STRUCT FUNCT OF NEW CLASS OF METALLOENZYMES FROM METHIONINE SALVAGE PATHWAY

一种具有两种不同异构体的新型金属酶 在肺炎克雷伯菌的蛋氨酸挽救途径中鉴定。 目前命名为E2的酶已被克隆并在E. Coli.令人惊讶的是，对克隆基因的诱导会导致 过表达两种不同的亚型，E2和ET，它们是 可通过层析分离的。这两种形式不同(至少在 组成)仅由它们的金属离子含量决定。这两种酶都能催化 1，2-二羟基-3-酮-5-甲硫戊烯的氧化降解 阴离子，但降解的产物取决于 结合在活性中心的金属离子。Ni12在活跃中跳跃 现场导致一氧化碳和甲酸盐的形成 退化过程。如果Fe‘2与酶结合，只有甲酸盐与酶结合 制作。我们已经准备了一个完全“C，”N富集物的样品 含镍，2_酶，并用该标准获得了光谱 三重共振实验曲目(HN(CO)CA、HNCA、HNCACB、 CbCa(CO)NH)和13C编辑实验(HCCH-TOCSY，13C-过滤 NOESY)。几乎完成了‘H，“C和”N的共振指定 为含有E2酶的天然镍“制造，并为 该酶已被测定。有初步证据表明 两种异构体之间的结构差异可能是由于 (在E2中)或(在E2‘中)存在或不存在二硫键 蛋白质中只有两种半胱氨酸。

A novel metalloenzyme with two distinct isoforms has been identified in the methionine salvage pathway of Klebsiella pneumoniae. The enzyme, presently named E2, has been cloned and expressed in E. coli. Surprisingly, induction of the cloned gene results in overexpression of two distinct isoforms, E2 and ET, which are separable by chromatography. The two forms differ (at least in composition) only by their metal ion contents. Both enzymes catalyze the oxidative degradation of 1,2-dihydroxy-3-keto-5-methylthiopentene anion, but the product of the degradation depends upon the identity of the metal ion bound in the active site. Ni 12 bound in the active site results in the formation of carbon monoxide and formate from the degradative process. If Fe'2 is bound to the enzyme, only formate is produced. We have prepared a sample of the fully "C,"N-enriched Ni,2_Containing enzyme and have acquired spectra using the standard repertoire of triple resonance experiments (HN(CO)CA, HNCA, HNCACB, CBCA(CO)NH) and 13C-edited experiments (HCCH-TOCSY, 13C-filtered NOESY). Nearly complete 'H, "C and "N resonance assignments have been made for the native Ni" containing E2 enzyme, and a global fold for the enzyme has been determined. There is preliminary evidence that structural differences between the two isoforms may be due to the presence (in E2) or absence (in E2') of a disulfide bridge between the only two cysteines in the protein.