Naslov
Structural characterization of extracellular lipase from Streptomyces rimosus: Assignment of disulfide bridges pattern by mass spectrometry

Autori
Leščić, Ivana ; Zehl, Martin ; Müller, Roland ; Vukelić, Bojana ; Abramić, Marija ; Pigac, Jasenka ; Allmaier, Günter ; Kojić-Prodić, Biserka

Izvornik
Biological chemistry (1431-6730) 385 (2004), 12; 1147-1156

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

Ključne riječi
amino-acid sequence; capillary electrophoresis-on-the-chip; GDSL lipolytic enzyme; MALDI; low energy CID; streptomycetes

Sažetak
Cloning, sequencing and high-level expression of the gene encoding extracellular lipase from Streptomyces rimosus R6-554W have been recently described, and primary structure of this gene product was deduced by bioinformatic approach. In this study, capillary electrophoresis-on-the-chip and mass spectrometry were used to characterize native and overexpressed extracellular lipase protein from Streptomyces rimosus. The exact molecular mass of the wild-type and the overexpressed lipase, determined by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was in excellent agreement (Δ m = 0.11 Da and Δ m = 0.26 Da, respectively) with the value 24165.76 Da calculated from the structure deduced from nucleotide sequence, considering mature enzyme with all six cysteines forming disulfide bridges. The primary structure derived from nucleotide sequence was completely verified by means of a combination of tryptic digestion and formic acid cleavage of the protein followed by obtaining the peptide mass fingerprints. 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 Streptomyces 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 following linkages: C27-C52, C93-C101 and C151-C198.

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Biologija

POVEZANOST RADA