Substitution of L133 with Methionine in GXSXG Domain Significantly Changed the Activity of Penicillium expansum Lipase

Bai, Qiaoyan; Cai, Yu-zhen; Yang, Long‐Qi; Wu, Qingqing; Su, Min; Tang, Lanfang

doi:10.1007/s10562-021-03795-2

Cited by 4 publications

(7 citation statements)

References 19 publications

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“…Ser119 and Phe120 are contained in the GXSXG pentapeptide, which is conserved among esterases and lipases , and serine hydrolases . It is known that GXSXG pentapeptide forms a “catalytic elbow” that is important for activity. , In addition, the carboxyl group of FA was found to be located inside the oxyanion hole , formed by the backbone NH groups of Leu50 and Phe120, with the nitrogen atoms being colored blue in Figure C.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Structure-Based Characterization and Improvement of an Enzymatic Activity of Acremonium alcalophilum Feruloyl Esterase

Phienluphon,

Kondo,

Mikami

et al. 2024

ACS Sustainable Chem. Eng.

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Bacteria and fungi utilize carbohydrate-active enzymes, such as feruloyl esterases (FAEs), to degrade lignocellulosic biomass. FAEs in subfamily 5 (SF5) of carbohydrate esterase family 1 target larger substrates, making them particularly interesting. However, their mechanisms are not well understood due to limited structural information. This study presents the first structure of the catalytic domain (CD) of an SF5 FAE from Acremonium alcalophilum (AaFaeD), both free and in a complex with ferulic acid (FA). FA binds within a hydrophobic cleft formed by two hydrophobic walls facing each other. Structure-based functional mutagenesis of key residues in these walls clarified their roles in catalysis. Notably, the F120Y mutant of the AaFaeD catalytic domain (AaFaeD-CD) showed a 1.5-fold increase in catalytic activity toward methyl ferulate compared with the wild type. Structural comparisons with SF2 and SF3 FAEs revealed a more open substrate-binding site in SF5. High-performance liquid chromatography and gas chromatography−mass spectrometry analysis of destarched wheat bran hydrolysis by AaFaeD-CD showed that SF5 FAEs can process both monomeric and dimeric phenolic substrates, like 5,5′-dehydrodiferulate, unlike SF2 and SF3 FAEs, which prefer monomeric substrates.

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Section: Resultsmentioning

confidence: 99%

“…15 It is known that GXSXG pentapeptide forms a "catalytic elbow" that is important for activity. 37,38 In addition, the carboxyl group of FA was found to be located inside the oxyanion hole 39,40 formed by the backbone NH groups of Leu50 and Phe120, with the nitrogen atoms being colored blue in Figure 3C.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

Structure-Based Characterization and Improvement of an Enzymatic Activity of Acremonium alcalophilum Feruloyl Esterase

Phienluphon,

Kondo,

Mikami

et al. 2024

ACS Sustainable Chem. Eng.

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“…This mutation affected the stability of the lipase at different pHs. Moreover, Bai (2022) [ 11 ] changed the X site in the GXSXG domain of Penicillium expansum lipase (PEL), and observed a decrease in the activity of the mutant lipase. Liu et al (2021) [ 37 ] found similar results in a hormone-sensitive lipase of Pseudomonas sp.…”

Section: Discussionmentioning

confidence: 99%

“…The pentapeptide is essential for lipase activity; however, little is known about its role in catalytic performance [ 11 ]. In order to explore its function, Hosseini et al (2013) [ 12 ] replaced the pentapeptide AHSMG in the lipase BTL2 of Bacillus thermocatenulatus with the pentapeptide GGSAG, found in CRL lipase of Candida rugosa .…”

Section: Introductionmentioning

confidence: 99%

Identification of a Novel Lipase with AHSMG Pentapeptide in Hypocreales and Glomerellales Filamentous Fungi

Gutiérrez-Domínguez

Chí-Manzanero

Rodríguez-Argüello

et al. 2022

IJMS

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Lipases are enzymes that hydrolyze triglycerides to fatty acids and glycerol. A typical element in lipases is a conserved motif of five amino acids (the pentapeptide), most commonly G-X-S-X-G. Lipases with the pentapeptide A-X-S-X-G are present in species of Bacillus, Paucimonas lemoignei, and the yeast Trichosporon asahii; they are usually thermotolerant and solvent resistant. Recently, while searching for true lipases in the Trichoderma harzianum genome, one lipase containing the pentapeptide AHSMG was identified. In this study, we cloned from T. harzianum strain B13-1 the lipase ID135964, renamed here as ThaL, which is 97.65% identical with the reference. We found that ThaL is a lid-containing true lipase of cluster III that belongs to a large family comprising highly conserved proteins in filamentous fungi in the orders Hypocreales and Glomerellales, in which predominantly pathogenic fungi are found. ThaL was expressed in conidia, as well as in T. harzianum mycelium, where it was cultured in liquid minimal medium. These results—together with the amino acid composition, absence of a signal peptide, mitochondrial sorting prediction, disordered regions in the protein, and lineage-specific phylogenetic distribution of its homologs—suggest that ThaL is a non-canonical effector. In summary, AHSMG-lipase is a novel lipase family in filamentous fungi, and is probably involved in pathogenicity.

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“…The optimal enzyme-substrate complex exhibits good binding affinity and a relatively low binding free energy ( Anuar et al, 2021 ). Therefore, a variety of computational programs have been developed to calculate the binding energy of protein and ligand complexes as a protein mutation design strategy to improve enzyme catalytic efficiency and stereoselectivity ( Kumari and Gupta, 2013 ; Zhang et al, 2013 ; Bai et al, 2022 ).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2023

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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.

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Substitution of L133 with Methionine in GXSXG Domain Significantly Changed the Activity of Penicillium expansum Lipase

Cited by 4 publications

References 19 publications

Structure-Based Characterization and Improvement of an Enzymatic Activity of Acremonium alcalophilum Feruloyl Esterase

Structure-Based Characterization and Improvement of an Enzymatic Activity of Acremonium alcalophilum Feruloyl Esterase

Identification of a Novel Lipase with AHSMG Pentapeptide in Hypocreales and Glomerellales Filamentous Fungi

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

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