Characterization of 1-Deoxy-D-xylulose 5-phosphate reductoisomerase, an enzyme involved in isopentenyl diphosphate biosynthesis, and identification of its catalytic amino acid residues
Characterization of 1-Deoxy-D-xylulose 5-phosphate reductoisomerase, an enzyme involved in isopentenyl diphosphate biosynthesis, and identification of its catalytic amino acid residues
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DOI：
10.1074/jbc.m001820200
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发表时间：
2000-06-30
影响因子：
4.8
通讯作者：
Seto, H
中科院分区：
文献类型：
--
作者：
Kuzuyama, T;Takahashi, S;Seto, H
1-脱氧-D-木酮糖-5-磷酸（DXP）还原异构酶（DXP reductoisomerase）是异戊烯基二磷酸生物合成途径中的一个关键酶，它催化DXP分子内重排和还原生成2-C-甲基-D-β-4-磷酸。编码这种酶的大肠杆菌的dxr基因被过表达为组氨酸标记的蛋白质，并进行了详细表征。对10株E. coli dxr缺陷突变体在四个点上显示碱基替换突变：两个无义突变和两个氨基酸替换（Gly（14）替换为Asp（14）和Glu（231）替换为Lys（231））。焦碳酸二乙酯处理使DXP还原异构酶失活，随后的羟胺处理恢复焦碳酸二乙酯处理的酶的活性。为了表征这些缺陷，我们过表达突变酶G14 D、E231 K、H153 Q、H209 Q和H257 Q。除G14 D外，所有这些突变酶均以可溶性蛋白质形式获得。虽然纯化的酶E231 K具有DXP和NADPH的野生型K-m值，但突变体酶具有小于0.24%的野生型k（cat）值。H153 Q、H209 Q和H257 Q对DXP的Km值分别增加至野生型值的3.5倍、7.6倍和19倍。这些结果表明，E.大肠杆菌DXP还原异构酶在DXP转化为2-C-甲基-D-C4-磷酸中起重要作用，His（153）、His（209）和His（257）部分与酶分子中的DXP结合有关。
1-Deoxy-D-xylulose 5-phosphate (DXP) reductoisomerase, which simultaneously catalyzes the intramolecular rearrangement and reduction of DXP to form 2-C-methyl-D-erythritol 4-phosphate, constitutes a key enzyme of an alternative mevalonate-independent pathway for isopentenyl diphosphate biosynthesis. The dxr gene encoding this enzyme from Escherichia coli was overexpressed as a histidine-tagged protein and characterized in detail. DNA sequencing analysis of the dxr genes from 10 E. coli dxr-deficient mutants revealed base substitution mutations at four points: two nonsense mutations and two amino acid substitutions (Gly(14) to Asp(14) and Glu(231) to Lys(231)), Diethyl pyrocarbonate treatment inactivated DXP reductoisomerase, and subsequent hydroxylamine treatment restored the activity of the diethyl pyrocarbonate-treated enzyme. To characterize these defects, we overexpressed the mutant enzymes G14D, E231K, H153Q, H209Q, and H257Q. All of these mutant enzymes except for G14D were obtained as soluble proteins. Although the purified enzyme E231K had wildtype K-m values for DXP and NADPH, the mutant enzyme had less than a 0.24% wild-type k(cat) value. K-m values of H153Q, H209Q, and H257Q for DXP increased to 3.5-, 7.6-, and 19-fold the wild-type value, respectively. These results indicate that Glu(231) of E. coli DXP reductoisomerase plays an important role(s) in the conversion of DXP to 2-C-methyl-D-erythritol 4-phosphate, and that His(153), His(209), and His(257), in part, associate with DXP binding in the enzyme molecule.