Biosynthesis of Novel Pyoverdines by Domain Substitution in a Nonribosomal Peptide Synthetase of Pseudomonas aeruginosa
Biosynthesis of Novel Pyoverdines by Domain Substitution in a Nonribosomal Peptide Synthetase of Pseudomonas aeruginosa
复制标题
DOI：
10.1128/aem.01453-14
复制
发表时间：
2014-09-01
影响因子：
4.4
通讯作者：
Ackerley, David F.
中科院分区：
文献类型：
--
作者：
Calcott, Mark J.;Owen, Jeremy G.;Ackerley, David F.
Pyoverdine 是一种由荧光假单胞菌产生的荧光非核糖体肽铁载体。铜绿假单胞菌非核糖体肽合成酶 (NRPS) PvdD 包含两个模块，每个模块在 Pyoverdine 的 C 末端包含一个 L-苏氨酸残基。为了生成修饰的吡弗丁肽，我们将替代底物指定腺苷酸化 (A) 和肽键催化缩合 (C) 结构域替换到 PvdD 的第二个模块中。当仅替换 A 结构域时，如果引入的 A 结构域指定了苏氨酸，则所得菌株仅产生高水平的野生型吡维丁，否则为痕量水平。每当引入指定苏氨酸的 A 结构域时，观察到高水平的吡弗定合成表明这些非天然 A 结构域能够与 PvdD C 结构域有效地通讯。此外，出乎意料的观察结果是，非苏氨酸特异性A结构域仍然将苏氨酸掺入到pyoverdine中，这表明天然PvdD C结构域对掺入的氨基酸底物表现出比这些A结构域更强的选择性（即，引入的A结构域对苏氨酸残基的错误激活比PvdD C结构域对非苏氨酸残基的错误掺入更频繁）。相反，PvdD的C和A结构域的取代在两种情况下产生了高产率的合理修饰的pyoverdine，这些pyoverdine具有赖氨酸或丝氨酸残基代替末端苏氨酸。然而，C-A 结构域取代更常见地产生截短的肽产物，这可能是由于非功能性重组 NRPS 模板上的合成停滞所致。
Pyoverdine is a fluorescent nonribosomal peptide siderophore made by fluorescent pseudomonads. The Pseudomonas aeruginosa nonribosomal peptide synthetase (NRPS) PvdD contains two modules that each incorporate an L-threonine residue at the C-terminal end of pyoverdine. In an attempt to generate modified pyoverdine peptides, we substituted alternative-substrate-specifying adenylation (A) and peptide bond-catalyzing condensation (C) domains into the second module of PvdD. When just the A domain was substituted, the resulting strains produced only wild-type pyoverdine-at high levels if the introduced A domain specified threonine or at trace levels otherwise. The high levels of pyoverdine synthesis observed whenever the introduced A domain specified threonine indicated that these nonnative A domains were able to communicate effectively with the PvdD C domain. Moreover, the unexpected observation that non-threonine-specifying A domains nevertheless incorporated threonine into pyoverdine suggests that the native PvdD C domain exhibited stronger selectivity than these A domains for the incorporated amino acid substrate (i.e., misactivation of a threonine residue by the introduced A domains was more frequent than mis-incorporation of a nonthreonine residue by the PvdD C domain). In contrast, substitution of both the C and A domains of PvdD generated high yields of rationally modified pyoverdines in two instances, these pyoverdines having either a lysine or a serine residue in place of the terminal threonine. However, C-A domain substitution more commonly yielded a truncated peptide product, likely due to stalling of synthesis on a nonfunctional recombinant NRPS template.