De Arpan, Lupidi Giulio, Petrelli Dezemona, Vitali Luca A
School of Pharmacy, University of Camerino, Camerino, Italy.
School of Biosciences and Veterinary Medicine, University of Camerino, Camerino, Italy.
FEMS Microbiol Lett. 2016 May;363(9). doi: 10.1093/femsle/fnw066. Epub 2016 Mar 23.
Streptococcus mutans harbours an intracellular, human DPP IV-analogous enzyme Xaa-Pro dipeptidyl-peptidase (EC 3.4.14.11). According to previous reports, an extracellular isozyme in S. gordonii and S. suis has been associated with virulence. Speculating that even an intracellular form may aid in virulence of S. mutans, we have tried to purify, characterize and evaluate enzyme inhibition by specific inhibitors. The native enzyme was partially purified by ion-exchange and gel filtration chromatography. Owing to low yield, the enzyme was overexpressed in Lactococcus lactis and purified by affinity chromatography. The recombinant enzyme (rSm-XPDAP) had a specific activity of 1070 U mg(-1), while the Vmax and Km were 7 μM min(-1) and 89 ± 7 μM (n = 3), respectively. The serine protease inhibitor phenylmethylsulphonyl fluoride and a DPP IV-specific inhibitor diprotin A proved to be active against rSm-XPDAP. As a novel approach, the evaluation of the effect of anti-human DPP IV (AHD) drugs on rSm-XPDAP activity found saxagliptin to be effective to some extent (Ki = 129 ± 16 μM), which may lead to the synthesis and development of a new class of antimicrobial agents.
变形链球菌含有一种细胞内的、与人二肽基肽酶IV类似的酶——Xaa-Pro二肽基肽酶（EC 3.4.14.11）。根据先前的报道，戈登链球菌和猪链球菌中的一种细胞外同工酶与毒力有关。推测即使是细胞内形式的酶也可能有助于变形链球菌的毒力，我们试图纯化、表征该酶并评估其被特异性抑制剂抑制的情况。天然酶通过离子交换和凝胶过滤色谱法进行部分纯化。由于产量较低，该酶在乳酸乳球菌中进行了过表达，并通过亲和色谱法进行纯化。重组酶（rSm-XPDAP）的比活性为1070 U mg(-1)，而Vmax和Km分别为7 μM min(-1)和89 ± 7 μM（n = 3）。丝氨酸蛋白酶抑制剂苯甲基磺酰氟和DPP IV特异性抑制剂二肽素A被证明对rSm-XPDAP有活性。作为一种新方法，评估抗人DPP IV（AHD）药物对rSm-XPDAP活性的影响发现，沙格列汀在一定程度上有效（Ki = 129 ± 16 μM），这可能会促使合成和开发一类新型抗菌剂。
