Cloning of amadoriase I isoenzyme from Aspergillus sp.: evidence of FAD covalently linked to Cys342.
Cloning of amadoriase I isoenzyme from Aspergillus sp.: evidence of FAD covalently linked to Cys342.
复制标题
从曲霉属中克隆阿马多里酶 I 同工酶：FAD 与 Cys342 共价连接的证据。
DOI：
10.1021/bi992031g
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发表时间：
2000
期刊：
影响因子：
2.9
通讯作者：
Monnier,VM
中科院分区：
文献类型：
--
作者：
Wu,X;Takahashi,M;Chen,SG;Monnier,VM
Amadoriases是一类新型的FAD酶，其催化糖化氨基酸的氧化去糖化以产生相应的氨基酸、葡萄糖酮和H2 O2。我们先前报道了从烟曲霉中分离纯化和鉴定两种阿马多里酶同工酶，并克隆了阿马多里酶II。为了鉴定Amadoriase I的一级结构，我们从烟曲霉中制备了cDNA文库，并根据肽图谱中的部分氨基酸序列设计引物，通过聚合酶链反应扩增探针，分离了一个克隆。从核苷酸序列推断的酶的一级结构包含445个氨基酸残基。该酶含有1 mol FAD作为辅因子，其与Cys 342共价连接，如通过诱变分析、基质辅助激光解吸/电离飞行时间质谱和电喷雾电离-碰撞活化解离串联质谱所确定。序列比对研究表明，amadoriase I与单体肌氨酸氧化酶具有22%的同源性，其中FAD也与同源Cys残基相连。Amadoriases作为解偶联高血糖和糖化反应的工具具有潜在的重要性，这些反应被认为在糖尿病并发症中起作用。
Amadoriases are a novel class of FAD enzymes which catalyze the oxidative deglycation of glycated amino acids to yield corresponding amino acids, glucosone, and H2O2. We previously reported the purification and characterization of two amadoriase isoenzymes fromAspergillus fumigatusand the molecular cloning of amadoriase II. To identify the primary structure of amadoriase I, we prepared a cDNA library fromAspergillus fumigatusand isolated a clone using a probe amplified by polymerase chain reaction with primers designed according to the partial amino acid sequences from peptide mapping. The primary structure of the enzyme deduced from the nucleotide sequence comprises 445 amino acid residues. The enzyme contains 1 mol of FAD as a cofactor, which is covalently linked to Cys342, as determined by mutagenesis analysis, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and electrospray ionization−collisional-activated dissociation tandem mass spectrometry. Sequence alignment studies show that amadoriase I has 22% homology with monomeric sarcosine oxidase in which FAD is also linked to a homologous Cys residue. Amadoriases are of potential importance as tools for uncoupling hyperglycemia and glycation reactions that are thought to play a role in diabetic complications.