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-N-epsilon-lysine was decreased 3-fold, thus resulting in a 3-fold improvement in specificity for fructosyl valine over fructosyl-N-epsilon-lysine.