Engineering a change in metal-ion specificity of the iron-dependent superoxide dismutase from Mycobacterium tuberculosis-- X-ray structure analysis of site-directed mutants.

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 Gln 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 Mycobacterium leprae, the only significant difference from the M. tuberculosis SOD within 10 A 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 A and 2.5 A, respectively, confirming that neither mutation has any gross effects on the conformation of the enzyme or the structure of the 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.

X-ray structure analysis of the iron-dependent superoxide dismutase from Mycobacterium tuberculosis at 2.0 Angstroms resolution reveals novel dimer-dimer interactions.

The X-ray structure of the tetrameric iron-dependent superoxide dismutase from Mycobacterium tuberculosis has been refined to an R-factor of 0.167 and a correlation coefficient of 0.954 at 2.0 A resolution. The crystals are monoclinic P2(1) and have four subunits related by strong non-crystallographic 222 (or D2) symmetry in the asymmetric unit. 198 of the 207 amino acids of each subunit are defined by the electron density which shows that they adopt the conserved fold of other iron- or manganese-dependent SODs. The structure can be divided into two domains, the N-terminal domain involving an extended region followed by two projecting antiparallel alpha-helices, and the C-terminal domain containing four more helical segments with a three-stranded antiparallel beta-sheet inserted sequentially between the fourth and fifth helices. The catalytic iron is co-ordinated by five ligands: three histidines (residues 28, 76 and 164), one aspartate (160) and a solvent molecule. The inferred positions of protons at the active site are consistent with the solvent ligand being a hydroxide ion. This ligand interacts with His145 in the Mycobacterium tuberculosis SOD. In the highly homologous Mycobacterium leprae Mn-SOD, the histidine is replaced by glutamine, this being the only significant residue difference within 10 A of the Fe3+. The nature of the amino acid at this position may influence the metal ion specificity of these enzymes. The subunits of the Mycobacterium tuberculosis SOD associate by polar contacts to form dimers, which closely resemble those of other dimeric or tetrameric Fe- or Mn-SODs. However, the dimer-dimer interactions within the tetramer are novel, being dominated by dimerisation of the 144 to 152 loop regions which connect the outer two beta-strands of the three-membered beta-sheet. This contrasts strongly with the other tetrameric Fe- or Mn-SODs where the dimer-dimer association is dominated by the projecting alpha alpha-turn in the N-terminal domain.