caa a22 4500 378895966 CHVBK 20180305123506.0 cr unu---uuuuu 161128e20041201xx s 000 0 eng 10.1515/BC.2004.148 doi (NATIONALLICENCE)gruyter-10.1515/BC.2004.148 Structural characterization of extracellular lipase from Streptomyces rimosus: assignment of disulfide bridge pattern by mass spectrometry [Elektronische Daten] [Ivana Leščić, Martin Zehl, Roland Müller, Bojana Vukelić, Marija Abramić, Jasenka Pigac, Günter Allmaier, Biserka Kojić-Prodić] 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=0.11Da and Δm=0.26Da, respectively) with a value of 24165.76Da 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. ©2004 by Walter de Gruyter Berlin New York Biochemistry nationallicence Molecular biology nationallicence Cellular biology nationallicence amino acid sequence nationallicence capillary electrophoresis-on-the-chip nationallicence GDSL lipolytic enzyme nationallicence low-energy collision-induced dissociation nationallicence matrix-assisted laser desorption/ionization nationallicence streptomycetes nationallicence Leščić Ivana Department of Physical Chemistry, Ruđer Bošković Institute, Bijenička cesta 54, HR-10002 Zagreb, Croatia aut Zehl Martin Institute of Chemical Technologies and Analysis, Vienna University of Technology, Getreidemarkt 9/164-IAC, A-1060 Vienna, Austria aut Müller Roland Institute of Chemical Technologies and Analysis, Vienna University of Technology, Getreidemarkt 9/164-IAC, A-1060 Vienna, Austria aut Vukelić Bojana Department of Organic Chemistry and Biochemistry, Ruđer Bošković Institute, Bijenička cesta 54, HR-10002 Zagreb, Croatia aut Abramić Marija Department of Organic Chemistry and Biochemistry, Ruđer Bošković Institute, Bijenička cesta 54, HR-10002 Zagreb, Croatia aut Pigac Jasenka Department of Physical Chemistry, Ruđer Bošković Institute, Bijenička cesta 54, HR-10002 Zagreb, Croatia aut Allmaier Günter Institute of Chemical Technologies and Analysis, Vienna University of Technology, Getreidemarkt 9/164-IAC, A-1060 Vienna, Austria aut Kojić-Prodić Biserka Department of Physical Chemistry, Ruđer Bošković Institute, Bijenička cesta 54, HR-10002 Zagreb, Croatia aut Biological Chemistry Walter de Gruyter 385/12(2004-12-01), 1147-1156 1431-6730 385:12<1147 2004 385 bchm https://doi.org/10.1515/BC.2004.148 text/html Onlinezugriff via DOI 1 research article jats NATIONALLICENCE 856 40 https://doi.org/10.1515/BC.2004.148 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Leščić Ivana Department of Physical Chemistry, Ruđer Bošković Institute, Bijenička cesta 54, HR-10002 Zagreb, Croatia aut NATIONALLICENCE 700 1- Zehl Martin Institute of Chemical Technologies and Analysis, Vienna University of Technology, Getreidemarkt 9/164-IAC, A-1060 Vienna, Austria aut NATIONALLICENCE 700 1- Müller Roland Institute of Chemical Technologies and Analysis, Vienna University of Technology, Getreidemarkt 9/164-IAC, A-1060 Vienna, Austria aut NATIONALLICENCE 700 1- Vukelić Bojana Department of Organic Chemistry and Biochemistry, Ruđer Bošković Institute, Bijenička cesta 54, HR-10002 Zagreb, Croatia aut NATIONALLICENCE 700 1- Abramić Marija Department of Organic Chemistry and Biochemistry, Ruđer Bošković Institute, Bijenička cesta 54, HR-10002 Zagreb, Croatia aut NATIONALLICENCE 700 1- Pigac Jasenka Department of Physical Chemistry, Ruđer Bošković Institute, Bijenička cesta 54, HR-10002 Zagreb, Croatia aut NATIONALLICENCE 700 1- Allmaier Günter Institute of Chemical Technologies and Analysis, Vienna University of Technology, Getreidemarkt 9/164-IAC, A-1060 Vienna, Austria aut NATIONALLICENCE 700 1- Kojić-Prodić Biserka Department of Physical Chemistry, Ruđer Bošković Institute, Bijenička cesta 54, HR-10002 Zagreb, Croatia aut NATIONALLICENCE 773 0- Biological Chemistry Walter de Gruyter 385/12(2004-12-01), 1147-1156 1431-6730 385:12<1147 2004 385 bchm CC0 http://creativecommons.org/publicdomain/zero/1.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-gruyter