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Reductive Alkylation Causes the Formation of a Molten Globule-Like Intermediate Structure in Geobacillus zalihae Strain T1 Thermostable Lipase

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Abstract

A thermostable lipase from Geobacillus zalihae strain T1 was chemically modified using propionaldehyde via reductive alkylation. The targeted alkylation sites were lysines, in which T1 lipase possessed 11 residues. Far-UV circular dichroism (CD) spectra of both native and alkylated enzyme showed a similar broad minimum between 208 and 222 nm, thus suggesting a substantial amount of secondary structures in modified enzyme, as compared with the corresponding native enzyme. The hydrolytic activity of the modified enzymes dropped drastically by nearly 15-fold upon chemical modification, despite both the native and modified form showed distinctive α-helical bands at 208 and 222 nm in CD spectra, leading us to the hypothesis of formation of a molten globule (MG)-like structure. As cooperative unfolding transitions were observed, the modified lipase was distinguished from the native state, in which the former possessed a denaturation temperature (T m) in lower temperature range at 61 °C while the latter at 68 °C. This was further supported by 8-anilino-1-naphthalenesulfonic acid (ANS) probed fluorescence which indicated higher exposure of hydrophobic residues, consequential of chemical modification. Based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, a small number of lysine residues were confirmed to be alkylated.

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Acknowledgments

The authors would like to extend their gratitude to Prof. Dr. Yasushi Kai and Dr. Hiroyoshi Matsumura for the protein structure determination, Prof. Dr. Ali A. Moosavi-Movahedi and Dr. Bimo Ario Tejo for their opinions, and the Ministry of Science, Technology and Innovation Malaysia for financial support (09-02-04-001). A special thank also to Prof. Dr. Tan Soon Guan for checking the manuscript.

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Authors and Affiliations

  1. Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia

    Kok Whye Cheong, Thean Chor Leow, Raja Noor Zaliha Raja Abd Rahman, Mahiran Basri, Mohd Basyaruddin Abdul Rahman & Abu Bakar Salleh

  2. Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia

    Thean Chor Leow

  3. Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia

    Raja Noor Zaliha Raja Abd Rahman

  4. Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia

    Abu Bakar Salleh

  5. Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia

    Mahiran Basri & Mohd Basyaruddin Abdul Rahman

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Cheong, K.W., Leow, T.C., Rahman, R.N.Z.R.A. et al. Reductive Alkylation Causes the Formation of a Molten Globule-Like Intermediate Structure in Geobacillus zalihae Strain T1 Thermostable Lipase. Appl Biochem Biotechnol 164, 362–375 (2011). https://doi.org/10.1007/s12010-010-9140-8

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