Espaillat Akbar, Carrasco-López Cesar, Bernardo-García Noelia, Rojas-Altuve Alzoray, Klett Javier, Morreale Antonio, Hermoso Juan A, Cava Felipe
Department of Molecular Biology and Laboratory for Molecular Infection Medicine Sweden, Umeå Centre for Microbial Research, Umeå University, Umeå, Sweden.
Department of Crystallography & Structural Biology, Institute of Physical-Chemistry "Rocasolano", CSIC, Madrid 28006, Spain.
Comput Struct Biotechnol J. 2021 Jan 26;19:1119-1126. doi: 10.1016/j.csbj.2021.01.031. eCollection 2021.
Broad-spectrum amino acid racemases (Bsrs) enable bacteria to generate non-canonical D-amino acids (NCDAAs), whose roles and impact on microbial physiology, including modulation of cell wall structure and dissolution of biofilms, are just beginning to be appreciated. Here we used a diverse array of structural, biochemical and molecular simulation studies to define and characterize how BsrV is post-translationally regulated. We discovered that contrary to alanine racemase AlrV highly compacted active site, BsrV's is broader and can be occupied by cell wall stem peptides. We found that peptidoglycan peptides modified with NCDAAs are better stabilized by BsrV's catalytic cavity and show better inhibitory capacity than canonical muropeptides. Notably, BsrV binding and inhibition can be recapitulated by undigested peptidoglycan sacculi as it exists in the cell. Docking simulations of BsrV binding the peptidoglycan polymer generate a model where the peptide stems are perfectly accommodated and stabilized within each of the dimeŕs active sites. Taking these biochemical and structural data together, we propose that inhibition of BsrV by peptidoglycan peptides underlies a negative regulatory mechanism to avoid excessive NCDAA production. Our results collectively open the door to use "à la carte" synthetic peptides as a tool to modulate DAAs production of Bsr enzymes.
广谱氨基酸消旋酶（Bsrs）使细菌能够产生非标准D-氨基酸（NCDAAs），其对微生物生理学的作用和影响，包括对细胞壁结构的调节和生物膜的溶解，才刚刚开始被认识。在这里，我们使用了一系列不同的结构、生化和分子模拟研究来定义和表征BsrV是如何进行翻译后调控的。我们发现，与丙氨酸消旋酶AlrV高度紧凑的活性位点相反，BsrV的活性位点更宽，可以被细胞壁茎肽占据。我们发现，用NCDAAs修饰的肽聚糖肽在BsrV的催化腔中更稳定，并且比标准的胞壁肽表现出更好的抑制能力。值得注意的是，BsrV的结合和抑制可以通过细胞中存在的未消化的肽聚糖囊泡来重现。BsrV与肽聚糖聚合物结合的对接模拟生成了一个模型，其中肽茎完美地容纳在每个二聚体活性位点内并得到稳定。综合这些生化和结构数据，我们提出肽聚糖肽对BsrV的抑制是一种负调控机制的基础，以避免过量产生NCDAAs。我们的研究结果共同为使用“定制”合成肽作为调节Bsr酶D-氨基酸产生的工具打开了大门。
