Program in Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, Viikinkaari 1, P.O. Box 65, FI-00014, Helsinki, Finland.
Antimicrobial Resistance Unit, Department of Infectious Disease Surveillance and Control, National Institute for Health and Welfare, P.O. Box 30, FI-00271, Helsinki, Finland.
Sci Rep. 2017 Jul 20;7(1):6020. doi: 10.1038/s41598-017-06135-w.
We introduce LytU, a short member of the lysostaphin family of zinc-dependent pentaglycine endopeptidases. It is a potential antimicrobial agent for S. aureus infections and its gene transcription is highly upregulated upon antibiotic treatments along with other genes involved in cell wall synthesis. We found this enzyme to be responsible for the opening of the cell wall peptidoglycan layer during cell divisions in S. aureus. LytU is anchored in the plasma membrane with the active part residing in the periplasmic space. It has a unique Ile/Lys insertion at position 151 that resides in the catalytic site-neighbouring loop and is vital for the enzymatic activity but not affecting the overall structure common to the lysostaphin family. Purified LytU lyses S. aureus cells and cleaves pentaglycine, a reaction conveniently monitored by NMR spectroscopy. Substituting the cofactor zinc ion with a copper or cobalt ion remarkably increases the rate of pentaglycine cleavage. NMR and isothermal titration calorimetry further reveal that, uniquely for its family, LytU is able to bind a second zinc ion which is coordinated by catalytic histidines and is therefore inhibitory. The pH-dependence and high affinity of binding carry further physiological implications.
我们介绍了 LytU，它是溶葡萄球菌素家族中一种短的锌依赖性五肽内肽酶。它是治疗金黄色葡萄球菌感染的潜在抗菌剂，其基因转录在抗生素治疗时会高度上调，同时还会上调其他参与细胞壁合成的基因。我们发现，这种酶在金黄色葡萄球菌的细胞分裂过程中负责打开细胞壁肽聚糖层。LytU 通过其活性部分位于周质空间的方式锚定在质膜上。它在位置 151 处具有独特的 Ile/Lys 插入，该插入位于催化位点邻近环中，对酶活性至关重要，但不影响溶葡萄球菌素家族共有的整体结构。纯化的 LytU 可裂解金黄色葡萄球菌细胞并切割五肽，该反应可通过 NMR 光谱方便地监测。用铜或钴离子替代辅助因子锌离子可显著提高五肽切割的速率。NMR 和等温滴定量热法进一步表明，LytU 能够独特地结合第二个锌离子，该锌离子由催化组氨酸配位，因此具有抑制作用。结合的 pH 依赖性和高亲和力具有进一步的生理意义。
