IDENTIFICATION OF CATALYTICALLY RELEVANT AMINO-ACIDS OF THE EXTRACELLULAR SERRATIA-MARCESCENS ENDONUCLEASE BY ALIGNMENT-GUIDED MUTAGENESIS
IDENTIFICATION OF CATALYTICALLY RELEVANT AMINO-ACIDS OF THE EXTRACELLULAR SERRATIA-MARCESCENS ENDONUCLEASE BY ALIGNMENT-GUIDED MUTAGENESIS
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DOI：
10.1093/nar/22.16.3280
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发表时间：
1994-08-25
影响因子：
14.9
通讯作者：
PINGOUD, A
中科院分区：
文献类型：
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作者：
FRIEDHOFF, P;GIMADUTDINOW, O;PINGOUD, A
通过胞外粘质沙雷氏菌核酸酶与三种相关核酸酶的序列比对，我们鉴定了在所有四个序列中保守的七个带电氨基酸残基。其中六个残基连同其他四个部分保守的 His 或 Asp 残基通过使用核酸酶基因变体的定点 PCR 介导的诱变被改变为丙氨酸，其中信号肽的编码序列被 N 端亲和标签 [Met(His)(6)GlySer] 的编码序列取代。其中四种突变蛋白的核酸酶活性几乎没有降低，但五种突变蛋白的活性降低了 10 至 1000 倍，其中一种 (His110Ala) 没有活性。基于这些结果表明，S.marcescens核酸酶采用了一种机制，其中His110与Mg2+离子和三个羧酸盐（Asp107、Glu148和Glu232）以及一个或两个碱性氨基酸残基（Arg108、Arg152）协同作用。
By sequence alignment of the extracellular Serratia marcescens nuclease with three related nucleases we have identified seven charged amino acid residues which are conserved in all four sequences. Six of these residues together with four other partially conserved His or Asp residues were changed to alanine by site-directed PCR-mediated mutagenesis using a variant of the nuclease gene in which the coding sequence of the signal peptide was replaced by the coding sequence for an N-terminal affinity tag [Met(His)(6)GlySer]. Four of the mutant proteins showed almost no reduction in nuclease activity but five displayed a 10- to 1000-fold reduction in activity and one (His110Ala) was inactive. Based upon these results it is suggested that the S.marcescens nuclease employs a mechanism in which His110 acts in concert with a Mg2+ ion and three carboxylates (Aspl07, Glu148 and Glu232) as well as one or two basic amino acid residues (Arg108, Arg152).