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. 2023 Jun 26:14:1193955.
doi: 10.3389/fmicb.2023.1193955. eCollection 2023.

N-terminal lid swapping contributes to the substrate specificity and activity of thermophilic lipase TrLipE

Yakun Fang  1   2 , Fan Liu  1   2 , Yi Shi  1   2 , Ting Yang  3 , Yu Xin  1   2 , Zhenghua Gu  1   2 , Guiyang Shi  1   2 , Liang Zhang  1   2
Affiliations

N-terminal lid swapping contributes to the substrate specificity and activity of thermophilic lipase TrLipE

Yakun Fang et al. Front Microbiol. .

Abstract

TrLipE is a thermophilic lipase that has potential commercial applications because of its catalytic ability under extreme conditions. Consistent with most lipases, the lid of TrLipE is located over the catalytic pocket, controls the substrate channel to the active center, and regulates the substrate specificity, activity, and stability of the enzyme through conformational changes. TrLipE from Thermomicrobium roseum has potential industrial applications, which is hindered by its weak enzymatic activity. Here, 18 chimeras (TrL1-TrL18) were reconstructed by N-terminal lid swapping between TrLipE and structurally similar enzymes. The results showed that the chimeras had a similar pH range and optimum pH as wild TrLipE but a narrower temperature range of 40-80°C, and TrL17 and the other chimeras showed lower optimum temperatures of 70°C and 60°C, respectively. In addition, the half-lives of the chimeras were lower than those of TrLipE under optimum temperature conditions. Molecular dynamics simulations indicated that chimeras had high RMSD, RMSF, and B-factor values. When p-nitrophenol esters with different chains were used as substrates, compared with TrLipE, most of the chimeras had a low Km and high kcat value. The chimeras TrL2, TrL3, TrL17, and TrL18 could specifically catalyze the substrate 4-nitrophenyl benzoate, with TrL17 showing the highest kcat/Km value of 363.88 ± 15.83 L⋅min-1⋅mmol-1. Mutants were then designed by investigating the binding free energies of TrL17 and 4-nitrophenyl benzoate. The results indicated that single, double, and triple substitution variants (M89W and I206N; E33W/I206M and M89W/I206M; and M89W/I206M/L21I and M89W/I206N/L21I, respectively) presented approximately 2- to 3-fold faster catalysis of 4-nitrophenyl benzoate than the wild TrL17. Our observations will facilitate the development of the properties and industrial applications of TrLipE.

Keywords: binding energy; lid swapping; molecular dynamics; substrate specificity; thermophilic lipase.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
The optimal temperature of chimeras. The chimeras had a low optimal temperature of 70°C for TrL17 and 60°C for the other chimeras, which was lower than that of TrLipE with an optimal temperature of 85°C.
FIGURE 2
FIGURE 2
The MD simulation result of TrLipE and chimeras. MD simulations were performed at 300 K, and the chimeras had higher RMSD, RMSF, and B-factor values than TrLipE.
FIGURE 3
FIGURE 3
The optimal pH of chimeras. The optimal pH for the chimeras was 8–8.5, and this value was slightly lower than the pH of 8.5–9 for TrLipE.
FIGURE 4
FIGURE 4
The relative enzyme activity of TrLipE and chimeras. (A) The relative enzyme activity of TrLipE and chimeras for 4-nitrophenol laurate; (B) The relative enzyme activity of chimeras TrL2, TrL3, TrL,17, and TrL18 for 4-nitrophenyl benzoate.
FIGURE 5
FIGURE 5
The heat map of the ratio of kcat/Km value between TrL17 and mutants. (A) The ratio of kcat/Km values between single mutants and TrL17; (B) The ratio of kcat/Km values between double, triple mutants, and TrL17.
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References

    1. Akoh C. C., Lee G. C., Shaw J. F. (2004). Protein engineering and applications of Candida rugosa lipase isoforms. Lipids 39 513–526. 10.1007/s11745-004-1258-7 - DOI - PubMed
    1. Almeida F., Travália B. M., Gonalves I. S., Forte M. B. S. (2021). Biodiesel production by lipase-catalyzed reactions: Bibliometric analysis and study of trends. Biofuel. Bioprod. Biorefin. 15 1141–1159.
    1. Alvarez Y., Esteban-Torres M., Cortés-Cabrera A., Gago F., Acebrón I., Benavente R., et al. (2014). Esterase LpEst1 from Lactobacillus plantarum: A novel and atypical member of the αβ hydrolase superfamily of enzymes. PLoS One 9:e92257. 10.1371/journal.pone.0092257 - DOI - PMC - PubMed
    1. Anobom C. D., Pinheiro A. S., De-Andrade R. A., Aguieiras E. C., Andrade G. C., Moura M. V., et al. (2014). From structure to catalysis: Recent developments in the biotechnological applications of lipases. Biomed Res. Int. 2014:684506. - PMC - PubMed
    1. Anuar N. F. S. K., Wahab R. A., Huyop F., Amran S. I., Hamid A. A. A., Halim K. B. A., et al. (2021). Molecular docking and molecular dynamics simulations of a mutant Acinetobacter haemolyticus alkaline-stable lipase against tributyrin. J. Biomol. Struct. Dyn. 39 2079–2091. 10.1080/07391102.2020.1743364 - DOI - PubMed