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Michael Waterman
Faculty Member
Last active: 2/12/2015

Folding requirements are different between sterol 14alpha-demethylase (CYP51) from Mycobacterium tuberculosis and human or fungal orthologs.

Lepesheva GI, Podust LM, Bellamine A, Waterman MR
J Biol Chem. 2001 276 (30): 28413-20

PMID: 11373285 · DOI:10.1074/jbc.M102767200

Upon sequence alignment of CYP51 sterol 14alpha-demethylase from animals, plants, fungi, and bacteria, arginine corresponding to Arg-448 of CYP51 in Mycobacterium tuberculosis (MT) is conserved near the C terminus of all family members. In MTCYP51 Arg-448 forms a salt bridge with Asp-287, connecting beta-strand 3-2 with helix J. Deletion of the three C-terminal residues of MTCYP51 has little effect on expression of P450 in Escherichia coli. However, truncation of the fourth amino acid (Arg-448) completely abolishes P450 expression. We have investigated whether Arg-448 has other structural or functional roles in addition to folding and whether its conservation reflects conservation of a common folding pathway in the CYP51 family. Characterization of wild type protein and three mutants, R448K, R448I, and R448A, including examination of catalytic activity, secondary and tertiary structure analysis by circular dichroism and tryptophan fluorescence, and studies of both equilibrium and temporal MTCYP51 unfolding behavior, shows that Arg-448 does not play any role in P450 function or maintenance of the native structure. C-terminal truncation of Candida albicans and human CYP51 orthologs reveals that, despite conservation in sequence, the requirement for arginine at the homologous C-terminal position in folding in E. coli is not conserved. Thus, despite similar spatial folds, functionally related but evolutionarily distinct P450s can follow different folding pathways.

MeSH Terms (28)

Amino Acid Sequence Candida albicans Catalysis Circular Dichroism Conserved Sequence Cytochrome P-450 Enzyme System Escherichia coli Evolution, Molecular Gene Deletion Humans Hydrogen-Ion Concentration Light Models, Molecular Molecular Sequence Data Mutagenesis, Site-Directed Mutation Mycobacterium tuberculosis Oxidoreductases Protein Conformation Protein Folding Protein Structure, Tertiary Sequence Alignment Species Specificity Spectrophotometry Sterol 14-Demethylase Time Factors Tryptophan Yeasts

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