Galactofuranose生物合成在大肠杆菌k - 12:识别和克隆UDP-galactopyranose变位酶。
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拿骚点,马丁SL,布朗,韦斯顿,Monsey D,邓肯。麦克内尔先生,K
Galactofuranose生物合成在大肠杆菌k - 12:识别和克隆UDP-galactopyranose变位酶。
J Bacteriol。1996年2月,178 (4):1047 - 52。
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8576037 (在PubMed]
- 文摘
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我们已经克隆两个开放阅读框架(orf6和orf8)大肠杆菌的k - 12 rfb地区。大肠杆菌表达的基因T7lac启动子的控制下,产生大量的重组蛋白,其中大部分积累在不溶性包涵体。然而,获得了足够的可溶性蛋白质,用于放射分析旨在检测UDP-galactopyranose变位酶活动(UDP-galactopyranose UDP-galactofuranose)的转换。分析是基于高压液相色谱分离糖磷酸盐释放两种形式的UDP-galactose磷酸二酯酶治疗。原油orf6基因产物UDP - [alpha-D-U-14C] -galactopyranose转化成产品,在磷酸二酯酶治疗了化合物的保留时间相同的合成alpha-galactofuranose-1-phosphate。没有变位酶活性提取物中发现细胞缺乏orf6表达质粒或从orf8-expressing细胞。orf6基因产物的纯化是阴离子交换色谱和疏水作用色谱法。原油中提取和纯化蛋白转化为6 9%的UDP-galactopyranose呋喃糖形式。所示的酶也催化逆反应;在这种情况下,一个大约86% furanose-to-pyranose转换观察。 These observations strongly suggest that orf6 encodes UDP-galactopyranose mutase (EC 5.4.99.9), and we propose that the gene be designated glf accordingly. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified UDP-galactopyranose mutase revealed one major band, and analysis by electrospray mass spectrometry indicated a single major species with a molecular weight of 42,960 +/- 8, in accordance with that calculated for the Glf protein. N-terminal sequencing revealed that the first 15 amino acids of the recombinant protein corresponded to those expected from the published sequence. UV-visible spectra of purified recombinant enzyme indicated that the protein contains a flavin cofactor, which we have identified as flavin adenine dinucleotide.