Expression and Characterization of Two Pathogenic Mutations in Human Electron Transfer Flavoprotein*
Expression and Characterization of Two Pathogenic Mutations in Human Electron Transfer Flavoprotein*
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DOI：
10.1074/jbc.272.42.26425
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发表时间：
1997-10
期刊：
影响因子：
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通讯作者：
D. Salazar;Lening Zhang;G. DeGala;F. Frerman
中科院分区：
文献类型：
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作者：
D. Salazar;Lening Zhang;G. DeGala;F. Frerman
电子转移黄素蛋白(ETF)或其电子受体电子转移黄素蛋白-泛醌氧化还原酶(ETF-QO)的缺陷导致人类遗传性代谢性疾病戊二酸II型。在这种疾病中，9种主要黄素蛋白脱氢酶到主呼吸链的电子传递受阻。在这些脱氢酶中，有四种脂肪酸β氧化的特定链长的黄素蛋白脱氢酶。在这项研究中，已在患者中发现的α亚单位的两个突变在大肠杆菌中表达。在αT266M和αG116R两个突变等位基因中，前者是在ETF缺乏症患者中发现的最常见的突变。人ETF的晶体结构表明，αG116位于α/β亚基界面的接触残基下的疏水口袋中，αT266的羟基氢与FAD的N(5)氢键；αT266的酰胺主链氢与黄素修复基的C(4)-O氢键(Roberts，D.L.，Frerman，F.E.和Kim，J-J)。P.(1996)论文集娜塔莉。阿卡德。SCI。美国第93号，14355-14360)。αG116RETF的稳定表达需要伴侣蛋白、GroEL和GroES的共同表达。αG116RETF折叠成与野生型不同的构象，在粗提物中没有催化活性。它不稳定，不能被广泛提纯。对αT266METF进行了纯化，并对粗提液中的蛋白质进行了稳定化处理。虽然该突变蛋白的整体结构没有改变，但其黄素环境发生了变化，吸收光谱和圆二色谱以及黄素从氧化和还原的蛋白质中释放的动力学表明。黄素N(5)位氢键的丧失和黄素结合的改变使黄素半喹酮的热力学稳定性比野生型ETF的半喹酮提高了10倍。该突变对肌氨酸和中链酰基辅酶A脱氢酶催化的ETF的还原半反应影响相对较小，这些酶可将黄素还原为半喹酮。但在以氧化αT266M ETF为底物的偶联酰辅酶A：泛醌还原酶分析中，ETF-QO的kcat/Km降低了33倍，这主要是由于ETF-QO催化αT266M ETF半醌歧化的速率降低所致。
Defects in electron transfer flavoprotein (ETF) or its electron acceptor, electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO), cause the human inherited metabolic disease glutaric acidemia type II. In this disease, electron transfer from nine primary flavoprotein dehydrogenases to the main respiratory chain is impaired. Among these dehydrogenases are the four chain length–specific flavoprotein dehydrogenases of fatty acid β-oxidation. In this investigation, two mutations in the α subunit that have been identified in patients were expressed inEscherichia coli. Of the two mutant alleles, αT266M and αG116R, the former is the most frequent mutation found in patients with ETF deficiency. The crystal structure of human ETF shows that αG116 lies in a hydrophobic pocket, under a contact residue of the α/β subunit interface, and that the hydroxyl hydrogen of αT266 is hydrogen-bonded to N(5) of the FAD; the amide backbone hydrogen of αT266 is hydrogen-bonded to C(4)-O of the flavin prosthetic group (Roberts, D. L., Frerman, F. E. and Kim, J-J. P. (1996)Proc. Natl. Acad. Sci. U. S. A. 93, 14355–14360). Stable expression of the αG116R ETF required coexpression of the chaperonins, GroEL and GroES. αG116R ETF folds into a conformation different from the wild type, and is catalytically inactive in crude extracts. It is unstable and could not be extensively purified. The αT266M ETF was purified and characterized after stabilization to proteolysis in crude extracts. Although the global structure of this mutant protein is unchanged, its flavin environment is altered as indicated by absorption and circular dichroism spectroscopy and the kinetics of flavin release from the oxidized and reduced protein. The loss of the hydrogen bond at N(5) of the flavin and the altered flavin binding increase the thermodynamic stability of the flavin semiquinone by 10-fold relative to the semiquinone of wild type ETF. The mutation has relatively little effect on the reductive half-reaction of ETF catalyzed by sarcosine and medium chain acyl-CoA dehydrogenases which reduce the flavin to the semiquinone. However, kcat/K m of ETF-QO in a coupled acyl-CoA:ubiquinone reductase assay with oxidized αT266M ETF as substrate is reduced 33-fold; this decrease is due in largest part to a decrease in the rate of disproportionation of the αT266M ETF semiquinone catalyzed by ETF-QO.