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首页> 外文期刊>Biochemistry >Escherichia coli dimethylallyl diphosphate : tRNAdimethylallyltransferase: Site-directed mutagenesis of highly conservedresidues
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Escherichia coli dimethylallyl diphosphate : tRNAdimethylallyltransferase: Site-directed mutagenesis of highly conservedresidues

机译：大肠杆菌二磷酸二甲基烯丙基酯：tRNA二甲基烯丙基转移酶：高度保守残基的定点诱变

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摘要

Dimethylallyl diphosphate:tRNA dimethylallyltransferase (DMAPP-tRNA transferase) catalyzes alkylation of the exocyclic amine of adenosine at position 37 in some tRNAs by the hydrocarbon moiety of dimethylallyl diphosphate (DMAPP). A multiple-sequence alignment of 28 gene sequences encoding DMAPP-tRNA transferases from various organisms revealed considerable homology, including II charged, 12 polar, and four aromatic amino acids that are highly conserved or conservatively substituted. Site-directed mutants were constructed for all of these amino acids, and a tripeptide Glu-Glu-Phe alpha -tubulin epitope was appended to the C-terminus of the protein to facilitate separation by immunoaffinity chromatography of overproduced mutant enzymes from coexpressed chromosomally encoded wild-type DMAPP-tRNA transferase. Steady-state kinetic constants were measured for wild-type DMAPP-tRNA transferase and the site-directed mutants using DMAPP and a 17-base RNA oligoribonucleotide corresponding to the stem-loop region of tRNA(Phe) as substrates. Substantial changes in k(cat), K-m(DMAPP), and/or K-m(RNA) were seen for several of the mutants, suggesting possible roles for these residues in substrate binding and catalysis.
机译：二磷酸二甲基烯丙酯：tRNA二甲基烯丙基转移酶（DMAPP-tRNA转移酶）通过二甲基烯丙基二磷酸酯（DMAPP）的烃基催化在某些tRNA中37位腺苷的环外胺烷基化。来自各种生物的编码DMAPP-tRNA转移酶的28个基因序列的多序列比对显示了相当的同源性，包括高度保守或保守取代的II带电荷，12极性和四个芳香族氨基酸。针对所有这些氨基酸构建定点突变体，并将三肽Glu-Glu-Pheα-微管蛋白表位附加到蛋白质的C末端，以利于通过免疫亲和层析从共表达的染色体编码野生物中分离过量生产的突变酶型DMAPP-tRNA转移酶。使用DMAPP和对应于tRNA（Phe）茎环区域的17个碱基的RNA寡核糖核苷酸作为底物，测量了野生型DMAPP-tRNA转移酶和定点突变体的稳态动力学常数。几个突变体的k（cat），K-m（DMAPP）和/或K-m（RNA）发生了实质性变化，表明这些残基在底物结合和催化中可能发挥作用。

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