具有不同底物特异性的氨肽酶参与细胞稳态的不同关键生理过程。它们在食品和制药行业也有广泛的应用。在细菌细胞内,广泛特异性氨肽酶主要通过降解由细胞ATP依赖性蛋白酶介导的初级蛋白水解产生的寡肽参与氨基酸的再循环。然而,在细菌中,一种真正广泛的特异性酶,可以分解酸性物质,基本的,Gly和疏水性氨基酸残基,极为罕见。这里,我们报告了一种推定的甘氨酰氨基肽酶(M61xc)的结构功能,该甘氨酰氨基肽酶来自Xanthomonaspvcampestris(Xcc),属于M61肽酶家族。该酶表现出广泛的特异性并切割Ala,Leu,Asp,Glu,Met,Ser,Phe,Tyr,Gly,Arg,和Lys在N终点站,最佳长度为3-7个氨基酸的肽。Further,我们报告了apo形式(2.1bias)和bestatin结合形式(1.95bias)的M61xc的高分辨率晶体结构,详细介绍其催化和底物偏好机制。对Xcc野生型和m61xc敲除突变株的粗细胞提取物中酶活性的比较分析阐明了M61xc的独特细胞内作用。该研究表明,M61xc是这些细菌中负责从肽的N-末端释放Asp/Glu残基的唯一酶。此外,鉴于其广泛的特异性和肽降解能力,它可以被认为等同于来自M17,M18,M42或S9等家族的M1或其他寡聚肽酶,它们在原核生物的蛋白酶体后蛋白降解中具有重要的辅助作用。
Aminopeptidases with varied substrate specificities are involved in different crucial physiological processes of cellular homeostasis. They also have wide applications in food and pharma industries. Within the bacterial cell, broad specificity aminopeptidases primarily participate in the recycling of amino acids by degrading oligopeptides generated via primary proteolysis mediated by cellular ATP-dependent proteases. However, in bacteria, a truly broad specificity enzyme, which can cleave off acidic, basic, Gly and hydrophobic amino acid residues, is extremely rare. Here, we report structure-function of a putative glycyl aminopeptidase (M61xc) from Xanthomonas campestris pv campestris (Xcc) belonging to the M61 peptidase family. The enzyme exhibits broad specificity and cleaves Ala, Leu, Asp, Glu, Met, Ser, Phe, Tyr, Gly, Arg, and Lys at the N terminus, optimally of peptides with a length of 3-7 amino acids. Further, we report the high-resolution crystal structure of M61xc in the apo form (2.1 Å) and bestatin-bound form (1.95 Å), detailing its catalytic and substrate preference mechanisms. Comparative analysis of enzyme activity in crude cell extracts from both wild-type and m61xc-knockout mutant strains of Xcc has elucidated the unique intracellular role of M61xc. This study suggests that M61xc is the exclusive enzyme in these bacteria that is responsible for liberating Asp/Glu residues from the N-termini of peptides. Also, in view of its broad specificity and peptide degradation ability, it could be considered equivalent to M1 or other oligomeric peptidases from families like M17, M18, M42 or S9, who have an important auxiliary role in post-proteasomal protein degradation in prokaryotes.