Characterization of a two-component alkanesulfonate monooxygenase from Escherichia coli
Characterization of a two-component alkanesulfonate monooxygenase from Escherichia coli
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DOI：
10.1074/jbc.274.38.26639
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发表时间：
1999-09-17
影响因子：
4.8
通讯作者：
Leisinger, T
中科院分区：
文献类型：
--
作者：
Eichhorn, E;van der Ploeg, JR;Leisinger, T
大肠杆菌ssuEADCB基因簇是利用链烷磺酸盐作为硫源所必需的，并且在硫酸盐或半胱氨酸饥饿的条件下表达。SsuD和SsuE蛋白过表达和表征。将SsuE纯化至均一，作为N-末端组氨酸标记的融合蛋白。天然SsuE是11，58，400的同二聚体酶，其催化FMN的NAD（P）H依赖性还原，但其也能够还原FAD或核黄素。使用阳离子交换、阴离子交换和疏水相互作用色谱法将SsuD蛋白纯化至> 98%纯度。该纯酶催化戊磺酸转化为亚硫酸盐和戊醛，并且能够磺化广泛的磺化底物，包括C-2至C-10未取代的线性烷烃磺酸盐、取代的乙磺酸和磺化缓冲液。SsuD催化是绝对依赖于FMNH 2和氧，并为2.1至4.2的SsuE/SsuD摩尔比在10 mM Tris-HCl，pH 9.1是最大的。天然SsuD是M-r 181，000的同源四聚体酶。这些结果表明，SsuD是一种宽范围的FMNH 2依赖性单加氧酶，催化烷磺酸盐氧解转化为亚硫酸盐和相应的醛，SsuE是为SsuD提供FMNH 2的FMN还原酶。
The Escherichia coli ssuEADCB gene cluster is required for the utilization of alkanesulfonates as sulfur sources, and is expressed under conditions of sulfate or cysteine starvation. The SsuD and SsuE proteins were overexpressed and characterized. SsuE was purified to homogeneity as an N-terminal histidine-tagged fusion protein. Native SsuE was a homodimeric enzyme of ill, 58,400, which catalyzed an NAD(P)H-dependent reduction of FMN, but it was also able to reduce FAD or riboflavin, The SsuD protein was purified to >98% purity using cation exchange, anion exchange, and hydrophobic interaction chromatography. The pure enzyme catalyzed the conversion of pentanesulfonic acid to sulfite and pentaldehyde and was able to desulfonate a wide range of sulfonated substrates including C-2 to C-10 unsubstituted linear alkanesulfonates, substituted ethanesulfonic acids and sulfonated buffers. SsuD catalysis was absolutely dependent on FMNH2 and oxygen, and was maximal for SsuE/SsuD molar ratios of 2.1 to 4.2 in 10 mM Tris-HCl, pH 9.1. Native SsuD was a homotetrameric enzyme of M-r 181,000. These results demonstrate that SsuD is a broad range FMNH2-dependent monooxygenase catalyzing the oxygenolytic conversion of alkanesulfonates to sulfite and the corresponding aldehydes, SsuE is the FMN reducing enzyme providing SsuD with FMNH2.