大肠杆菌丙酮酸甲酸裂解酶的失活：AdhE和小分子的作用
Inactivation of E. coli pyruvate formate-lyase: role of AdhE and small molecules.
作者信息
Nnyepi Mbako R, Peng Yi, Broderick Joan B
机构信息
Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59718, USA.
出版信息
Arch Biochem Biophys. 2007 Mar 1;459(1):1-9. doi: 10.1016/j.abb.2006.12.024. Epub 2007 Jan 12.
Abstract
Escherichia coli AdhE has been reported to harbor three distinct enzymatic activities: alcohol dehydrogenase, acetaldehyde-CoA dehydrogenase, and pyruvate formate-lyase (PFL) deactivase. Herein we report on the cloning, expression, and purification of E. coli AdhE, and the re-investigation of its purported enzymatic activities. While both the alcohol dehydrogenase and acetaldehyde-CoA dehydrogenase activities were readily detectable, we were unable to obtain any evidence for catalytic deactivation of PFL by AdhE, regardless of whether the reported cofactors for deactivation (Fe(II), NAD, and CoA) were present. Our results demonstrate that AdhE is not a PFL deactivating enzyme. We have also examined the potential for deactivation of active PFL by small-molecule thiols. Both beta-mercaptoethanol and dithiothreitol deactivate PFL efficiently, with the former providing quite rapid deactivation. PFL deactivated by these thiols can be reactivated, suggesting that this deactivation is non-destructive transfer of an H atom equivalent to quench the glycyl radical.
摘要
据报道，大肠杆菌AdhE具有三种不同的酶活性：乙醇脱氢酶、乙醛辅酶A脱氢酶和丙酮酸甲酸裂解酶（PFL）失活酶。在此，我们报告了大肠杆菌AdhE的克隆、表达和纯化，以及对其所谓酶活性的重新研究。虽然乙醇脱氢酶和乙醛辅酶A脱氢酶的活性很容易检测到，但无论是否存在报道的失活辅因子（Fe(II)、NAD和辅酶A），我们都无法获得任何证据证明AdhE对PFL有催化失活作用。我们的结果表明，AdhE不是一种PFL失活酶。我们还研究了小分子硫醇使活性PFL失活的可能性。β-巯基乙醇和二硫苏糖醇都能有效地使PFL失活，前者失活速度相当快。被这些硫醇失活的PFL可以重新激活，这表明这种失活是通过相当于淬灭甘氨酰自由基的氢原子的非破坏性转移实现的。
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本文引用的文献
1
S-Adenosylmethionine: a wolf in sheep's clothing, or a rich man's adenosylcobalamin?S-腺苷甲硫氨酸：披着羊皮的狼，还是富人的腺苷钴胺素？
Chem Rev. 2003 Jun;103(6):2129-48. doi: 10.1021/cr020422m.
2
Expression of the Escherichia coli yfiD gene responds to intracellular pH and reduces the accumulation of acidic metabolic end products.大肠杆菌yfiD基因的表达对细胞内pH作出响应，并减少酸性代谢终产物的积累。
Microbiology (Reading). 2002 Apr;148(Pt 4):1015-1026. doi: 10.1099/00221287-148-4-1015.
3
Adenosylmethionine as a source of 5'-deoxyadenosyl radicals.作为5'-脱氧腺苷自由基来源的腺苷甲硫氨酸。
Curr Opin Chem Biol. 2001 Oct;5(5):506-11. doi: 10.1016/s1367-5931(00)00237-4.
4
Adenosylmethionine-dependent iron-sulfur enzymes: versatile clusters in a radical new role.依赖腺苷甲硫氨酸的铁硫酶：具有全新作用的多功能簇
J Biol Inorg Chem. 2001 Mar;6(3):209-26. doi: 10.1007/s007750100210.
5
Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods.自由基S-腺苷甲硫氨酸，一个将常见生物合成途径中未解决的步骤与自由基机制联系起来的新型蛋白质超家族：使用新的分析和信息可视化方法进行功能表征。
Nucleic Acids Res. 2001 Mar 1;29(5):1097-106. doi: 10.1093/nar/29.5.1097.
6
The anaerobic ribonucleotide reductase from Escherichia coli. The small protein is an activating enzyme containing a [4fe-4s](2+) center.
J Biol Chem. 1999 Oct 29;274(44):31291-6. doi: 10.1074/jbc.274.44.31291.
7
The aerobic/anaerobic interface.
Curr Opin Microbiol. 1999 Apr;2(2):181-7. doi: 10.1016/S1369-5274(99)80032-0.
8
Activation of the anaerobic ribonucleotide reductase from Escherichia coli. The essential role of the iron-sulfur center for S-adenosylmethionine reduction.大肠杆菌厌氧核糖核苷酸还原酶的激活。铁硫中心在S-腺苷甲硫氨酸还原中的关键作用。
J Biol Chem. 1997 Sep 26;272(39):24216-23. doi: 10.1074/jbc.272.39.24216.
9
The free radical of the anaerobic ribonucleotide reductase from Escherichia coli is at glycine 681.来自大肠杆菌的厌氧核糖核苷酸还原酶的自由基位于甘氨酸681处。
J Biol Chem. 1996 Mar 22;271(12):6827-31.
10
An iron-sulfur center and a free radical in the active anaerobic ribonucleotide reductase of Escherichia coli.大肠杆菌活性厌氧核糖核苷酸还原酶中的一个铁硫中心和一个自由基。
J Biol Chem. 1993 Feb 5;268(4):2296-9.
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