Bal Naresh C, Agrawal Himanshu, Meher Akshaya K, Arora Ashish
Molecular and Structural Biology, Central Drug Research Institute, Lucknow, India.
Biol Chem. 2007 May;388(5):467-79. doi: 10.1515/BC.2007.057.
The enzyme peptidyl-tRNA hydrolase (Pth, EC 3.1.1.29) is essential for the viability of bacteria. The ORF Rv1014c of Mycobacterium tuberculosis H37Rv, designated as the mtpth gene, was cloned and over-expressed and the product was purified. Generation of polyclonal antibodies against the purified recombinant protein, termed MtPth, facilitated detection of endogenously expressed MtPth in M. tuberculosis H37Rv cell lysate. MtPth could release diacetyl-[(3)H]-lysine from diacetyl-[(3)H]-lysyl-tRNA(Lys) with Michaelis-Menten kinetic parameters of K (m)=0.7+/-0.2 microM and k (cat)=1.22+/-0.2 s(-1). Transformation of a pTrc99c/mtpth vector allowed the growth of E. coli thermosensitive Pth(ts) mutant strain AA7852 at the non-permissive temperature of 42 degrees C, demonstrating the in vivo activity of MtPth. In addition, at 39 degrees C, over-expression of MtPth in AA7852 cells allowed the cells to remain viable in the presence of up to 200 microg/ml erythromycin. A 3D fold based on NMR and a structural model based on the E. coli Pth crystal structure were generated for MtPth. The essential nature of conserved active-site residues N12, H22 and D95 of MtPth for catalysis was demonstrated by mutagenesis and complementation in E. coli mutant strain AA7852. Thermal and urea/guanidinium chloride (GdmCl)-induced unfolding curves for MtPth indicate a simple two-state unfolding process without any intermediates.
肽基 - tRNA水解酶（Pth，EC 3.1.1.29）对细菌的生存至关重要。结核分枝杆菌H37Rv的开放阅读框Rv1014c，命名为mtpth基因，被克隆并过量表达，其产物得到纯化。针对纯化的重组蛋白（称为MtPth）产生的多克隆抗体，有助于检测结核分枝杆菌H37Rv细胞裂解物中内源性表达的MtPth。MtPth能够从二乙酰 - [³H] - 赖氨酰 - tRNA（Lys）中释放二乙酰 - [³H] - 赖氨酸，其米氏动力学参数为K（m）=0.7±0.2微摩尔，k（cat）=1.22±0.2秒⁻¹。pTrc99c/mtpth载体的转化使大肠杆菌温度敏感型Pth（ts）突变株AA7852在42℃的非允许温度下生长，证明了MtPth的体内活性。此外，在39℃时，AA7852细胞中MtPth的过量表达使细胞在存在高达200微克/毫升红霉素的情况下仍能存活。基于核磁共振生成了MtPth的三维折叠结构，并基于大肠杆菌Pth晶体结构生成了结构模型。通过在大肠杆菌突变株AA7852中的诱变和互补，证明了MtPth保守活性位点残基N12、H22和D95催化的本质。MtPth的热诱导和尿素/氯化胍（GdmCl）诱导的解折叠曲线表明是一个简单的两态解折叠过程，没有任何中间体。
