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

High-resolution matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to characterize the primary structure of the lev ansucrase (EC 2.4.1.10) secreted by Acetobacter diazotropicus SRT4. The tec hnique 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-te rminal blocking group and the position of a disulfide bridge between Cys(30 9) and Cys(365) among the three Cys residues. A free cysteine (Cys(127)) wa s identified by modifying an intact molecule with a sulfhydryl reagent, 5-( octyldithio)-2-nitrobenzoic acid, under non-reducing conditions. In additio n, the enzyme obtained by site-directed mutagenesis at Asp(279) to Asn(279) was also identified by the above methods. Post-source decay analysis of th e tryptic peptide containing the mutation site unequivocally revealed an As n residue at position 279, Copyright (C) 1999 John Wiley & Sons Ltd.