Structural characterization ofAcetobacter diazotropicus levansucrase by matrix-assisted laser desorption/ionization mass spectrometry: identification of an N-terminal blocking group and a free-thiol cysteine residue
✍ Scribed by Betancourt, Lazaro; Takao, Toshifumi; Hernandez, Lazaro; Padron, Gabriel; Shimonishi, Yasutsugu
- Publisher
- John Wiley and Sons
- Year
- 1999
- Tongue
- English
- Weight
- 140 KB
- Volume
- 34
- Category
- Article
- ISSN
- 1076-5174
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✦ Synopsis
High-resolution matrix-assisted laser desorption/ionization time-of-Ñight mass spectrometry was used to characterize the primary structure of the levansucrase (EC 2.4.1.10) secreted by Acetobacter diazotropicus SRT4. The technique permitted not only the reading frame of this enzyme, the amino acid sequence of which was deduced from DNA, but also the elucidation of an N-terminal blocking group and the position of a disulÐde bridge between Cys309 and Cys365 among the three Cys residues. A free cysteine (Cys127) was identiÐed by modifying an intact molecule with a sulfhydryl reagent, 5-(octyldithio)-2-nitrobenzoic acid, under non-reducing conditions. In addition, the enzyme obtained by site-directed mutagenesis at Asp279 to Asn279 was also identiÐed by the above methods. Post-source decay analysis of the tryptic peptide containing the mutation site unequivocally revealed an Asn residue at position 279.