UBC Theses and Dissertations
Structural basis for heme degradation in Staphylococcus aureus Ukpabi, Georgia Nonye
IsdG and IsdI are heme degrading enzymes from the bacterium Staphylococcus aureus. Heme degrading enzymes use heme as a substrate and cofactor to degrade the porphyrin ring and release the central iron atom. Previous work on the structure of these enzymes showed metal coordination by a conserved His residue in the heme complex of the inactive IsdG-N7A variant and in the cobalt protoporphyrin IX complex of IsdI. In these structures, the porphyrin ring is highly distorted from planarity resulting in the β- and δ-meso carbons being displaced toward the distal side of the heme pocket and the α- and γ-meso carbons towards the His proximal side. This heme distortion is described as ruffling and is not present in the classical family of heme degrading enzymes called the heme oxygenases (HO) that bind heme in a planar manner. Thus, heme ruffling is proposed to be an important structural feature for the reaction mechanism of IsdG and IsdI. The role of heme ruffling in IsdI activity was examined in this study. For the first time, IsdI was cocrystallized in complex with its true substrate, heme, and the structure was solved to 1.50 Å resolution. The structure revealed extensive heme ruffling of 2.1 Å as determined by normal-coordinate analysis. IsdI was then engineered to adopt a flatter heme by mutating a conserved tryptophan residue, W66, in the heme pocket to the less bulky side chains of tyrosine, phenylalanine, leucine and alanine. Of the IsdI variants tested, only W66Y was amenable to X-ray structure solution. Heme ruffling in the variant was lessened to about 1.4 Å, demonstrating that W66 is an important contributor to heme distortion in IsdI. The activity of the W66Y variant was reduced to half that of wild-type suggesting a link between heme distortion and enzyme activity in IsdI.
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