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A Commensal Dipeptidyl Aminopeptidase with Specificity for N-Terminal Glycine Degrades Human-Produced Antimicrobial Peptides in Vitro.
ACS Chemical Biology ( IF 4 ) Pub Date : 2018-08-23 , DOI: 10.1021/acschembio.8b00420
Janice H Xu 1 , Zhenze Jiang 2 , Angelo Solania 1 , Sandip Chatterjee 1 , Brian Suzuki 2 , Christopher B Lietz 2 , Vivian Y H Hook 2 , Anthony J O'Donoghue 2 , Dennis W Wolan 1
Affiliation  

Proteases within the C1B hydrolase family are encoded by many organisms. We subjected a putative C1B-like cysteine protease secreted by the human gut commensal Parabacteroides distasonis to mass spectrometry-based substrate profiling to find preferred peptide substrates. The P. distasonis protease, which we termed Pd_dinase, has a sequential diaminopeptidase activity with strong specificity for N-terminal glycine residues. Using the substrate sequence information, we verified the importance of the P2 glycine residue with a panel of fluorogenic substrates and calculated kcat and KM for the dipeptide glycine-arginine-AMC. A potent and irreversible dipeptide inhibitor with a C-terminal acyloxymethyl ketone warhead, glycine-arginine- AOMK, was then synthesized and demonstrated that the Pd_dinase active site requires a free N-terminal amine for potent and rapid inhibition. We next determined the homohexameric Pd_dinase structure in complex with glycine-arginine- AOMK and uncovered unexpected active site features that govern the strict substrate preferences and differentiate this protease from members of the C1B and broader papain-like C1 protease families. We finally showed that Pd_dinase hydrolyzes several human antimicrobial peptides and therefore posit that this P. distasonis enzyme may be secreted into the extracellular milieu to assist in gut colonization by inactivation of host antimicrobial peptides.

中文翻译:

具有特异性的N末端甘氨酸的商业化二肽氨基肽酶会体外降解人为产生的抗菌肽。

C1B水解酶家族中的蛋白酶由许多生物体编码。我们对人类肠道共副细菌副细菌分泌的一种假定的C1B样半胱氨酸蛋白酶进行了基于质谱的底物谱分析,以发现优选的肽底物。我们将P.distasonis蛋白酶称为Pd_dinase,它具有顺序的二氨基肽酶活性,对N端甘氨酸残基具有很强的特异性。使用底物序列信息,我们用一组荧光底物验证了P2甘氨酸残基的重要性,并计算了二肽甘氨酸-精氨酸-AMC的kcat和KM。一种有效且不可逆的二肽抑制剂,带有C末端的酰氧基甲基酮战斗部,甘氨酸-精氨酸-AOMK,然后合成,并证明了Pd_dinase活性位点需要游离的N末端胺来有效且快速地抑制。接下来,我们确定了与甘氨酸-精氨酸-AOMK复杂的同六聚体Pd_dinase结构,并发现了意想不到的活性位点特征,这些特征控制着严格的底物偏好,并使该蛋白酶与C1B和更广泛的木瓜蛋白酶样C1蛋白酶家族的成员区分开。我们最终表明,Pd_dinase水解了几种人类抗菌肽,因此我们认为,这种Distasonis酶可能会分泌到细胞外环境中,以通过灭活宿主抗菌肽来协助肠道定植。接下来,我们确定了与甘氨酸-精氨酸-AOMK复杂的同六聚体Pd_dinase结构,并发现了意想不到的活性位点特征,这些特征控制着严格的底物偏好,并使该蛋白酶与C1B和更广泛的木瓜蛋白酶样C1蛋白酶家族的成员区分开。我们最终表明,Pd_dinase水解了几种人类抗菌肽,因此我们认为,这种Distasonis酶可能会分泌到细胞外环境中,以通过灭活宿主抗菌肽来协助肠道定植。接下来,我们确定了与甘氨酸-精氨酸-AOMK复杂的同六聚体Pd_dinase结构,并发现了意想不到的活性位点特征,这些特征控制着严格的底物偏好,并使该蛋白酶与C1B和更广泛的木瓜蛋白酶样C1蛋白酶家族的成员区分开。我们最终表明,Pd_dinase水解了几种人类抗菌肽,因此我们认为,这种Distasonis酶可能会分泌到细胞外环境中,以通过灭活宿主抗菌肽来协助肠道定植。
更新日期:2018-08-07
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