Gil Filipa, Catalão Maria João, Moniz-Pereira José, Leandro Paula, McNeil Michael, Pimentel Madalena
Unidade dos Retrovirus e Infecções Associadas, Centro de Patogénese Molecular, Faculty of Pharmacy, University of Lisbon, Portugal.
Unidade de Biologia Molecular e Biopatologia Experimental, iMed, Faculty of Pharmacy, University of Lisbon, Portugal.
Microbiology (Reading). 2008 May;154(Pt 5):1364-1371. doi: 10.1099/mic.0.2007/014621-0.
dsDNA bacteriophages use the dual system endolysin-holin to achieve lysis of their bacterial host. In addition to these two essential genes, some bacteriophages encode additional proteins within their lysis module. In this report, we describe the activity of a protein encoded by gene lysB from the mycobacteriophage Ms6. lysB is localized within the lysis cassette, between the endolysin gene (lysA) and the holin gene (hol). Analysis of the deduced amino acid sequence of LysB revealed the presence of a conserved motif (Gly-Tyr-Ser-Gln-Gly) characteristic of enzymes with lipolytic activity. A blast search within the sequences of protein databases revealed significant similarities to other putative proteins that are encoded by mycobacteriophages only, indicating that LysB and those proteins may be specific to their mycobacterial hosts. A screening for His(6)-LysB activity on esterase and lipase substrates confirmed the lipolytic activity. Examination of the kinetic parameters of recombinant His(6)-LysB for the hydrolysis of p-nitrophenyl esters indicated that although this protein could use a wide range of chain length substrates (C(4)-C(18)), it presents a higher affinity for p-nitrophenyl esters of longer chain length (C(16) and C(18)). Using p-nitrophenyl butyrate as a substrate, the enzyme showed optimal activity at 23 degrees C and pH 7.5-8.0. Activity was increased in the presence of Ca(2+) and Mn(2+). To the best of our knowledge, this is the first description of a protein with lipolytic activity encoded within a bacteriophage.
双链DNA噬菌体利用溶菌酶 - 穿孔素双系统实现对其细菌宿主的裂解。除了这两个必需基因外，一些噬菌体在其裂解模块中还编码额外的蛋白质。在本报告中，我们描述了分枝杆菌噬菌体Ms6的lysB基因编码的一种蛋白质的活性。lysB位于裂解盒内，在溶菌酶基因（lysA）和穿孔素基因（hol）之间。对LysB推导的氨基酸序列分析揭示了存在具有脂解活性的酶所特有的保守基序（甘氨酸 - 酪氨酸 - 丝氨酸 - 谷氨酰胺 - 甘氨酸）。在蛋白质数据库序列中进行的Blast搜索显示与仅由分枝杆菌噬菌体编码的其他推定蛋白质有显著相似性，表明LysB和那些蛋白质可能对其分枝杆菌宿主具有特异性。对His(6)-LysB在酯酶和脂肪酶底物上的活性进行筛选证实了其脂解活性。对重组His(6)-LysB水解对硝基苯酯的动力学参数进行检测表明，尽管该蛋白质可以使用多种链长的底物（C(4)-C(18)），但它对较长链长（C(16)和C(18)）的对硝基苯酯具有更高的亲和力。以对硝基苯丁酸为底物时，该酶在23℃和pH 7.5 - 8.0时表现出最佳活性。在Ca(2+)和Mn(2+)存在下活性增强。据我们所知，这是首次对噬菌体中编码的具有脂解活性的蛋白质进行描述。
