Structural characterization of extracellular lipase from Streptomyces rimosus: assignment of disulfide bridge pattern by mass spectrometry.
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 (Deltam=0.11 Da and Deltam=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.
Lescić
Ivana
I
Department of Physical Chemistry, Ruder Bosković Institute, Bijenicka cesta 54, HR-10002 Zagreb, Croatia.
Zehl
Martin
M
Müller
Roland
R
Vukelić
Bojana
B
Abramić
Marija
M
Pigac
Jasenka
J
Allmaier
Günter
G
Kojić-Prodić
Biserka
B
D000595:Amino Acid Sequence; D004220:Disulfides / Q000737:chemistry*; D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel; D005110:Extracellular Space / Q000201:enzymology*; D005561:Formates / Q000737:chemistry; D006868:Hydrolysis; D007202:Indicators and Reagents; D007525:Isoelectric Focusing; D008049:Lipase / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D040081:Protein Array Analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013302:Streptomyces / Q000737:chemistry / Q000201:enzymology*; D014357:Trypsin / Q000737:chemistry
D004220:Disulfides; D005561:Formates; D007202:Indicators and Reagents; C030544:formic acid; D008049:Lipase; D014357:Trypsin