Influence of mutations at different distances from the active center on the activity and stability of laccase 13B22

Abstract

Laccases with high catalytic efficiency and high thermostability can drive a broader application scope. However, the structural distribution of key amino acids capable of significantly influencing the performance of laccases has not been explored in depth. Thirty laccase 13B22 mutants with changes in amino acids at distances of 5 Å (first shell), 5–8 Å (second shell), and 8–12 Å (third shell) from the active center were validated experimentally with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as substrate. Twelve of these mutants (first shell, 1; second shell, 4; third shell, 7) showed higher catalytic efficiency than the wild-type enzyme. Mutants D511E and I88L-D511E showed 5.36- and 10.58-fold increases in k_cat/K_m, respectively, with increases in optimal temperature of 15 °C and optimal pH from 7.0 to 8.0. Furthermore, both mutants exhibited greater thermostability compared to the wild-type, with increases of 3.33 °C and 5.06 °C in T_m and decreases of 0.39 J and 0.59 J in total structure energy, respectively. The D511E mutation resides in the third shell, while I88L is in the second shell, and their performance enhancements were attributed to alterations in the rigidity or flexibility of specific protein structural domains. Both mutants showed enhanced degradation efficiency for benzo[a]pyrene and zearalenone. These findings highlight the importance of the residues located far from the active center in the function of laccase (second shell and third shell), suggesting broader implications for enzyme optimization and biotechnological applications.

Keywords

Laccase / Catalytic activity / Structural distance / Mutant / Thermostability / Biological Sciences / Biochemistry and Cell Biology

References

[1] AgrawalK, BhardwajN, KumarB, ChaturvediV, VermaP. Process optimization, purification and characterization of alkaline stable white laccase from myrothecium verrucaria ITCC-8447 and its application in delignification of agroresidues. Int J Biol Macromol, 2019, 125: 1042-1055.

[2] AliM, BhardwajP, IshqiHM, ShahidM, IslamA. Laccase engineering: redox potential is not the only Activity-Determining feature in the metalloproteins. Molecules, 2023, 28: 17.

[3] AnishchenkoI, OvchinnikovS, KamisettyH, BakerD. Origins of Coevolution between residues distant in protein 3D structures. Proc Natl Acad Sci U S A, 2017, 114349122-9127.

[4] BourbonnaisR, PaiceMG, ReidID, LanthierP, YaguchiM. Lignin oxidation by laccase isozymes from trametes versicolor and role of the mediator 2,2’-azinobis(3-ethylbenzthiazoline-6-sulfonate) in kraft lignin depolymerization. Appl Environ Microbiol, 1995, 6151876-1880.

[5] BrodkinHR, NovakWR, MilneAC, D’AquinoJA, KarabacakNM, GoldbergIG, AgarJN, PayneMS, PetskoGA, OndrechenMJ, RingeD. Evidence of the participation of remote residues in the catalytic activity of Co-type nitrile hydratase from Pseudomonas putida. Biochemistry, 2011, 50224923-4935.

[6] ChauhanPS, GoradiaB, SaxenaA. Bacterial Laccase: recent update on production, properties and industrial applications. 3 Biotech, 2017, 75323.

[7] ChenC, GuanB-H, GengQ, ZhengY-C, ChenQ, PanJ, XuJ-H. Enhanced thermostability of Candida ketoreductase by Computation-Based Cross-Regional combinatorial mutagenesis. ACS Catal, 2023, 13117407-7416.

[8] De la MoraE, LovettJE, BlanfordCF, GarmanEF, ValderramaB, Rudino-PineraE. Structural changes caused by radiation-induced reduction and radiolysis: the effect of X-ray absorbed dose in a fungal multicopper oxidase. Acta Crystallogr D Biol Crystallogr, 2012, 68Pt 5564-577.

[9] DingZ, GuanF, XuG, WangY, YanY, ZhangW, WuN, YaoB, HuangH, TullerT, TianJ. MPEPE, a predictive approach to improve protein expression in E. coli based on deep learning. Comput Struct Biotechnol J, 2022, 20: 1142-1153.

[10] DingZ, XuG, MiaoR, WuN, ZhangW, YaoB, GuanF, HuangH, TianJ. Rational redesign of thermophilic PET hydrolase LCCICCG to enhance hydrolysis of high crystallinity polyethylene terephthalates. J Hazard Mater, 2023, 453: 131386.

[11] DurãoP, ChenZ, FernandesAT, HildebrandtP, MurgidaDH, TodorovicS, PereiraMM, MeloEP, MartinsLO. Copper incorporation into Recombinant CotA laccase from Bacillus subtilis: characterization of fully copper loaded enzymes. J Biol Inorg Chem, 2008, 132183-193.

[12] GuJ, XuY, NieY. Role of distal sites in enzyme engineering. Biotechnol Adv, 2023, 63: 108094.

[13] GuanZB, LuoQ, WangHR, ChenY, LiaoXR. Bacterial laccases: promising biological green tools for industrial applications. Cell Mol Life Sci, 2018, 75193569-3592.

[14] JanuszG, PawlikA, Świderska-BurekU, PolakJ, SulejJ, Jarosz-WilkołazkaA, PaszczyńskiA. Laccase properties, physiological functions, and evolution. Int J Mol Sci, 2020, 213966.

[15] JumperJ, EvansR, PritzelA, GreenT, FigurnovM, RonnebergerO, TunyasuvunakoolK, BatesR, ŽídekA, PotapenkoA, BridglandA, MeyerC, KohlSAA, BallardAJ, CowieA, Romera-ParedesB, NikolovS, JainR, AdlerJ, BackT, PetersenS, ReimanD, ClancyE, ZielinskiM, SteineggerM, PacholskaM, BerghammerT, BodensteinS, SilverD, VinyalsO, SeniorAW, KavukcuogluK, KohliP, HassabisD. Highly accurate protein structure prediction with alphafold. Nature, 2021, 5967873583-589.

[16] KataokaK, KogiH, TsujimuraS, SakuraiT. Modifications of laccase activities of copper efflux oxidase, CueO by synergistic mutations in the first and second coordination spheres of the type I copper center. Biochem Biophys Res Commun, 2013, 4313393-397.

[17] KirschRD, JolyE. An improved PCR-mutagenesis strategy for two-site mutagenesis or sequence swapping between related genes. Nucleic Acids Res, 1998, 2671848-1850.

[18] KoschorreckK, SchmidRD, UrlacherVB. Improving the functional expression of a Bacillus licheniformis laccase by random and site-directed mutagenesis. BMC Biotechnol, 2009, 9: 12.

[19] Kumar V, Patel SKS, Gupta RK, Otari SV, Gao H, Lee JK, Zhang L (2019) Enhanced saccharification and fermentation of rice straw by reducing the concentration of phenolic compounds using an immobilized enzyme cocktail. Biotechnol J 14 (6), 1800468

[20] LuoQ, ChenY, XiaJ, WangKQ, CaiYJ, LiaoXR, GuanZB. Functional expression enhancement of Bacillus pumilus CotA-laccase mutant WLF through site-directed mutagenesis. Enzyme Microb Technol, 2018, 109: 11-19.

[21] MacKerellAD, BashfordD, BellottM, DunbrackRL, EvanseckJD, FieldMJ, FischerS, GaoJ, GuoH, HaS, Joseph-McCarthyD, KuchnirL, KuczeraK, LauFT, MattosC, MichnickS, NgoT, NguyenDT, ProdhomB, ReiherWE, RouxB, SchlenkrichM, SmithJC, StoteR, StraubJ, WatanabeM, Wiórkiewicz-KuczeraJ, YinD, KarplusM. All-atom empirical potential for molecular modeling and dynamics studies of proteins. J Phys Chem B, 1998, 102183586-3616.

[22] Madzak C, Otterbein L, Chamkha M, Moukha S, Asther M, Gaillardin C, Beckerich JM (2005) Heterologous production of a laccase from the basidiomycete Pycnoporus cinnabarinus in the dimorphic yeast Yarrowia lipolytica. FEMS Yeast Res 5 (6–7), 635–646.

[23] MaoG, WangK, WangF, LiH, ZhangH, XieH, WangZ, WangF, SongA. An engineered thermostable laccase with great ability to decolorize and detoxify malachite green. Int J Mol Sci, 2021, 22: 21.

[24] MateDM, EvaG-R, SusanaC, VSV, MagnusF, SergeyS, AlcaldeM. Switching from blue to yellow: altering the spectral properties of a high redox potential laccase by directed evolution. Biocatal Biotransform, 2013, 3118-21.

[25] MengX, YangL, LiuH, LiQ, XuG, ZhangY, GuanF, ZhangY, ZhangW, WuN, TianJ. Protein engineering of stable IsPETase for PET plastic degradation by premuse. Int J Biol Macromol, 2021, 180: 667-676.

[26] MollaniaN, KhajehK, RanjbarB, HosseinkhaniS. Enhancement of a bacterial laccase thermostability through directed mutagenesis of a surface loop. Enzyme Microb Technol, 2011, 495446-452.

[27] NguyenJT, FongJ, FongD, FongT, LuceroRM, GallimoreJM, BurataOE, ParungaoK, RascónAAJr. Soluble expression of Recombinant midgut Zymogen (native propeptide) proteases from the Aedes aegypti mosquito utilizing E. coli as a host. BMC Biochem, 2018, 19112.

[28] OsipovE, PolyakovK, KittlR, ShleevS, DorovatovskyP, TikhonovaT, HannS, LudwigR, PopovV. Effect of the L499M mutation of the ascomycetous Botrytis Aclada laccase on redox potential and catalytic properties. Acta Crystallogr D Biol Crystallogr, 2014, 70Pt 112913-2923.

[29] PardoI, Rodríguez-EscribanoD, AzaP, de SalasF, MartínezAT, CamareroS. A highly stable laccase obtained by swapping the second cupredoxin domain. Sci Rep, 2018, 8115669.

[30] PearceR, HuangX, SetiawanD, ZhangY. EvoDesign: designing Protein-Protein binding interactions using evolutionary interface profiles in conjunction with an optimized physical energy function. J Mol Biol, 2019, 431132467-2476.

[31] PearlmanSM, SerberZ, FerrellJE. A mechanism for the evolution of phosphorylation sites. Cell, 2011, 1474934-946.

[32] PhillipsJC, BraunR, WangW, GumbartJ, TajkhorshidE, VillaE, ChipotC, SkeelRD, KaléL, SchultenK. Scalable molecular dynamics with NAMD. J Comput Chem, 2005, 26161781-1802.

[33] PleadinJ, FreceJ, MarkovK. Mycotoxins in food and feed. Adv Food Nutr Res, 2019, 89: 297-345.

[34] Ropejko K, Twarużek M (2021) Zearalenone and its Metabolites-General overview, occurrence, and toxicity. Toxins (Basel) 13 (1) ：35-47

[35] Sambrook J (2016) D. R., Molecular Cloning: A Laboratory Manual (Third Edition). Molecular Cloning: A Laboratory Manual (Third Edition)

[36] SinghG, AryaSK. Utility of laccase in pulp and paper industry: A progressive step towards the green technology. Int J Biol Macromol, 2019, 134: 1070-1084.

[37] SongX, ShanY, CaoL, ZhongX, WangX, GaoY, WangK, WangW, ZhuT. Decolorization and detoxication of malachite green by engineered Saccharomyces cerevisiae expressing novel thermostable laccase from trametes trogii. Bioresour Technol, 2024, 399: 130591.

[38] Sun F, Yu D, Zhou H, Lin H, Yan Z, Wu A (2023) CotA laccase from Bacillus licheniformis ZOM-1 effectively degrades Zearalenone, aflatoxin B1 and alternariol. Food Control

[39] Sun Z, You Y, Xu H, You Y, He W, Wang Z, Li A, Xia Y (2024) Food-Grade expression of two laccases in Pichia pastoris and study on their enzymatic degradation characteristics for Mycotoxins. J Agric Food Chem 72(16):9365–9375

[40] SunB, SunH, ZhangL, HuW, WangX, BrennanCS, HanD, WuG, YiY, LüX. Characterization and rational engineering of a novel laccase from Geobacillus thermocatenulatus M17 for improved lignin degradation activity. Int J Biol Macromol, 2025, 292: 138856.

[41] TiwariMK, KaliaVC, KangYC, LeeJK. Role of a remote leucine residue in the catalytic function of polyol dehydrogenase. Mol Biosyst, 2014, 10123255-3263.

[42] WangR, ChengY, XieY, LiJ, ZhangY, FangZ, FangW, ZhangX, XiaoY. Statistical coupling analysis uncovers sites crucial for the proton transfer in laccase Lac15. Biochem Biophys Res Commun, 2019, 5194894-900.

[43] WangC, TangK, DaiY, JiaH, LiY, GaoZ, WuB. Identification, characterization, and Site-Specific mutagenesis of a thermostable ω-Transaminase from Chloroflexi bacterium. ACS Omega, 2021, 62617058-17070.

[44] WiśniewskaKM, Twarda-ClapaA, BiałkowskaAM. Novel Cold-Adapted Recombinant laccase KbLcc1 from Kabatiella bupleuri G3 IBMiP as a green catalyst in biotransformation. Int J Mol Sci, 2021, 22: 17.

[45] WooleryGL, PowersL, PeisachJ, SpiroTG. X-ray absorption study of Rhus Laccase: evidence for a copper-copper interaction, which disappears on type 2 copper removal. Biochemistry, 1984, 23153428-3434.

[46] XIEY, WANGR, LIZ, LIJ, FANGZ, FANGW, ZHANGX, XIAOY. Semi-rational engineering of microbial laccase Lac15 for enhanced activity. Acta Microbiol Sinica, 2022, 6241501-1512

[47] YangH, LiuL, WangM, LiJ, WangNS, DuG, ChenJ. Structure-based engineering of methionine residues in the catalytic cores of alkaline amylase from Alkalimonas amylolytica for improved oxidative stability. Appl Environ Microbiol, 2012, 78217519-7526.

[48] YangC, SongG, LimW. Effects of mycotoxin-contaminated feed on farm animals. J Hazard Mater, 2020, 389: 122087.

Funding

the National Key R&D Program of China(2022YFC2105500)

the National Natural Science Foundation of China(32202720)

the Key R&D Program of Shandong province(2022CXGC020801)

the China Agriculture Research System of MOF and MARA(CARS-41)

the Central Public-interest Scientific Institution Basal Research Fund(Y2025YC66 and 1610392024004)

RIGHTS & PERMISSIONS

The Author(s)