Christensen Lise Friis, Høie Magnus Haraldson, Bang-Berthelsen Claus Heiner, Marcatili Paolo, Hansen Egon Bech
National Food Institute, Technical University of Denmark, Kemitorvet, DK-2800 Kongens Lyngby, Denmark.
Department of Health Technology, Technical University of Denmark, Ørsteds Plads, DK-2800 Kongens Lyngby, Denmark.
Microorganisms. 2023 Sep 8;11(9):2256. doi: 10.3390/microorganisms11092256.
Lactic acid bacteria (LAB) have an extracellular proteolytic system that includes a multi-domain, cell envelope protease (CEP) with a subtilisin homologous protease domain. These CEPs have different proteolytic activities despite having similar protein sequences. Structural characterization has previously been limited to CEP homologs of dairy- and human-derived LAB strains, excluding CEPs of plant-derived LAB strains. CEP structures are a challenge to determine experimentally due to their large size and attachment to the cell envelope. This study aims to clarify the prevalence and structural diversity of CEPs by using the structure prediction software AlphaFold 2. Domain boundaries are clarified based on a comparative analysis of 21 three-dimensional structures, revealing novel domain architectures of CEP homologs that are not necessarily restricted to specific LAB species or ecological niches. The C-terminal flanking region of the protease domain is divided into fibronectin type-III-like domains with various structural traits. The analysis also emphasizes the existence of two distinct domains for cell envelope attachment that are preceded by an intrinsically disordered cell wall spanning domain. The domain variants and their combinations provide CEPs with different stability, proteolytic activity, and potentially adhesive properties, making CEPs targets for steering proteolytic activity with relevance for both food development and human health.
乳酸菌（LAB）具有一种细胞外蛋白水解系统，该系统包括一个具有枯草杆菌蛋白酶同源蛋白酶结构域的多结构域细胞包膜蛋白酶（CEP）。尽管这些CEP具有相似的蛋白质序列，但它们具有不同的蛋白水解活性。以前，结构表征仅限于源自乳制品和人类的LAB菌株的CEP同源物，不包括源自植物的LAB菌株的CEP。由于CEP的尺寸较大且与细胞膜相连，因此通过实验确定其结构具有挑战性。本研究旨在使用结构预测软件AlphaFold 2来阐明CEP的普遍性和结构多样性。基于对21个三维结构的比较分析，明确了结构域边界，揭示了CEP同源物的新型结构域架构，这些架构不一定局限于特定的LAB物种或生态位。蛋白酶结构域的C端侧翼区域被分为具有各种结构特征的纤连蛋白III型样结构域。分析还强调存在两个不同的细胞膜附着结构域，在其之前有一个内在无序的细胞壁跨膜结构域。这些结构域变体及其组合为CEP提供了不同的稳定性、蛋白水解活性和潜在的粘附特性，使CEP成为调控蛋白水解活性的靶点，这与食品开发和人类健康都相关。
