Meinnel T, Lazennec C, Blanquet S
Laboratoire de Biochimie Unité de Recherche Associée no. 1970 du Centre National de la Recherche Scientifique Ecole Polytechnique, Palaiseau, France.
J Mol Biol. 1995 Nov 24;254(2):175-83. doi: 10.1006/jmbi.1995.0609.
A set of 50 site-directed mutants of the Escherichia coli fms gene was constructed to delineate the residues of the active site of peptide deformylase, including the ligands of the zinc ion. In particular, because zinc is usually coordinate by Asp, Cys, Glu or His residues, all the corresponding codons were individually changed. The functional consequence of the substitutions was assessed by complementation of a fms-null strain with the help of vectors expressing the mutate genes. In addition to the mutations of the Cys90 codon, only those of the three conserved residues of the 132HEXXH136 motif of peptide deformylase prevented the indicator strain growing. Most enzyme variants were purified to homogeneity in a second step. Their characterization in vitro showed that the defects in complementation as observed in vivo corresponded to huge decreases of deformylation efficiency. The change of Glu88 also led to a significant decrease in catalytic rate. Unexpectedly, upon substitutions of Glu79 or of Glu83, the enzymes exhibited a strongly increased catalytic efficiency. The measurement of the content of zinc in each purified variant indicated that Cys90, His132 and His136 bound the metal ion. Zinc-free variants mutated at these positions were obtained and shown to display an increased sensitivity to proteolytic attack. Altogether, the data showed that both the presence of zinc and the conserved residues of the HEXXH motif were crucial for the activity of deformylase. This behaviour identified the enzyme as a member of the zinc metalloproteases superfamily. However, the unexpected participation in the binding of the zinc atom of Cys90, upstream from the HEXXH motif, suggested that peptide deformylase could be representative of a new sub-family, distinct from those of thermolysin and astacin.
构建了一组50个大肠杆菌fms基因的定点突变体，以确定肽脱甲酰基酶活性位点的残基，包括锌离子的配体。特别地，由于锌通常由天冬氨酸、半胱氨酸、谷氨酸或组氨酸残基配位，所有相应的密码子都被单独改变。通过在表达突变基因的载体的帮助下对fms缺失菌株进行互补来评估替换的功能后果。除了半胱氨酸90密码子的突变外，只有肽脱甲酰基酶的132HEXXH136基序的三个保守残基的突变阻止了指示菌株的生长。大多数酶变体在第二步中被纯化至同质。它们的体外特性表明，体内观察到的互补缺陷对应于脱甲酰基效率的大幅下降。谷氨酸88的变化也导致催化速率显著下降。出乎意料的是，在替换谷氨酸79或谷氨酸83后，这些酶表现出催化效率大幅提高。对每个纯化变体中锌含量的测量表明，半胱氨酸90、组氨酸132和组氨酸136与金属离子结合。获得了在这些位置发生突变的无锌变体，并显示出对蛋白水解攻击的敏感性增加。总之，数据表明锌的存在和HEXXH基序的保守残基对脱甲酰基酶的活性都至关重要。这种行为确定该酶为锌金属蛋白酶超家族的成员。然而，HEXXH基序上游的半胱氨酸90意外地参与锌原子的结合，这表明肽脱甲酰基酶可能代表一个新的亚家族，不同于嗜热菌蛋白酶和虾红素蛋白酶的亚家族。
