Active site analysis of fructosyl amine oxidase using homology modeling and site-directed mutagenesis
Miura, S.; Ferri, S.; Tsugawa, W.; Kim, S.; Sode, K.
Biotechnology Letters 28(23): 1895-1900
2006
ISSN/ISBN: 1573-6776 PMID: 17043907 DOI: 10.1007/s10529-006-9173-9
Accession: 029880858
A three-dimensional structural model of fructosyl amine oxidase from the marine yeast Pichia N1-1 was generated using the crystal structure of monomeric sarcosine oxidase from Bacillus sp. B-0618 as template. The putative active site region was investigated by site-directed mutagenesis, identifying several amino acid residues likely playing important roles in the enzyme reaction. Asn354 was identified as a residue that plays an important role in substrate recognition and that can be substituted in order to change substrate specificity while maintaining high catalytic activity. While the Asn354Ala substitution had no effect on the V (max) K (m) (-1) value for fructosyl valine, the V (max) K (m) (-1) value for fructosyl-(epsilon) N-lysine was decreased 3-fold, thus resulting in a 3-fold improvement in specificity for fructosyl valine over fructosyl-(epsilon) N-lysine.