| An essential aspartate from B. subtilis levansucrase was directly identified by chemically trapping a denatured [14C] labeled covalent enzyme-fructose intermediate using a catalysis dependent method. The intermediate was digested with pepsin and the radiolabeled peptide was purified using HPLC reverse phase chromatography. Primary structure of the active site peptide was analyzed by direct sequencing from the N-terminus and confirmed by electrospray mass spectrometry; the amino acid sequence determined was WDSWPLQN (Trp,Asp,Ser,Trp,Pro,Leu,Gln,Asn). Only one aspartate, positioned 86 amino acids from the N-terminus, was found in the levansucrase active site peptide; thus the location of the corresponding catalytically essential aspartate from S. mutans fructosyltransferase, positioned 248 amino acids from the N-terminus, was indirectly identified (WDSWPVQD) by sequence alignment with the levansucrase amino acid sequence. In order to determine the catalytic role of the indirectly identified S. mutans fructosyltransferase aspartate, a missense mutation was created at the wild type fructosyltransferase aspartate, (position 1422 in the nucleotide sequence), substituting aspartate with asparagine by changing base G to an A. Both wild type and mutant strains were analyzed by an inulin synthesis assay to determine enzyme activity. Wild type fructosyltransferase demonstrated enzyme activity; in contrast, no measurable enzyme activity was detected from the mutant.;Additionally, known beta-D-fructosyltransferase sequences from different organisms with unknown catalytic sites were aligned with the directly and indirectly identified essential aspartate from S. mutans and B. subtilis respectively. The amino acid sequence surrounding the aspartate was conserved; 50--100% identity was found, suggesting a common ancestor through horizontal gene transfer. As a result, putative catalytic amino acids were indirectly identified in B. amyloliquefaciens, B. stearothermophilus, Z. mobilis, A. diazotrophicus and S. salivarius levansucrases. Finally, only aspartate was found conserved among beta-fructosyltransferases and related beta-fructofuranosidases. |
