Grangemard I, Peypoux F, Wallach J, Das B C, Labbé H, Caille A, Genest M, Maget-Dana R, Ptak M, Bonmatin J M
Laboratoire de Biochimie Analytique et Synthèse Bioorganique, Université Claude Bernard Lyon, France.
J Pept Sci. 1997 Mar-Apr;3(2):145-54. doi: 10.1002/(sici)1099-1387(199703)3:2<145::aid-psc96>3.0.co;2-y.
The biosynthesis of bacterial isoleucyl-rich surfactins was controlled by supplementation of L-isoleucine to the culture medium. Two new variants, the [Ile4,7]- and [Ile2,4,7]surfactins, were thus produced by Bacillus subtilis and their separation was achieved by reverse-phase HPLC. Amino acids of the heptapeptide moiety were analysed by chemical methods, and the lipid moiety was identified by beta-hydroxy anteiso pentadecanoic acid by combined GC/MS. Sequences were established on the basis of two-dimensional NMR data. Because conformational parameters issuing from NMR spectra suggested that the cyclic backbone fold was globally conserved in the new variants, structure-activity relationships were discussed in details on the basis of the three-dimensional model of surfactin in solution. Indeed, both variants have increased surface properties compared with that of surfactin, and this improvement is assigned to an increase of the hydrophobicity of the apolar domain favouring micellization. Furthermore, the additional Leu-to-Ile substitution at position 2 in the [Ile2,4,7]surfactin leads to a substantial increase of its affinity for calcium, when compared with that of [Ile4,7]surfactin or surfactin. This effect is assigned, from the model, to an increase in the accessibility of the acidic side chains constituting the calcium binding site. Thus, the propensities of such active lipopeptides for both hydrophobic and electrostatic interactions were improved, further substantiating that they can be rationally designed.
通过向培养基中添加L-异亮氨酸来控制细菌富含异亮氨酸的表面活性素的生物合成。枯草芽孢杆菌由此产生了两种新变体，即[Ile4,7]-和[Ile2,4,7]表面活性素，并通过反相高效液相色谱法实现了它们的分离。通过化学方法分析了七肽部分的氨基酸，并通过气相色谱/质谱联用鉴定了脂质部分为β-羟基异十五烷酸。基于二维核磁共振数据确定了序列。由于核磁共振谱得出的构象参数表明新变体中的环状主链折叠在整体上是保守的，因此在表面活性素溶液三维模型的基础上详细讨论了构效关系。实际上，与表面活性素相比，这两种变体都具有增强的表面性质，这种改善归因于有利于胶束化的非极性结构域疏水性的增加。此外，与[Ile4,7]表面活性素或表面活性素相比，[Ile2,4,7]表面活性素在第2位额外的亮氨酸到异亮氨酸取代导致其对钙的亲和力大幅增加。根据模型，这种效应归因于构成钙结合位点的酸性侧链可及性的增加。因此，此类活性脂肽对疏水和静电相互作用的倾向都得到了改善，进一步证实了它们可以进行合理设计。
