Food Technology Division, Bhabha Atomic Research Centre, Mumbai 400085, India; Life Sciences Department, Homi Bhabha National Institute, Mumbai 400094, India.
Beamline Development and Application Section, Bhabha Atomic Research Centre, Mumbai 400085, India; Life Sciences Department, Homi Bhabha National Institute, Mumbai 400094, India.
Biochim Biophys Acta Gen Subj. 2024 May;1868(5):130598. doi: 10.1016/j.bbagen.2024.130598. Epub 2024 Mar 16.
Products of microbial protein metabolism in the gut can influence the health of the host in many ways. Members of the Bacteriodales, major commensals of the human colon have been associated with long-term intake of high-protein diets. Undigested proteins or peptides that reach the colon can be hydrolyzed by extra-cellular proteases found in some Bacteroides species into amino acids and peptides which can be further catabolized. In this communication, we have characterized one such secreted aminopeptidase (BfAP) from Bacteroides fragilis belonging to the M28 family which is capable of degrading peptides released from soybean protein after predigestion in the small intestine. The BfAP enzyme was cloned, expressed in E. coli, and purified to homogeneity. It is a metallopeptidase requiring Co ion for optimum activity at 55 °C and pH 8 and preferentially cleaves neutral aliphatic (Met/Leu) and positively charged (Arg/Lys) amino acids from the N-terminus of peptides. It showed high specificity for long peptides as well as proteins like β-casein. Structural analysis of BfAP and its orthologues using AlphaFold2 reveal a shared highly conserved M28 domain, but vary with respect to their N-terminal region with some of them possessing an additional cap domain which may be important for regulation of substrate binding. Although BfAP lacks the typical cap domain, it shows small extensions that can form a loop adjacent to the proposed active site and may affect substrate binding. We suggest that this secreted enzyme may play an important role in protein metabolism in the colon where Bacteroides species are abundant.
肠道中微生物蛋白质代谢产物可以在许多方面影响宿主的健康。拟杆菌门的成员是人类结肠的主要共生菌，与长期摄入高蛋白饮食有关。未被消化的蛋白质或肽到达结肠后，可以被某些拟杆菌物种分泌的细胞外蛋白酶水解成氨基酸和肽，然后进一步分解代谢。在本通讯中，我们从脆弱拟杆菌中鉴定了一种属于 M28 家族的分泌型氨肽酶（BfAP），它能够降解在小肠中预先消化的大豆蛋白释放的肽。BfAP 酶被克隆、在大肠杆菌中表达并纯化至均一性。它是一种金属蛋白酶，在 55°C 和 pH8 下需要 Co 离子才能达到最佳活性，优先从肽的 N 端切割中性脂肪族（Met/Leu）和带正电荷的（Arg/Lys）氨基酸。它对长肽以及β-酪蛋白等蛋白质具有很高的特异性。使用 AlphaFold2 对 BfAP 和其同源物进行结构分析表明，它们具有共享的高度保守的 M28 结构域，但在 N 端区域存在差异，其中一些具有额外的帽结构域，这可能对底物结合的调节很重要。尽管 BfAP 缺乏典型的帽结构域，但它显示出小的延伸部分，可以形成与假定的活性位点相邻的环，可能影响底物结合。我们认为这种分泌酶可能在富含拟杆菌的结肠中发挥重要作用，参与蛋白质代谢。
