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Engineering a Putative Zinc Binding Site in SOD5
Author(s) -
Romero Claudia Moreno,
Peterson Ryan L.,
Taylor Alexander B.,
Holloway Stephen P.,
Galaleldeen Ahmad,
Culotta Valeria Cizewski,
Hart P. John
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.836.7
Subject(s) - superoxide dismutase , candida albicans , reactive oxygen species , zinc , superoxide , chemistry , hydrogen peroxide , sod1 , mutant , binding site , yeast , biochemistry , active site , enzyme , corpus albicans , catalase , microbiology and biotechnology , biology , gene , organic chemistry
Cu‐Zn Superoxide Dismutase (SOD1) is an enzyme that catalyzes the disproportionation of superoxide into molecular oxygen and hydrogen peroxide at nearly diffusion‐controlled rates. SOD5 represents a new class of Cu‐only superoxide dismutases that catalyzes the same reaction close to the same rate. SOD5 is upregulated during Candida albicans infection. Candida albicans is a diploid fungus that is a causal agent of oral and genital yeast infections in humans. Systemic infections of C. albicans can be lethal in immunocompromised individuals. Competent immune systems can cure the infections easily as the host macrophage engulfs the fungus and releases reactive oxygen species (ROS) that aid in killing it. SOD5 differs from SOD1 in that it is monomeric and possesses a copper binding site but lacks a zinc binding site. In this study, we mutated residues V101 and E110 to generate a putative zinc binding site. The double mutant, similar to wild‐type SOD5, is insoluble. The protein was expressed, denatured, refolded, metallated with copper and zinc, and crystallized. The structure of the double mutant reveals that mutating residues 101 and 110 is insufficient to create a functional zinc‐binding site.