Nguyen Kiet T, Hu Xubo, Colton Craig, Chakrabarti Ratna, Zhu Michael X, Pei Dehua
Department of Chemistry, and Ohio State Biochemistry Program, The Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210, USA.
Biochemistry. 2003 Aug 26;42(33):9952-8. doi: 10.1021/bi0346446.
Ribosomal protein synthesis in eubacteria and eukaryotic organelles initiates with an N-formylmethionyl-tRNA(i), resulting in N-terminal formylation of all nascent polypeptides. Peptide deformylase (PDF) catalyzes the subsequent removal of the N-terminal formyl group from the majority of bacterial proteins. Deformylation was for a long time thought to be a feature unique to the prokaryotes, making PDF an attractive target for designing novel antibiotics. However, recent genomic sequencing has revealed PDF-like sequences in many eukaryotes, including man. In this work, the cDNA encoding Homo sapiens PDF (HsPDF) has been cloned and a truncated form that lacks the N-terminal 58-amino-acid targeting sequence was overexpressed in Escherichia coli. The recombinant, Co(2+)-substituted protein is catalytically active in deformylating N-formylated peptides, shares many of the properties of bacterial PDF, and is strongly inhibited by specific PDF inhibitors. Expression of HsPDF fused to the enhanced green fluorescence protein in human embryonic kidney cells revealed its location in the mitochondrion. However, HsPDF is much less active than its bacterial counterpart, providing a possible explanation for the apparent lack of deformylation in the mammalian mitochondria. The lower catalytic activity is at least partially due to mutation of a highly conserved residue (Leu-91 in E. coli PDF) in mammalian PDF. PDF inhibitors had no detectable effect on two different human cell lines. These results suggest that HsPDF is likely an evolutional remnant without any functional role in protein formylation/deformylation and validates PDF as an excellent target for antibacterial drug design.
真细菌和真核细胞器中的核糖体蛋白质合成起始于N-甲酰甲硫氨酰-tRNA(i)，导致所有新生多肽的N端甲酰化。肽脱甲酰基酶（PDF）催化随后从大多数细菌蛋白质中去除N端甲酰基。长期以来，脱甲酰化一直被认为是原核生物特有的特征，这使得PDF成为设计新型抗生素的一个有吸引力的靶点。然而，最近的基因组测序在包括人类在内的许多真核生物中发现了类似PDF的序列。在这项工作中，编码人类PDF（HsPDF）的cDNA已被克隆，并且在大肠杆菌中过表达了一种缺少N端58个氨基酸靶向序列的截短形式。重组的、Co(2+)取代的蛋白质在使N-甲酰化肽脱甲酰基方面具有催化活性，具有许多细菌PDF的特性，并受到特定PDF抑制剂的强烈抑制。在人胚肾细胞中表达与增强型绿色荧光蛋白融合的HsPDF揭示了其在线粒体中的定位。然而，HsPDF的活性远低于其细菌对应物，这为哺乳动物线粒体中明显缺乏脱甲酰化现象提供了一种可能的解释。较低的催化活性至少部分是由于哺乳动物PDF中一个高度保守的残基（大肠杆菌PDF中的Leu-91）发生了突变。PDF抑制剂对两种不同的人类细胞系没有可检测到的影响。这些结果表明，HsPDF可能是一种进化残余物，在蛋白质甲酰化/脱甲酰化过程中没有任何功能作用，并验证了PDF作为抗菌药物设计的一个优秀靶点。
