Department of Biophysics, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110 029, India.
Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, India.
Biochem J. 2018 Feb 9;475(3):547-560. doi: 10.1042/BCJ20170666.
Peptidyl-tRNA hydrolase (Pth) catalyzes the breakdown of peptidyl-tRNA into peptide and tRNA components. Pth from (Pth) was cloned, expressed, purified and crystallized in a native unbound (Pth-N) state and in a bound state with the phosphate ion and cytosine arabinoside (cytarabine) (Pth-C). Structures of Pth-N and Pth-C were determined at 1.36 and 1.10 Å resolutions, respectively. The structure of Pth-N showed that the active site is filled with water molecules. In the structure of Pth-C, a phosphate ion is present in the active site, while cytarabine is bound in a cleft which is located away from the catalytic site. The cytarabine-binding site is formed with residues: Gln19, Trp27, Glu30, Gln31, Lys152, Gln158 and Asp162. In the structure of Pth-N, the side chains of two active-site residues, Asn70 and Asn116, were observed in two conformations. Upon binding of the phosphate ion in the active site, the side chains of both residues were ordered to single conformations. Since Trp27 is present at the cytarabine-binding site, the fluorescence studies were carried out which gave a dissociation constant () of 3.3 ± 0.8 × 10 M for cytarabine. The binding studies using surface plasmon resonance gave a value of 3.7 ± 0.7 × 10 M. The bacterial inhibition studies using the agar diffusion method and the biofilm inhibition assay established the strong antimicrobial potential of cytarabine. It also indicated that cytarabine inhibited Gram-negative bacteria more profoundly when compared with Gram-positive bacteria in a dose-dependent manner. Cytarabine was also effective against the drug-resistant bacteria both alone as well as in combination with other antibiotics.
肽基-tRNA 水解酶 (Pth) 催化肽基-tRNA 分解为肽和 tRNA 成分。 从 (Pth) 克隆、表达、纯化并结晶为天然未结合态 (Pth-N) 和与磷酸离子和胞嘧啶阿拉伯糖苷 (阿糖胞苷) (Pth-C) 结合态。 Pth-N 和 Pth-C 的结构分别在 1.36 和 1.10 Å 分辨率下确定。 Pth-N 的结构表明活性位点充满了水分子。在 Pth-C 的结构中，磷酸离子存在于活性位点，而阿糖胞苷结合在远离催化位点的裂缝中。阿糖胞苷结合位点由残基形成：Gln19、Trp27、Glu30、Gln31、Lys152、Gln158 和 Asp162。在 Pth-N 的结构中，两个活性位点残基 Asn70 和 Asn116 的侧链观察到两种构象。在活性位点结合磷酸离子后，两个残基的侧链都被有序排列成单一构象。由于 Trp27 存在于阿糖胞苷结合位点，进行了荧光研究，得到阿糖胞苷的离解常数 () 为 3.3 ± 0.8 × 10 M。使用表面等离子体共振的结合研究给出了 3.7 ± 0.7 × 10 M 的值。琼脂扩散法和生物膜抑制测定的细菌抑制研究确立了阿糖胞苷具有很强的抗菌潜力。它还表明，阿糖胞苷以剂量依赖的方式更强烈地抑制革兰氏阴性细菌，而不是革兰氏阳性细菌。阿糖胞苷单独使用以及与其他抗生素联合使用对耐药细菌也有效。
