Cysteine sulfinate desulfinase, a NIFS-like protein of Escherichia coli with selenocysteine lyase and cysteine desulfurase activities - Gene cloning, purification, and characterization of a novel pyridoxal enzyme
Cysteine sulfinate desulfinase, a NIFS-like protein of Escherichia coli with selenocysteine lyase and cysteine desulfurase activities - Gene cloning, purification, and characterization of a novel pyridoxal enzyme
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DOI：
10.1074/jbc.272.36.22417
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发表时间：
1997-09-05
影响因子：
4.8
通讯作者：
Esaki, N
中科院分区：
文献类型：
--
作者：
Mihara, H;Kurihara, T;Esaki, N
硒半胱氨酸裂解酶(EC 4.4.1.16)专一地将硒半胱氨酸分解为丙氨酸和元素硒，而棕色固氮菌的半胱氨酸脱硫酶(NIF蛋白)不分青红皂白地作用于半胱氨酸和硒半胱氨酸，分别产生元素硫和硒，以及丙氨酸。这些蛋白质显示出一定的序列同源性，大肠杆菌基因组包含三个与NIFS序列同源的基因。我们克隆了定位于染色体63.4min的该基因，并对该基因产物进行了表达、纯化和鉴定。该酶由两个相同的亚基组成，有401个氨基酸残基(M-r43,238)，含有5‘-磷酸吡哆醛作为辅酶。该酶催化从L-半胱氨酸、L-半胱氨酸、L-硒半胱氨酸和L-硒半胱氨酸中脱除元素硫和硒原子，生成L-丙氨酸。由于L-半胱氨酸亚磺酸被脱硫生成L-丙氨酸，因此我们将这种新的酶命名为半胱氨酸亚硫酸盐脱硫酶。用丙氨酸取代四个半胱氨基残基(Cys-100、Cys-176、Cys-323和Cys-358)的突变酶都是活性的，Cys-358对应于A.vinelandii NIFS的Cys-325，它在所有NIFS样蛋白中保守，在催化上是必需的(郑，L.，White，R.H.，Cash，V.L.和Dean，D.R.(1994)BioChemical 33,4714-4720)，不是半胱氨酸亚磺酸盐脱硫酶所必需的。因此，该酶在这方面不同于A.vinelandii NIFS。
Selenocysteine lyase (EC 4.4.1.16) exclusively decomposes selenocysteine to alanine and elemental selenium, whereas cysteine desulfurase (NIFS protein) of Azotobacter vinelandii acts indiscriminately on both cysteine and selenocysteine to produce elemental sulfur and selenium respectively, and alanine. These proteins exhibit some sequence homology, The Escherichia coli genome contains three genes with sequence homology to nifS. We have cloned the gene mapped at 63.4 min in the chromosome and have expressed, purified to homogeneity, and characterized the gene product, The enzyme comprises two identical subunits with 401 amino acid residues (M-r 43,238) and contains pyridoxal 5'-phosphate as a coenzyme. The enzyme catalyzes the removal of elemental sulfur and selenium atoms from L-cysteine, L-cystine, L-selenocysteine, and L-selenocystine to produce L-alanine, Because L-cysteine sulfinic acid was desulfinated to form L-alanine as the preferred substrate, we have named this new enzyme cysteine sulfinate desulfinase. Mutant enzymes having alanine substituted for each of the four cysteinyl residues (Cys-100, Cys-176, Cys-323, and Cys-358) were all active, Cys-358 corresponds to Cys-325 of A. vinelandii NIFS, which is conserved among all NIFS-like proteins and catalytically essential (Zheng, L., White, R. H., Cash, V. L., and Dean, D. R. (1994) Biochemistry 33, 4714-4720), is not required for cysteine sulfinate desulfinase. Thus, the enzyme is distinct from A. vinelandii NIFS in this respect.