Structural characterization of Acetobacter diazotropicus levansucrase by matrix-assisted laser desorption/ionization mass spectrometry : Identification of an N-terminal blocking group and a free-thiol cysteine residue

摘要: High-resolution matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to characterize the primary structure of levansucrase (EC 2.4.1.10) secreted by Acetobacter diazotropicus SRT4. The technique permitted not only reading frame this enzyme, amino acid sequence which deduced from DNA, but also elucidation an N-terminal blocking group and position a disulfide bridge between Cys309 Cys365 among three Cys residues. A free cysteine (Cys127) identified modifying intact molecule with sulfhydryl reagent, 5-(octyldithio)-2-nitrobenzoic acid, under non-reducing conditions. In addition, enzyme obtained site-directed mutagenesis at Asp279 Asn279 above methods. Post-source decay analysis tryptic peptide containing mutation site unequivocally revealed Asn residue 279. Copyright © 1999 John Wiley & Sons Ltd.