The Role of Solvent-Accessible Leu-208 of Cold-Active Pseudomonas fluorescens Strain AMS8 Lipase in Interfacial Activation, Substrate Accessibility and Low-Molecular Weight Esterification in the Presence of Toluene
The alkaline cold-active lipase from Pseudomonas fluorescens AMS8 undergoes major structural changes when reacted with hydrophobic organic solvents. In toluene, the AMS8 lipase catalytic region is exposed by the moving hydrophobic lid 2 (Glu-148 to Gly-167). Solvent-accessible surface area analysis...
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MDPI AG
2017-08-01
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| Series: | Molecules |
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| author | Norhayati Yaacob Nor Hafizah Ahmad Kamarudin Adam Thean Chor Leow Abu Bakar Salleh Raja Noor Zaliha Raja Abd Rahman Mohd Shukuri Mohamad Ali |
| author_facet | Norhayati Yaacob Nor Hafizah Ahmad Kamarudin Adam Thean Chor Leow Abu Bakar Salleh Raja Noor Zaliha Raja Abd Rahman Mohd Shukuri Mohamad Ali |
| author_sort | Norhayati Yaacob |
| collection | DOAJ |
| description | The alkaline cold-active lipase from Pseudomonas fluorescens AMS8 undergoes major structural changes when reacted with hydrophobic organic solvents. In toluene, the AMS8 lipase catalytic region is exposed by the moving hydrophobic lid 2 (Glu-148 to Gly-167). Solvent-accessible surface area analysis revealed that Leu-208, which is located next to the nucleophilic Ser-207 has a focal function in influencing substrate accessibility and flexibility of the catalytic pocket. Based on molecular dynamic simulations, it was found that Leu-208 strongly facilitates the lid 2 opening via its side-chain. The KM and Kcat/KM of L208A mutant were substrate dependent as it preferred a smaller-chain ester (pNP-caprylate) as compared to medium (pNP-laurate) or long-chain (pNP-palmitate) esters. In esterification of ethyl hexanoate, L208A promotes a higher ester conversion rate at 20 °C but not at 30 °C, as a 27% decline was observed. Interestingly, the wild-type (WT) lipase’s conversion rate was found to increase with a higher temperature. WT lipase AMS8 esterification was higher in toluene as compared to L208A. Hence, the results showed that Leu-208 of AMS8 lipase plays an important role in steering a broad range of substrates into its active site region by regulating the flexibility of this region. Leu-208 is therefore predicted to be crucial for its role in interfacial activation and catalysis in toluene. |
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| spelling | doaj.art-63c1180622ef4faa9537ef1ce20ccce22022-12-22T02:01:41ZengMDPI AGMolecules1420-30492017-08-01228131210.3390/molecules22081312molecules22081312The Role of Solvent-Accessible Leu-208 of Cold-Active Pseudomonas fluorescens Strain AMS8 Lipase in Interfacial Activation, Substrate Accessibility and Low-Molecular Weight Esterification in the Presence of TolueneNorhayati Yaacob0Nor Hafizah Ahmad Kamarudin1Adam Thean Chor Leow2Abu Bakar Salleh3Raja Noor Zaliha Raja Abd Rahman4Mohd Shukuri Mohamad Ali5Enzyme Technology/Molecular Biomedicine Laboratory, Enzyme and Microbial Technology Research Centre, Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, MalaysiaEnzyme and Microbial Technology Research Centre, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 Serdang, MalaysiaEnzyme and Microbial Technology Research Centre, Department of Cell Biology and Molecule, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, MalaysiaEnzyme and Microbial Technology Research Centre, Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, MalaysiaEnzyme and Microbial Technology Research Centre, Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, MalaysiaEnzyme and Microbial Technology Research Centre, Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, MalaysiaThe alkaline cold-active lipase from Pseudomonas fluorescens AMS8 undergoes major structural changes when reacted with hydrophobic organic solvents. In toluene, the AMS8 lipase catalytic region is exposed by the moving hydrophobic lid 2 (Glu-148 to Gly-167). Solvent-accessible surface area analysis revealed that Leu-208, which is located next to the nucleophilic Ser-207 has a focal function in influencing substrate accessibility and flexibility of the catalytic pocket. Based on molecular dynamic simulations, it was found that Leu-208 strongly facilitates the lid 2 opening via its side-chain. The KM and Kcat/KM of L208A mutant were substrate dependent as it preferred a smaller-chain ester (pNP-caprylate) as compared to medium (pNP-laurate) or long-chain (pNP-palmitate) esters. In esterification of ethyl hexanoate, L208A promotes a higher ester conversion rate at 20 °C but not at 30 °C, as a 27% decline was observed. Interestingly, the wild-type (WT) lipase’s conversion rate was found to increase with a higher temperature. WT lipase AMS8 esterification was higher in toluene as compared to L208A. Hence, the results showed that Leu-208 of AMS8 lipase plays an important role in steering a broad range of substrates into its active site region by regulating the flexibility of this region. Leu-208 is therefore predicted to be crucial for its role in interfacial activation and catalysis in toluene.https://www.mdpi.com/1420-3049/22/8/1312molecular dynamicscold-active lipasesubstrate-accessibilityinterfacial activationhydrolysisesterification |
| spellingShingle | Norhayati Yaacob Nor Hafizah Ahmad Kamarudin Adam Thean Chor Leow Abu Bakar Salleh Raja Noor Zaliha Raja Abd Rahman Mohd Shukuri Mohamad Ali The Role of Solvent-Accessible Leu-208 of Cold-Active Pseudomonas fluorescens Strain AMS8 Lipase in Interfacial Activation, Substrate Accessibility and Low-Molecular Weight Esterification in the Presence of Toluene Molecules molecular dynamics cold-active lipase substrate-accessibility interfacial activation hydrolysis esterification |
| title | The Role of Solvent-Accessible Leu-208 of Cold-Active Pseudomonas fluorescens Strain AMS8 Lipase in Interfacial Activation, Substrate Accessibility and Low-Molecular Weight Esterification in the Presence of Toluene |
| title_full | The Role of Solvent-Accessible Leu-208 of Cold-Active Pseudomonas fluorescens Strain AMS8 Lipase in Interfacial Activation, Substrate Accessibility and Low-Molecular Weight Esterification in the Presence of Toluene |
| title_fullStr | The Role of Solvent-Accessible Leu-208 of Cold-Active Pseudomonas fluorescens Strain AMS8 Lipase in Interfacial Activation, Substrate Accessibility and Low-Molecular Weight Esterification in the Presence of Toluene |
| title_full_unstemmed | The Role of Solvent-Accessible Leu-208 of Cold-Active Pseudomonas fluorescens Strain AMS8 Lipase in Interfacial Activation, Substrate Accessibility and Low-Molecular Weight Esterification in the Presence of Toluene |
| title_short | The Role of Solvent-Accessible Leu-208 of Cold-Active Pseudomonas fluorescens Strain AMS8 Lipase in Interfacial Activation, Substrate Accessibility and Low-Molecular Weight Esterification in the Presence of Toluene |
| title_sort | role of solvent accessible leu 208 of cold active pseudomonas fluorescens strain ams8 lipase in interfacial activation substrate accessibility and low molecular weight esterification in the presence of toluene |
| topic | molecular dynamics cold-active lipase substrate-accessibility interfacial activation hydrolysis esterification |
| url | https://www.mdpi.com/1420-3049/22/8/1312 |
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