Rationally introducing non-canonical amino acids to enhance catalytic activity of LmrR for Henry reaction

Abstract

The use of enzymes to catalyze Henry reaction has advantages of mild reaction conditions and low contamination, but low enzyme activity of promiscuous catalysis limits its application. Here, rational design was first performed to identify the key amino acid residues in Henry reaction catalyzed by Lactococcal multidrug resistance Regulator (LmrR). Further, non-canonical amino acids were introduced into LmrR, successfully obtaining variants that enhanced the catalytic activity of LmrR. The best variant, V15CNF, showed a 184% increase in enzyme activity compared to the wild type, and was 1.92 times more effective than the optimal natural amino acid variant, V15F. Additionally, this variant had a broad substrate spectrum, capable of catalyzing reactions between various aromatic aldehydes and nitromethane, with product yielded ranging from 55 to 99%. This study improved enzymatic catalytic activity by enhancing affinity between the enzyme and substrates, while breaking limited types of natural amino acid residues by introducing non-canonical amino acids into the enzyme, providing strategies for molecular modifications.

Keywords

Henry reaction / Catalytic activity / Rational design / Non-canonical amino acid / Molecular dynamics simulations

References

[1] Agustiandari H, Lubelski J, Den Berg V, Van Saparoea HB, Kuipers OP, Driessen AJM. LmrR is a transcriptional repressor of expression of the multidrug ABC transporter LmrCD in Lactococcus lactis. J Bacteriol, 2008, 190(2): 759-763.

[2] Agustiandari H, Peeters E, de Wit JG, Charlier D, Driessen AJM. LmrR-mediated gene regulation of multidrug resistance in Lactococcus lactis. Microbiology, 2011, 157(5): 1519-1530.

[3] Anastas P, Eghbali N. Green chemistry: principles and practice. ChemInform, 2010

[4] Bos J, Fusetti F, Driessen AJM, Roelfes G. Enantioselective artificial metalloenzymes by creation of a novel active site at the protein dimer interface. Angew Chem Int Ed, 2012, 51(30): 7472-7475.

[5] Bos J, García-Herraiz A, Roelfes G. An enantioselective artificial metallo-hydratase. Chem Sci, 2013

[6] Bos J, García-Herraiz A, Roelfes G. An enantioselective artificial metallo-hydratase. Chem Sci, 2013, 4(9): 3578-3582.

[7] Bos J, Browne WR, Driessen AJM, Roelfes G. Supramolecular assembly of artificial metalloenzymes based on the dimeric protein lmrr as promiscuous scaffold. J Am Chem Soc, 2015, 137(31): 9796-9799.

[8] Chatterjee A, Xiao H, Bollong M, Ai HW, Schultz PG. Efficient viral delivery system for unnatural amino acid mutagenesis in mammalian cells. Proc Natl Acad Sci USA, 2013, 110(29): 11803-11808.

[9] Chin JW, Santoro SW, Martin AB, King DS, Wang L, Schultz PG. Addition of p-azido-L-phenylalanine to the genetic code of Escherichia coli. J Am Chem Soc, 2002, 124(31): 9026-9027.

[10] Ching WM, Kallen RG. Mechanism of carbanion addition to carbonyl compounds. J Am Chem Soc, 1978, 100(19): 6119-6124.

[11] Drienovská I, Alonso-Cotchico L, Vidossich P, Lledós A, Maréchal JD, Roelfes G. Design of an enantioselective artificial metallo-hydratase enzyme containing an unnatural metal-binding amino acid. Chem Sci, 2017, 8(10): 7228-7235.

[12] Dunitz JD, Lehn JM, Wipff G. Stereochemistry of reaction paths at carbonyl centres. Tetrahedron, 1974, 30(12): 1563-1572.

[13] Ferrara S, Mejias SH, Liutkus M, Renno G, Stella F, Kociolek I, Fuenzalida-Werner JP, Barolo C, Coto PB, Cortajarena AL, Costa RD. Designing artificial fluorescent proteins: Squaraine-LmrR biophosphors for high performance deep-red biohybrid light-emitting diodes. Adv Funct Mater, 2022

[14] Fuhshuku K, Asano Y. Synthesis of (R)-β-nitro alcohols catalyzed by R-selective hydroxynitrile lyase from Arabidopsis thaliana in the aqueous–organic biphasic system. J Biotechnol, 2011, 153(3): 153-159.

[15] Georgescu R, Bandara G, Sun L. Arnold FH, Georgiou G. Saturation mutagenesis. Directed evolution library creation: methods and protocols, 2003, Totowa, NJ: Humana Press, 75-83.

[16] Green AP, Hayashi T, Mittl PRE, Hilvert D. A chemically programmed proximal ligand enhances the catalytic properties of a Heme enzyme. J Am Chem Soc, 2016, 138(35): 11344-11352.

[17] Kühbeck D, Dhar BB, Schön EM, Cativiela C, Díaz DD. C-C Bond formation catalyzed by natural gelatin and collagen proteins. Beilstein J Org Chem, 2013, 9(1): 1111-1118.

[18] Laureanti JA, Ginovska B, Buchko GW, Schenter GK, Hebert M, Zadvornyy OA, Peters JW, Shaw WJ. A positive charge in the outer coordination sphere of an artificial enzyme increases CO2 hydrogenation. Organometallics, 2020, 39(9): 1532-1544.

[19] Leveson-Gower RB, Zhou Z, Drienovská I, Roelfes G. Unlocking iminium catalysis in artificial enzymes to create a Friedel-crafts alkylase. ACS Catal, 2021, 11(12): 6763-6770.

[20] Leveson-Gower RB, de Boer RM, Roelfes G. Tandem Friedel-crafts-alkylation-enantioselective-protonation by artificial enzyme iminium catalysis. ChemCatChem, 2022, 14(8

[21] Liu Y, Xu G, Zhou J, Ni J, Zhang L, Hou X, Yin D, Rao Y, Zhao Y-L, Ni Y. Structure-guided engineering of D-carbamoylase reveals a key loop at substrate entrance tunnel. ACS Catal, 2020, 10(21): 12393-12402.

[22] Luzzio FA. The Henry reaction: recent examples. Tetrahedron, 2001, 57(6): 915-945.

[23] Madoori PK, Agustiandari H, Driessen AJM, Thunnissen A-MWH. Structure of the transcriptional regulator LmrR and its mechanism of multidrug recognition. EMBO J, 2008, 28(2): 156-166.

[24] Marcelli T, van der Haas RNS, van Maarseveen JH, Hiemstra H. Asymmetric organocatalytic Henry reaction. Angew Chem Int Ed, 2006, 45(6): 929-931.

[25] Mehl RA, Anderson JC, Santoro SW, Wang L, Martin AB, King DS, Horn DM, Schultz PG. Generation of a bacterium with a 21 amino acid genetic code. J Am Chem Soc, 2003, 125(4): 935-939.

[26] Petukh M, Dai L, Alexov E. SAAMBE: webserver to predict the charge of binding free energy caused by amino acids mutations. Int J Mol Sci, 2016

[27] Roelfes G. LmrR: a privileged scaffold for artificial metalloenzymes. Acc Chem Res, 2019, 52(3): 545-556.

[28] Sánchez DA, Tonetto GM, Ferreira ML. Burkholderia cepacia lipase: a versatile catalyst in synthesis reactions. Biotechnol Bioeng, 2018, 115(1): 6-24.

[29] Schultz KC, Supekova L, Ryu Y, Xie J, Perera R, Schultz PG. A genetically encoded infrared probe. J Am Chem Soc, 2006, 128(43): 13984-13985.

[30] Simoni D, Invidiata FP, Manfredini S, Ferroni R, Lampronti I, Roberti M, Pollini GP. Facile synthesis of 2-nitroalkanols by tetramethylguanidine (TMG)-catalyzed addition of primary nitroalkanes to aldehydes and alicyclic ketones. Tetrahedron Lett, 1997, 38(15): 2749-2752.

[31] Sinéad EM, Moody TS, Maguire AR. Biocatalytic approaches to the Henry (Nitroaldol) reaction. Eur J Org Chem, 2012, 16: 3059-3067.

[32] Takeuchi K, Tokunaga Y, Imai M, Takahashi H, Shimada I. Dynamic multidrug recognition by multidrug transcriptional repressor LmrR. Sci Rep, 2014

[33] Tang RC, Guan Z, He YH, Zhu W. Enzyme-catalyzed Henry (nitroaldol) reaction. J Mol Catal B Enzymatic, 2010, 63(1): 62-67.

[34] Teng H, Chen K, Wang L, Wu J, Xu G. Rational immobilization of l-threonine aldolase from Bacillus nealsonii for efficiently synthesis of l-syn-3-[4- (methylsulfonyl)] phenylserine. Process Biochem, 2023, 130: 685-694.

[35] Trost BM, Yeh VSC, Ito H, Bremeyer N. Effect of ligand structure on the zinc-catalyzed Henry reaction. Asymmetric syntheses of (−)-denopamine and (−)-arbutamine. Org Lett, 2002, 4(16): 2621-2623.

[36] Van Der Berg JP, Madoori PK, Komarudin AG, Thunnissen AM, Driessen AJM, Korolev S. Binding of the lactococcal drug dependent transcriptional regulator LmrR to its ligands and responsive promoter regions. PLoS ONE, 2015, 10(8

[37] Villarino L, Splan KE, Reddem E, Alonso-Cotchico L, Gutiérrez de Souza C, Lledós A, Maréchal JD, Thunnissen AMWH, Roelfes G. An artificial heme enzyme for cyclopropanation reactions. Angew Chem Int Ed, 2018, 57(26): 7785-7789.

[38] Villarino L, Chordia S, Alonso-Cotchico L, Reddem E, Zhou Z, Thunnissen AMWH, Maréchal J-D, Roelfes G. Cofactor binding dynamics influence the catalytic activity and selectivity of an artificial metalloenzyme. ACS Catal, 2020, 10(20): 11783-11790.

[39] Xia Y, Jiang M, Liu M, Zhang Y, Qu H, Xiong T, Huang H, Cheng D, Chen F. Catalytic Syn-selective nitroaldol approach to amphenicol antibiotics: evolution of a unified asymmetric synthesis of (−)-chloramphenicol, (−)-azidamphenicol, (+)-thiamphenicol, and (+)-florfenicol. J Org Chem, 2021, 86(17): 11557-11570.

[40] Yu Y, Cui C, Wang J, Lu Y. Biosynthetic approach to modeling and understanding metalloproteins using unnatural amino acids. Sci China Chem, 2017, 60(2): 188-200.

[41] Yu Z, Zhang Q, Tang H, Xu G. Rationally design and chemical modification: Getting a new and efficient biocatalyst for Henry reaction. Enzyme Microb Technol, 2020, 142.

[42] Zheng W, Chen K, Wang Z, Cheng X, Xu G, Yang L, Wu J. Construction of a highly diastereoselective aldol reaction system with I-threonine aldolase by computer-assisted rational molecular modification and medium engineering. Org Lett, 2020, 22(15): 5763-5767.

[43] Zheng W, Yu H, Fang S, Chen K, Wang Z, Cheng X, Xu G, Yang L, Wu J. Directed evolution of I-threonine aldolase for the diastereoselective synthesis of -hydroxy-α-amino acids. ACS Catal, 2021, 11(6): 3198-3205.

[44] Zhou Z, Roelfes G. Synergistic catalysis in an artificial enzyme by simultaneous action of two abiological catalytic sites. Nat Catal, 2020, 3(3): 289-294.

[45] Zong Z, Gao L, Cai W, Yu L, Cui C, Chen S, Zhang D. Computer-assisted rational modifications to improve the thermostability of β-glucosidase from Penicillium piceum H16. BioEnergy Res, 2015, 8(3): 1384-1390.