The measurement of glycated hemoglobin A1c (HbA1c) has important implications for diagnosis of diabetes and assessment of treatment effectiveness. We proposed specific sequence motifs to identify enzymes that oxidize glycated compounds from genome database searches. The gene encoding a putative fructosyl amino acid oxidase was found in the Phaeosphaeria nodorum SN15 genome and successfully expressed in Escherichia coli. The recombinant protein (XP_001798711) was confirmed to be a novel fructosyl peptide oxidase (FPOX) with high specificity for a-glycated compounds, such as HbA1c model compounds fructosyl-N-alpha-valine (f-(alpha)Val) and fructosyl-N-alpha-valyl-histidine (f-(alpha)Val-His). Unlike previously reported FPOXs, the P. nodorum FPOX has a K-m value for f-(alpha)Val-His (0.185 mM) that is considerably lower than that for f-(alpha)Val (0.458 mM). Based on amino acid sequence alignment, three dimensional structural modeling, and site-directed mutagenesis, Gly60 was found to be a determining residue for the activity towards f-(alpha)Val-His. A flexible surface loop region was also found to likely play an important role in accepting f-(alpha)Val-His. Biotechnol. Bioeng. 2010;106: 358-366. (C) 2010 Wiley Periodicals, Inc.