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已被发现， 在肺炎克雷伯氏菌的蛋氨酸补救途径中鉴定。 该酶目前命名为E2，已在大肠杆菌中克隆和表达。 杆菌 令人惊讶的是，克隆基因的诱导导致 两种不同的亚型E2和ET的过度表达， 可通过色谱法分离。 这两种形式不同（至少在 其金属离子含量。 两种酶都催化 2-二羟基-3-酮基-5-甲硫基戊烯氧化降解 阴离子，但降解产物取决于 结合在活性部位的金属离子。 Ni 12结合在活性 现场的结果，在形成一氧化碳和甲酸盐从 降解过程。 如果Fe '2与酶结合，则只有甲酸盐与酶结合。 制作。 我们准备了一份富含碳氮的 含Ni，2_的酶，并使用标准品获得光谱 三重共振实验（HN（CO）CA，HNCA，HNCACB， CBCA（CO）NH）和13 C编辑实验（HCCH-TOCSY，13 C过滤 NOESY）。 几乎完成了“H”，“C”和“N”共振分配， 对于天然的含Ni”的E2酶， 酶已被确定。 初步证据表明 两种异构体之间的结构差异可能是由于 存在（在E2中）或不存在（在E2 '中）二硫键， 只有两个半胱氨酸

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.