Exploitation of the selectivity-conferring code of nonribosomal peptide synthetases for the rational design of novel peptide antibiotics
Exploitation of the selectivity-conferring code of nonribosomal peptide synthetases for the rational design of novel peptide antibiotics
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DOI：
10.1021/bi0259406
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发表时间：
2002-07-30
期刊：
影响因子：
2.9
通讯作者：
Marahiel, MA
中科院分区：
文献类型：
--
作者：
Eppelmann, K;Stachelhaus, T;Marahiel, MA
最近，苯丙氨酸激活腺苷酸化（A）结构域的晶体结构的解析揭示了非核糖体肽合成酶（NRPS）中同源底物氨基酸的特异性识别的结构基础。通过增加序列比较和同源性建模，我们成功地利用这些信息来破译NRPS的选择性赋予代码。每个密码子组合了NRPS A结构域的10个氨基残基，推测其构建底物结合口袋。在这项研究中，破译的代码是第一次合理地改变整个NRPS模块在体外和体内的底物特异性。首先，将表面活性素合成酶A的L-Glu活化起始模块C-A（Glu）-PCP的单残基Lys 239突变为Gln 239，以实现与假定的L-Gln活化结合口袋的完美匹配。突变蛋白C-A（Glu）-PCP（Lys 239-> Gln）的生化表征揭示了假定的底物特异性从L-Glu到L-Gln的改变，而催化效率没有降低。第二，根据赋予选择性的密码，L-Asp和L-Asn激活A结构域的结合口袋在三个位置上不同：分别是Val 299对Ile，His 322对Glu，和Ile 330对瓦尔。因此，来自表面活性素合成酶B的第二模块的重组A结构域AspA的结合口袋逐步适应于识别L-Asn。单，双，和三重突变体的生化表征显示，His 322代表一个关键位置，其突变足以引起预期的选择性开关。随后，将编码单突变体AspA（His 322-> Glu）的基因片段引入回表面活性素生物合成基因簇中。发现所得枯草芽孢杆菌C，C：1菌株产生预期的迄今未知的脂七肽[Asn（5）]表面活性素。这表明，定点诱变，指导NRPS A结构域的选择性赋予代码，代表了一个强大的替代NRPS生物合成模板的遗传操作和合理设计的新型肽抗生素。
Recently, the solved crystal structure of a phenylalanine-activating adenylation (A) domain enlightened the structural basis for the specific recognition of the cognate substrate amino acid in nonribosomal peptide synthetases (NRPSs). By adding sequence comparisons and homology modeling, we successfully used this information to decipher the selectivity-conferring code of NRPSs. Each codon combines the 10 amino residues of a NRPS A domain that are presumed to build up the substrate-binding pocket. In this study, the deciphered code was exploited for the first time to rationally alter the substrate specificity of whole NRPS modules in vitro and in vivo. First, the single-residue Lys239 of the L-Glu-activating initiation module C-A(Glu)-PCP Of the surfactin synthetase A was mutated to Gln239 to achieve a perfect match to the postulated L-Gln-activating binding pocket. Biochemical characterization of the mutant protein C-A(Glu)-PCP(Lys239 --> Gln) revealed the postulated alteration in substrate specificity from L-Glu to L-Gln without decrease in catalytic efficiency. Second, according to the selectivity-conferring code, the binding pockets Of L-Asp and L-Asn-activating A domains differs in three positions: Val299 versus Ile, His322 versus Glu, and Ile330 versus Val, respectively. Thus, the binding pocket of the recombinant A domain AspA, derived from the second module of the surfactin synthetases B, was stepwisely adapted for the recognition Of L-Asn. Biochemical characterization of single, double, and triple mutants revealed that His322 represents a key position, whose mutation was sufficient to give rise to the intended selectivity-switch. Subsequently, the gene fragment encoding the single-mutant AspA(His322 --> Glu) was introduced back into the surfactin biosynthetic gene cluster. The resulting Bacillus subtilis C, C:1 strain was found to produce the expected so far unknown lipoheptapeptide [Asn(5)]surfactin. This indicates that site-directed mutagenesis, guided by the selectivity-conferring code of NRPS A domains, represents a powerful alternative for the genetic manipulation of NRPS biosynthetic templates and the rational design of novel peptide antibiotics.