Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei-shi, Japan.
Biotechnol Bioeng. 2010 Jun 15;106(3):358-66. doi: 10.1002/bit.22710.
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 alpha-glycated compounds, such as HbA1c model compounds fructosyl-(alpha)N-valine (f-(alpha)Val) and fructosyl-(alpha)N-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.
糖化血红蛋白 A1c（HbA1c）的测定对糖尿病的诊断和治疗效果评估具有重要意义。我们从基因组数据库搜索中提出了特定的序列基序，以鉴定氧化糖化化合物的酶。在被孢霉 SN15 基因组中发现了编码假定的果糖基氨基酸氧化酶的基因，并在大肠杆菌中成功表达。重组蛋白（XP_001798711）被确认为一种新型的果糖肽氧化酶（FPOX），对α-糖化化合物具有高度特异性，如 HbA1c 模型化合物果糖基（α）N-缬氨酸（f-（α）Val）和果糖基（α）N-缬氨酰-组氨酸（f-（α）Val-His）。与先前报道的 FPOX 不同，被孢霉 FPOX 对 f-（α）Val-His 的 K(m)值（0.185mM）明显低于 f-（α）Val（0.458mM）。基于氨基酸序列比对、三维结构建模和定点突变，发现 Gly60 是对 f-（α）Val-His 活性起决定作用的残基。还发现一个柔性表面环区可能在接受 f-（α）Val-His 方面发挥重要作用。
