Structural characterization of extracellular lipase from Streptomyces rimosus: assignment of disulfide bridge pattern by mass spectrometry
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Ivana Leščić
Abstract
The cloning, sequencing and high-level expression of the gene encoding extracellular lipase from Streptomyces rimosus R6-554W have been recently described, and the primary structure of this gene product was deduced using a bioinformatic approach. In this study, capillary electrophoresis-on-the-chip and mass spectrometry were used to characterize native and overexpressed extracellular lipase protein from S. rimosus. The exact molecular mass of the wild-type and the overexpressed lipase, determined by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, were in excellent agreement (Δm=0.11 Da and Δm=0.26 Da, respectively) with a value of 24165.76 Da calculated from the structure deduced from the nucleotide sequence, considering the mature enzyme with all six cysteines forming disulfide bridges. 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. Selected peptides were further investigated by MALDI low-energy collision-induced dissociation hybrid tandem mass spectrometry, allowing the unambiguous determination of their predicted amino acid sequence. No post-translational modifications of mature S. rimosus lipase were detected. Comparison of the peptide mass fingerprints from the reduced and non-reduced overexpressed enzyme unequivocally revealed three intramolecular disulfide bonds with the following linkages: C27-C52, C93-C101 and C151-C198.
References
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©2004 by Walter de Gruyter Berlin New York
Articles in the same Issue
- Structural flexibility of small GTPases. Can it explain their functional versatility?
- Neuroendocrine regulation of salivary IgA synthesis and secretion: implications for oral health
- Structural characterization of extracellular lipase from Streptomyces rimosus: assignment of disulfide bridge pattern by mass spectrometry
- Thermal unfolding of ribonuclease T1 studied by multi-dimensional NMR spectroscopy
- Conserved asparagine residue 54 of α-sarcin plays a role in protein stability and enzyme activity
- Replacement of the interchain disulfide bridge-forming amino acids A7 and B7 by glutamate impairs the structure and activity of insulin
- Stereospecificity of horseradish peroxidase
- Novel thioredoxin targets in Dictyostelium discoideum identified by two-hybrid analysis: interactions of thioredoxin with elongation factor 1α and yeast alcohol dehydrogenase
- Functional characterization of the postulated intramolecular sphingolipid activator protein domain of human acid sphingomyelinase
- St. John's wort (Hypericum perforatum) counteracts cytokine-induced tryptophan catabolism in vitro
- Nuclear fibroblast growth factor-2 interacts specifically with splicing factor SF3a66
- Content Index
- Author Index
- Subject Index
Articles in the same Issue
- Structural flexibility of small GTPases. Can it explain their functional versatility?
- Neuroendocrine regulation of salivary IgA synthesis and secretion: implications for oral health
- Structural characterization of extracellular lipase from Streptomyces rimosus: assignment of disulfide bridge pattern by mass spectrometry
- Thermal unfolding of ribonuclease T1 studied by multi-dimensional NMR spectroscopy
- Conserved asparagine residue 54 of α-sarcin plays a role in protein stability and enzyme activity
- Replacement of the interchain disulfide bridge-forming amino acids A7 and B7 by glutamate impairs the structure and activity of insulin
- Stereospecificity of horseradish peroxidase
- Novel thioredoxin targets in Dictyostelium discoideum identified by two-hybrid analysis: interactions of thioredoxin with elongation factor 1α and yeast alcohol dehydrogenase
- Functional characterization of the postulated intramolecular sphingolipid activator protein domain of human acid sphingomyelinase
- St. John's wort (Hypericum perforatum) counteracts cytokine-induced tryptophan catabolism in vitro
- Nuclear fibroblast growth factor-2 interacts specifically with splicing factor SF3a66
- Content Index
- Author Index
- Subject Index