ENGINEERING A CHANGE IN METAL-ION SPECIFICITY OF THE IRON-DEPENDENT SUPEROXIDE-DISMUTASE FROM MYCOBACTERIUM-TUBERCULOSIS X-RAY STRUCTURE-ANALYSIS OF SITE-DIRECTED MUTANTS

Citation
K. Bunting et al., ENGINEERING A CHANGE IN METAL-ION SPECIFICITY OF THE IRON-DEPENDENT SUPEROXIDE-DISMUTASE FROM MYCOBACTERIUM-TUBERCULOSIS X-RAY STRUCTURE-ANALYSIS OF SITE-DIRECTED MUTANTS, European journal of biochemistry, 251(3), 1998, pp. 795-803
Citations number
47
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biology
ISSN journal
0014-2956
Volume
251
Issue
3
Year of publication
1998
Pages
795 - 803
Database
ISI
SICI code
0014-2956(1998)251:3<795:EACIMS>2.0.ZU;2-I
Abstract
We have refined the X-ray structures of two site-directed mutants of t he iron-dependent superoxide dismutase (SOD) from Mycobacterium tuberc ulosis, These mutations which affect residue 145 in the enzyme (H145Q and H145E) were designed to alter its metal-ion specificity. This resi due 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-c hain helps to coordinate the metal ion via a solvent molecule which is thought to be a hydroxide ion. The mutations were based on the observ ation that in the closely homologous manganese dependent SOD from Myco bacteriurm leprae, the only significant difference from the M. tubercu losis SOD within 10 Angstrom of the metal-binding site is the substitu tion of Gln for His at position 145. Hence an H145Q mutant of the M. t uberculosis (TB) SOD was engineered to investigate this residue's role in metal ion dependence and an isosteric H145E mutant was also expres sed. The X-ray structures of the H145Q and H145E mutants have been sol ved at resolutions of 4.0 Angstrom and 2.5 Angstrom, respectively, con firming that neither mutation has any gross effects on the conformatio n of the enzyme or the structure of che active site. The residue subst itutions 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 m utant SOD has manganese bound to it whereas the H145Q mutant SOD retai ns iron as the active-site metal. This alteration in metal specificity may reflect on the preference of manganese ions for anionic ligands.