engleski

Characterization of an extracellular lipase from Streptomyces rimosus by MALDI-TOF and MALDI-IT/RTOF multistage mass spectrometry: assignment of disulfide bridge pattern and localization of the active site serine

Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3), especially those from microorganisms, have found wide biotechnological application. However, natural enzymes, including lipases, do not always show satisfactory performance for efficient use as biocatalysts. Therefore, efforts are made to optimize their enzymatic properties (activity, stability and, most importantly, enantioselectivity), e.g. by rational protein design. This requires the detailed characterization of the protein structure, including the topology of the enzyme active site. The cloning, sequencing and high-level expression of the gene encoding extracellular lipase from Streptomyces rimosus R6-554W has been recently described. The gene product, which has been isolated and characterized by biochemical and bioinformatical methods, seems to differ in structure from most other known lipases and exhibits several properties that indicate its significant biotechnological potential. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and MALDI - ion trap - reflection time-of-flight (MALDI-QIT/RTOF) MS was used to characterize the S. rimosus lipase at the protein level. The primary structure, derived from the nucleotide sequence, was completely verified using a combination of tryptic digestion and formic acid cleavage of the protein, followed by peptide mass fingerprinting and fragmentation of selected peptides by low-energy collision-induced dissociation (LE CID). The enzyme contains six cysteines. All of them were shown to form intramolecular disulfide bonds, which were elucidated, and therefore contribute to the observed pronounced thermal stability of this enzyme and its ruggedness towards organic solvents. A chemical modification approach combined with MALDI-MS was used to identify the active site serine residue. The lipase was covalently modified by mechanism-based inhibition with 3, 4-dichloroisocoumarin. Exclusively one molecule of inhibitor was found to bind to the lipase and the site specificity of the inhibition reaction was confirmed. Exact localization of the nucleophilic amino acid residue in the active site of the enzyme was accomplished by LE CID sequencing of the modified peptide. The identification of the serine at position 10 as a member of the catalytic triad is in agreement with the previously obtained data from multiple sequence alignment of S. rimosus lipase with different esterases and experimentally proves the classification of this protein as GDS(L) lipolytic enzyme. The described MS-based method represents a fast and accurate alternative to the more common site-directed mutagenesis approach.

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