Sánchez-Hidalgo Marina, Martínez-Bueno Manuel, Fernández-Escamilla Ana M, Valdivia Eva, Serrano Luis, Maqueda Mercedes
Departamento de Microbiología, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain.
J Antimicrob Chemother. 2008 Jun;61(6):1256-65. doi: 10.1093/jac/dkn126. Epub 2008 Apr 1.
Bacteriocins are antimicrobial peptides produced by bacteria and have a relatively narrow range of activity against closely related strains. AS-48 is a circular bacteriocin produced by Enterococcus faecalis that acts against many gram-positive and some gram-negative bacteria, and could well serve as a natural food preservative and antimicrobial agent. The structure of AS-48 is a five-helix bundle in which a hypothetical plane containing the C(alpha) atoms of E4, E20, E49 and E58 segregates a patch of positively charged residues from the rest of the hydrophobic or uncharged surface residues.
The aim of this study is to investigate the significance of the four glutamic residues with regard to the potency, stability and functionality of enterocin AS-48.
Four genetically engineered variants of AS-48 were obtained by replacing each glutamic residue with alanine by site-directed mutagenesis. Each mutant peptide was purified from E. faecalis cultures. The activity of highly concentrated samples and the MIC were determined against nine bacterial strains by the spot-assay method. Structural studies were made with circular dichroism (CD) spectroscopy.
Occasional alterations to the net charge of AS-48 did not significantly affect its activity when high concentrations of bacteriocin were used. Nevertheless, according to the MIC values, three of the four mutated peptides showed weaker activity against the majority of the gram-positive bacteria tested. CD spectroscopy showed that the derivatives were well structured, in a similar way to those of the native molecule, with no modifications in their helix content.
The spatial location of the Glu residues rather than their negative charge played a critical role in AS-48 target-cell specificity and bactericidal activity, because the replacement of Glu with Ala modify the interactions between neighbouring residues through their side chains and the interaction to the solvent affecting the protein stability and causing variations in the activity levels against identical organisms.
细菌素是由细菌产生的抗菌肽，对亲缘关系较近的菌株具有相对较窄的活性范围。AS - 48是一种由粪肠球菌产生的环状细菌素，对许多革兰氏阳性菌和一些革兰氏阴性菌都有作用，有望作为一种天然食品防腐剂和抗菌剂。AS - 48的结构是一个五螺旋束，其中包含E4、E20、E49和E58的Cα原子的假想平面将一片带正电荷的残基与其余疏水或不带电荷的表面残基分隔开。
本研究旨在探讨四个谷氨酸残基对肠球菌素AS - 48的效力、稳定性和功能的重要性。
通过定点诱变将每个谷氨酸残基替换为丙氨酸，获得了四种AS - 48的基因工程变体。每个突变肽从粪肠球菌培养物中纯化。通过斑点试验法测定了高浓度样品对九种细菌菌株的活性和最低抑菌浓度（MIC）。用圆二色性（CD）光谱进行结构研究。
当使用高浓度细菌素时，AS - 48净电荷的偶尔改变对其活性没有显著影响。然而，根据MIC值，四个突变肽中的三个对大多数测试的革兰氏阳性菌显示出较弱的活性。CD光谱表明，这些衍生物结构良好，与天然分子相似，其螺旋含量没有改变。
谷氨酸残基的空间位置而非其负电荷在AS - 48的靶细胞特异性和杀菌活性中起关键作用，因为用丙氨酸取代谷氨酸会通过其侧链改变相邻残基之间的相互作用以及与溶剂的相互作用，影响蛋白质稳定性并导致对相同生物体的活性水平发生变化。
