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