Purification and characterization of TDP-D-glucose 4,6-dehydratase from anthracycline-producing streptomycetes.
Purification and characterization of TDP-D-glucose 4,6-dehydratase from anthracycline-producing streptomycetes.
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产蒽环类链霉菌 TDP-D-葡萄糖 4,6-脱水酶的纯化和表征。
DOI：
10.1099/00221287-138-4-779
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发表时间：
1992
期刊：
影响因子：
--
通讯作者：
Floss,HG
中科院分区：
文献类型：
--
作者：
Thompson,MW;Strohl,WR;Floss,HG
TDP-d-葡萄糖 4,6-脱水酶可将 TDP-d-葡萄糖转化为 TDP-d-4-酮-6-脱氧葡萄糖，从产生柔红霉素和鲍霉素的生物链霉菌中纯化至接近均质。 C5（968 倍纯化，回收率 41%），来自柔红霉素生产者Streptomyces peucetiusATCC 29050（1000 倍纯化，回收率 37%）。来自链霉菌的 TDP-d-葡萄糖 4,6-脱水酶。 C5和S。经SDS-PAGE和HPLC凝胶过滤确定鲫鱼为同源二聚体，亚基相对分子质量分别为39000和36000。对于来自两种生物体的酶，在不添加 NAD+ 的情况下，或者当 ADP-葡萄糖、ADP-甘露糖、GDP-甘露糖、UDP-葡萄糖或 UDP-半乳糖替代 TDP-d-葡萄糖作为底物时，观察到可忽略不计的活性。对于来自链霉菌属的酶。 C5，NAD+和TDP-d-葡萄糖的K'm值分别为19·2μm和31·3μm。 TDP-d-葡萄糖的 V'max 为 309 nmol min−1（mg 蛋白质）−1。对于S。 pucetiusenzyme，NAD+ 和 TDP-d-葡萄糖的 K'm 值分别为 20·1 μm34·7 μm。NAD+ 的 Vmax 值为 180 nmol min−1（mg 蛋白质）−1，TDP-d-葡萄糖的 Vmax 值为 201 nmol min−1（mg 蛋白质）−1。 TDP 是两种生物体中 TDP-d-葡萄糖 4,6-脱水酶的良好抑制剂。来自S.的TDP-d-葡萄糖4,6-脱水酶的N端氨基酸序列。 pucetius和来自红霉素生产者Saccharopolyspora erythraea的酶是相似的，而来自Streptomycesp的酶是相似的。 C5 含有与其他两种酶不同的 N 端氨基酸序列。
TDP-d-glucose 4,6-dehydratase, which converts TDP-d-glucose to TDP-d-4-keto-6-deoxyglucose, was purified to near-homogeneity from the daunorubicin and baumycin-producing organismStreptomycessp. C5 (968-fold purification with a 41% recovery), and from the daunorubicin producerStreptomyces peucetiusATCC 29050 (1000-fold purification with a 37% recovery). The TDP-d-glucose 4,6-dehydratases fromStreptomycessp. C5 andS. peucetiuswere determined by SDS-PAGE and HPLC gel filtration to be homodimers with subunit relative molecular masses of 39000 and 36000, respectively. For the enzymes from both organisms, negligible activity was observed in the absence of added NAD+, or when ADP-glucose, ADP-mannose, GDP-mannose, UDP-glucose or UDP-galactose was substituted for TDP-d-glucose as substrate. For the enzyme fromStreptomycessp. C5, theK'mvalues for NAD+and TDP-d-glucose were 19·2 μmand 31·3 μm, respectively. TheV'maxfor TDP-d-glucose was 309 nmol min−1(mg protein)−1. For theS. peucetiusenzyme, theK'mvalues for NAD+and TDP-d-glucose were 20·1 μm34·7 μm, respectively.Vmaxvalues were 180 nmol min−1(mg protein)−1for NAD+and 201 nmol min−1(mg protein)−1for TDP-d-glucose. TDP was a good inhibitor of TDP-d-glucose 4,6-dehydratase from both organisms. The N-terminal amino acid sequence of the TDP-d-glucose 4,6-dehydratase fromS. peucetiusand from the erythromycin producer,Saccharopolyspora erythraea, were similar, whereas the enzyme fromStreptomycessp. C5 contained a different N-terminal amino acid sequence from either of the other two enzymes.