Engineering a change in metal‐ion specificity of the iron‐dependent superoxide dismutase from Mycobacterium tuberculosis

Abstract

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 Å 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 Å and 2.5 Å , 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.Keywords : superoxide dismutase; Mycobacterium tuberculosis; X-ray structure; site-directed mutagenesis.The Mycobacterium tuberculosis superoxide dismutase at an approximate distance of 18 Å . The structure of the M. tu-(SOD) is a 207-residue enzyme with a subunit molecular mass berculosis SOD has recently been determined at 2.0-Å resolution of 23 kDa . It is one of a homologous series establishing that it is a compact tetramer. of superoxide dismutases with Aϩβ tertiary structure which have A diagram of the tertiary structure of the M. tuberculosis SOD either iron or manganese located at the catalytic centre and can is shown in Fig. 1 where the metal-binding site is indicated. be either dimeric or tetrameric. X-ray structures for several other Comparison of the dimer-dimer contacts within the tetrameric members of this family are available: human (Borgstahl et al., members of this family show that they vary greatly from enzyme 1992), Bacillus stearothermophilus (Parker and Blake, 1988a), to enzyme (Wagner et al., 1993;Cooper et al., 1995) giving rise Escherichia coli (Lah et al., 1995), Pseudomonas ovalis (Stod-to a range of quaternary structures despite strong conservation dard et al., 1990) and Thermus thermophilus (Ludwig et al., of the dimer structure. The active-site residues of Fe-SOD and 1991). These enzymes are structurally distinct from the Cu-Zn Mn-SOD are almost completely ...

Elemental Analysis ofMycobacterium avium-,Mycobacterium tuberculosis-, andMycobacterium smegmatis-Containing Phagosomes Indicates Pathogen-Induced Microenvironments within the Host Cell’s Endosomal System

“…However, the fact that a similar increase in the zinc concentration was observed in the presence of TNF-␣ or IFN-␥ in infected macrophages implies specific regulatory mechanisms for the delivery of this element to the mycobacterial phagosome. In this respect, the observation that a Cu,Zn-SOD contributes to the resistance of M. tuberculosis against oxidative burst products generated by activated macrophages is intriguing (14).…”

“…Not only is iron important for growth but either iron or manganese is also part of the active center of stress-resistance proteins such as superoxide dismutase (SOD), 5 a known scavenger of oxygen radicals. SOD has been described in pathogenic M. avium and M. tuberculosis (14,15). However, it is unclear whether the iron concentration in the mycobacterial phagosome in macrophages is limiting, as documented previously for Salmonella-containing vacuoles in epithelial cells.…”

The Single Superoxide Dismutase of Rhodobacter capsulatus Is a Cambialistic, Manganese-Containing Enzyme

“…Standard errors in metal determination and activity measurements were less than 5 and 10%, respectively. has been ascribed to the oxidation of an essential tryptophan residue in the vicinity of the iron binding site (4,42,46), although the actual identity of the reactive amino acid is still a matter of debate (7,27,37,45). A recombinant SOD Rc reconstituted with Mn was virtually insensitive to H 2 O 2 , whereas Fe incorporation led to a holoenzyme that was inhibited 60 to 70% by a similar treatment (data not shown).…”

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“…Extracellular proteins of M. tuberculosis are of great interest because of the likelihood that these proteins play critical roles in host-pathogen interaction, because these proteins are leading candidates for incorporation into subunit vaccines, and because these proteins are prime targets for the development of new antimycobacterial drugs (1)(2)(3)(4).…”

“…Superoxide dismutase has been characterized at the DNA and protein level in several mycobacterial species in addition to M. tuberculosis. All of these enzymes are members of a family of homologous superoxide dismutases with similar biochemical properties and tertiary structure, combining either two or four subunits to form the active enzyme and coordinating either iron or manganese ions at the catalytic site (4,8,9). A feature of the M. tuberculosis superoxide dismutase that distinguishes it from the other mycobacterial enzymes studied is that it binds iron as its metal ion ligand instead of manganese (4).…”