Engineering a change in metal-ion specificity of the iron-dependent superoxide dismutase from Mycobacterium tuberculosis X-Ray structure analysis of site-directed mutants
Engineering a change in metal-ion specificity of the iron-dependent superoxide dismutase from Mycobacterium tuberculosis X-Ray structure analysis of site-directed mutants
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DOI：
10.1046/j.1432-1327.1998.2510795.x
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发表时间：
1998-02-01
期刊：
影响因子：
--
通讯作者：
Young, D
中科院分区：
文献类型：
--
作者：
Bunting, K;Cooper, JB;Young, D
我们改进了结核分枝杆菌铁依赖性超氧化物歧化酶（SOD）的两种定点突变体的X射线结构，这些影响酶中残基145的突变（H145 Q和H145 E）旨在改变其金属离子特异性。该残基是同源SOD酶中的Gin或His，并且先前已显示在活性位点相互作用中起作用，因为其侧链有助于通过溶剂分子（被认为是氢氧根离子）来配位金属离子。这些突变是基于观察到麻风分枝杆菌的锰依赖性SOD与M.结核病SOD在10埃的金属结合位点内的一个特征是在位置145处用Gln取代His。因此，M.结核病（TB）SOD的基因工程研究这个残基的作用，金属离子的依赖性和等量的H145 E突变体也表达。H145Q和H145E突变体的X射线结构分别在4.0埃和2.5埃的分辨率下解析，证实了两种突变对酶的构象或活性位点的结构都没有任何明显的影响。残基取代被容纳在酶的三维结构中的小的局部构象变化。过氧化物抑制实验和原子吸收光谱法令人惊讶地建立了H145E突变体SOD具有与其结合的锰，而H145Q突变体SOD保留铁作为活性位点金属。这种金属特异性的改变可能反映了锰离子对阴离子配体的偏好。
We have refined the X-ray structures of two site-directed mutants of the iron-dependent superoxide dismutase (SOD) from Mycobacterium tuberculosis, These mutations which affect residue 145 in the enzyme (H145Q and H145E) were designed to alter its metal-ion specificity. This residue is either Gin or His in homologous SOD enzymes and has previously been shown to play a role in active-site interactions since its side-chain helps to coordinate the metal ion via a solvent molecule which is thought to be a hydroxide ion. The mutations were based on the observation that in the closely homologous manganese dependent SOD from Mycobacteriurm leprae, the only significant difference from the M. tuberculosis SOD within 10 Angstrom of the metal-binding site is the substitution of Gln for His at position 145. Hence an H145Q mutant of the M. tuberculosis (TB) SOD was engineered to investigate this residue's role in metal ion dependence and an isosteric H145E mutant was also expressed. The X-ray structures of the H145Q and H145E mutants have been solved at resolutions of 4.0 Angstrom and 2.5 Angstrom, respectively, confirming that neither mutation has any gross effects on the conformation of the enzyme or the structure of che active site. The residue substitutions are accommodated in the enzyme's three-dimensional structure by small local conformational changes. Peroxide inhibition experiments and atomic absorption spectroscopy establish surprisingly the H145E mutant SOD has manganese bound to it whereas the H145Q mutant SOD retains iron as the active-site metal. This alteration in metal specificity may reflect on the preference of manganese ions for anionic ligands.