Structural, functional and chemical changes in Pseudozyma antarctica lipase B on exposure to hydrogen peroxide.
The effect on primary, secondary, tertiary and quaternary structure of Pseudozyma (formerly Candida) antarctica lipase B (PalB) on exposure to hydrogen peroxide was investigated using nano-electrospray ionization-mass spectrometry (nano-ESI-MS), liquid chromatography tandem mass spectrometry (LC/MS/MS), circular dichroism (CD), and dynamic light scattering (DLS). Treatment with hydrogen peroxide generated heavier protein variants, with a mass gain that increased with increasing incubation time. Furthermore, elevated concentration of H(2)O(2) was shown to result in partial fragmentation of the protein. Proteolytic digestion of the enzyme gave primary sequence coverage of more than 90%, revealing oxidation of methionine, tryptophan and cystine residues. The active site histidine was not observed in oxidized form in any of the experiments. However, oxidation of cystine to cysteic acid indicated disruption of disulphide bridges, and CD evaluations confirmed that severe changes to the secondary structure towards random coil had occurred. The structural changes could be an effect of the observed amino acid side chain oxidations, and was correlated with deactivation of the lipase. From DLS experiments, it was seen that the lipase exposed to both high temperature and H(2)O(2) formed large and intermediate sized aggregates, not observed for the heat-treated enzyme. The findings reported here could lay the basis for developing enzyme variants with higher oxidative stability.
Törnvall
Ulrika
U
Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden. ulrika.tornvall@biotek.lu.se
Hedström
Martin
M
Schillén
Karin
K
Hatti-Kaul
Rajni
R
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D004795:Enzyme Stability / Q000187:drug effects; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008049:Lipase / Q000737:chemistry* / Q000378:metabolism; D008715:Methionine / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D017433:Protein Structure, Secondary / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects; D021241:Spectrometry, Mass, Electrospray Ionization; D013696:Temperature; D013997:Time Factors
D005656:Fungal Proteins; D016877:Oxidants; D008715:Methionine; D006861:Hydrogen Peroxide; D008049:Lipase; C431349:lipase B, Candida antarctica