017, 8(1): 2106.

[17] Harder E, Damm W, Maple J, Wu C, Reboul M, Xiang JY, Wang L, Lupyan D, Dahlgren MK, Knight JL, Kaus JW, Cerutti DS, Krilov G, Jorgensen WL, Abel R, Friesner RA. OPLS3: a force field providing broad coverage of drug-like small molecules and proteins. J Chem Theory Comput, 2016, 12(1): 281-296.

[18] Hegde K, Dasu VV. Structural stability and unfolding properties of cutinases from Thermobifida fusca. Appl Biochem Biotechnol, 2014, 174(2): 803-819.

[19] Huang J, Rauscher S, Nawrocki G, Ran T, Feig M, de Groot BL, Grubmuller H, MacKerell AD Jr. CHARMM36m: an improved force field for folded and intrinsically disordered proteins. Nat Methods, 2017, 14(1): 71-73.

[20] Humphrey W, Dalke A, Schulten K (1996). VMD: visual molecular dynamics. J Mol Graph 14(1), 33–38, 27–38

[21] Joo S, Cho IJ, Seo H, Son HF, Sagong HY, Shin TJ, Choi SY, Lee SY, Kim KJ. Structural insight into molecular mechanism of poly(ethylene terephthalate) degradation. Nat Commun, 2018, 9(1): 382.

[22] Kitadokoro K, Thumarat U, Nakamura R, Nishimura K, Karatani H, Suzuki H, Kawai F. Crystal structure of cutinase Est119 from Thermobifida alba AHK119 that can degrade modified polyethylene terephthalate at 1.76Å resolution. Polym Degrad Stabil, 2012, 97(5): 771-775.

[23] Kolattukudy PE. Polyesters in higher plants. Adv Biochem Eng Biotechnol, 2001, 71: 1-49.

[24] Liu Z, Gosser Y, Baker PJ, Ravee Y, Lu Z, Alemu G, Li H, Butterfoss GL, Kong XP, Gross R, Montclare JK. Structural and functional studies of Aspergillus oryzae cutinase: enhanced thermostability and hydrolytic activity of synthetic ester and polyester degradation. J Am Chem Soc, 2009, 131(43): 15711-15716.

[25] Martinez C, Nicolas A, van Tilbeurgh H, Egloff MP, Cudrey C, Verger R, Cambillau C. Cutinase, a lipolytic enzyme with a preformed oxyanion hole. Biochemistry, 1994, 33(1): 83-89.

[26] Nyyssola A. Which properties of cutinases are important for applications?. Appl Microbiol Biotechnol, 2015, 99(12): 4931-4942.

[27] Oda M, Yamagami Y, Inaba S, Oida T, Yamamoto M, Kitajima S, Kawai F. Enzymatic hydrolysis of PET: functional roles of three Ca(2+) ions bound to a cutinase-like enzyme, Cut190*, and its engineering for improved activity. Appl Microbiol Biotechnol, 2018, 102(23): 10067-10077.

[28] Otwinowski Z, Minor W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol, 1997, 276: 307-326.

[29] Pronk S, Pall S, Schulz R, Larsson P, Bjelkmar P, Apostolov R, Shirts MR, Smith JC, Kasson PM, van der Spoel D, Hess B, Lindahl E. GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit. Bioinformatics, 2013, 29(7): 845-854.

[30] Purdy RE, Kolattukudy PE. Depolymerization of a hydroxy fatty acid biopolymer, cutin, by an extracellular enzyme from Fusarium solani f. pisi: isolation and some properties of the enzyme. Arch Biochem Biophys, 1973, 159(1): 61-69.

[31] Purdy RE, Kolattukudy PE. Hydrolysis of plant cuticle by plant pathogens. Properties of cutinase I, cutinase II, and a nonspecific esterase isolated from Fusarium solani pisi. Biochemistry, 1975, 14(13): 2832-2840.

[32] Ribitsch D, Acero EH, Greimel K, Eiteljoerg I, Trotscha E, Freddi G, Schwab H, Guebitz GM. Characterization of a new cutinase from Thermobifida alba for PET-surface hydrolysis. Biocatal Biotransform, 2011, 30(1): 2-9.

[33] Ribitsch D, Altijana HA, Zitzenbacher S, Zartl B, Gamerith C, Pellis A, Jungbauer A, Łyskowski A, Steinkellner G, Gruber K, Tscheliessnig R, Acero EH, Guebitz GM. Small cause, large effect: structural characterization of cutinases from Thermobifida cellulosilytica. Biotechnol Bioeng, 2017, 114(11): 2481-2488.

[34] Saavedra JM, Azocar MA, Rodriguez V, Ramirez-Sarmiento CA, Andrews BA, Asenjo JA, Parra LP. Relevance of local flexibility near the active site for enzymatic catalysis: biochemical characterization and engineering of cellulase Cel5A from Bacillus agaradherans. Biotechnol J, 2018, 13(8): e1700669.

[35] Schrödinger LS, LLC (2015) Maestro. Schrödinger, LLC, New York

[36] Silva C, Da S, Silva N, Matama T, Araujo R, Martins M, Chen S, Chen J, Wu J, Casal M, Cavaco-Paulo A. Engineered Thermobifida fusca cutinase with increased activity on polyester substrates. Biotechnol J, 2011, 6(10): 1230-1239.

[37] Sirin S, Kumar R, Martinez C, Karmilowicz MJ, Ghosh P, Abramov YA, Martin V, Sherman W. A computational approach to enzyme design: predicting omega-aminotransferase catalytic activity using docking and MM-GBSA scoring. J Chem Inf Model, 2014, 54(8): 2334-2346.

[38] Sondergaard CR, Olsson MH, Rostkowski M, Jensen JH. Improved treatment of ligands and coupling effects in empirical calculation and rationalization of pKa values. J Chem Theory Comput, 2011, 7(7): 2284-2295.

[39] Tournier V, Topham CM, Gilles A, David B, Folgoas C, Moya-Leclair E, Kamionka E, Desrousseaux ML, Texier H, Gavalda S, Cot M, Guemard E, Dalibey M, Nomme J, Cioci G, Barbe S, Chateau M, Andre I, Duquesne S, Marty A. An engineered PET depolymerase to break down and recycle plastic bottles. Nature, 2020, 580(7802): 216-219.

[40] Vanommeslaeghe K, Raman EP, MacKerell AD Jr. Automation of the CHARMM General Force Field (CGenFF) II: assignment of bonded parameters and partial atomic charges. J Chem Inf Model, 2012, 52(12): 3155-3168.

[41] Wang Q, Fan X, Gao W, Chen J. Characterization of bioscoured cotton fabrics using FT-IR ATR spectroscopy and microscopy techniques. Carbohydr Res, 2006, 341(12): 2170-2175.

[42] Wei Y, Swenson L, Castro C, Derewenda U, Minor W, Arai H, Aoki J, Inoue K, Servin-Gonzalez L, Derewenda ZS. Structure of a microbial homologue of mammalian platelet-activating factor acetylhydrolases: Streptomyces exfoliatus lipase at 1.9 A resolution. Structure, 1998, 6(4): 511-519.

[43] Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, Keegan RM, Krissinel EB, Leslie AG, McCoy A, McNicholas SJ, Murshudov GN, Pannu NS, Potterton EA, Powell HR, Read RJ, Vagin A, Wilson KS. Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr, 2011, 67(Pt 4): 235-242.

[44] Yuan S, Chan HCS, Filipek S, Vogel H. PyMOL and inkscape bridge the data and the data visualization. Structure, 2016, 24(12): 2041-2042.

[45] Zhang Y, Chen S, Xu M, Cavaco-Paulo A, Wu J, Chen J. Characterization of Thermobifida fusca cutinase-carbohydrate-binding module fusion proteins and their potential application in bioscouring. Appl Environ Microbiol, 2010, 76(20): 6870-6876.

[46] Zhang Y, Chen S, He M, Wu J, Chen J, Wang Q. Effects of Thermobifida fusca cutinase-carbohydrate-binding module fusion proteins on cotton bioscouring. Biotechnol Bioprocess Eng, 2011, 16(4): 645-653.

Funding

Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences(XDB20000000)

Program of Shanghai Academic Research Leader(19XD1404800)

National Natural Science Foundation of China(11811530637)