Project Grant R35GM151876

STRUCTURAL REQUIREMENTS FOR STEROL 14A-DEMETHYLASES - PROJECT SUMMARY CYTOCHROMES P450 (CYP) FORM ONE OF THE LARGEST ENZYME SUPERFAMILIES ON EARTH AND CAN BE DIVIDED INTO TWO GROUPS BASED ON THEIR SUBSTRATE PREFERENCES: THOSE METABOLIZING A VAST ARRAY OF XENOBIOTIC MOLECULES (E.G., DRUGS AND POLLUTANTS) AND THOSE WITH VERY STRICT REQUIREMENTS TOWARDS THEIR PHYSIOLOGICAL SUBSTRATES (E.G., STEROIDS AND VITAMINS). STEROL 14A-DEMETHYLASES (CYP51) REPRESENT A VERY SPECIAL P450 FAMILY, WHICH IS CURRENTLY REGARDED AS THE EVOLUTIONARY PROGENITOR FOR ALL EXISTING P450S. CYP51 ENZYMES ARE FOUND IN ALL KINGDOMS OF LIFE. WITH SEQUENCE IDENTITIES ACROSS PHYLA AS LOW AS 22-25%, THEY CATALYZE THE SAME THREE-STEP REGIO- AND STEREO-SPECIFIC REACTION THAT INVOLVES TWO SUCCESSIVE HYDROXYLATIONS FOLLOWED BY THE C-C BOND CLEAVAGE. THE CYP51 REACTION IS AN ESSENTIAL STEP IN THE BIOSYNTHESIS OF STEROLS AND SERVES AS THE MAJOR DRUG TARGET FOR THE TREATMENT OF FUNGAL INFECTIONS IN HUMANS AND PLANTS. WE HAVE VALIDATED CYP51 DRUGGABILITY ACROSS PHYLOGENY, INCLUDING PROTOZOA (TRYPANOSOMES, LEISHMANIA, AMOEBAS) AND HUMAN. WE DETERMINED 35 CYP51 X-RAY STRUCTURES AND SUCCESSFULLY APPLIED STRUCTURE-BASED DRUG DESIGN TO BUILD AN IN-HOUSE CHEMICAL LIBRARY OF NOVEL PATHOGEN-SELECTIVE INHIBITORS, WHICH ARE NON-TOXIC AND HAVE FAVORABLE PHARMACOKINETICS. OUR WORK HAS REVEALED THE CONSERVED MOLECULAR BASIS FOR CYP51 CATALYSIS, SPECIES-SPECIFIC VARIATIONS SUBSTRATE PREFERENCES AND SENSITIVITY TO INHIBITION, AND HAS ENABLED IDENTIFICATION OF CYP51 ORTHOLOGS IN >1000 BACTERIAL ORGANISMS, JUSTIFYING PROKARYOTIC ANCESTRY OF THE ENZYME. WE HAVE ALSO DISCOVERED THAT, AS PART OF CONSERVATION FROM BACTERIA TO HUMAN, CYP51 CATALYSIS INVOLVES LARGE-SCALE CONFORMATIONAL REARRANGEMENTS IN THE P450 MOLECULE. THE GOALS OF OUR RESEARCH UNDER MIRA SUPPORT WILL BE TO COMBINE THE STRENGTH OF DIFFERENT TECHNIQUES OF BIOCHEMISTRY, MOLECULAR AND STRUCTURAL BIOLOGY (CRYSTALLOGRAPHY, CRYO-ELECTRON MICROSCOPY, COMPUTATIONAL APPROACHES) IN ORDER TO UNDERSTAND HOW CONFORMATIONAL DYNAMICS GOVERNS CYP51 THROUGHOUT THE CATALYTIC CYCLE, MEDIATES ALLOSTERIC REGULATION, SPECIFICITY IN MOLECULAR RECOGNITION AND INTERMOLECULAR ASSEMBLIES OF MULTI-COMPONENT ELECTRON TRANSFER SYSTEMS. WE WILL GAIN NEW MECHANISTIC INSIGHTS INTO CYP51 ENZYME FUNCTION AND INHIBITION, AS WELL AS UNCOVER THE STRUCTURAL BASIS FOR ITS POSSIBLE ROLE IN P450 DIVERSIFICATION. THE CURRENT SET OF PROTEINS AVAILABLE FOR COMPARATIVE ANALYSIS COMPRISE CYP51S FORM ENDOPLASMIC RETICULUM OF HIGHER AND LOWER EUKARYOTES (VERTEBRATES, FUNGI, AND PROTOZOA), THEIR ENDOGENOUS ELECTRON DONOR PARTNERS CYTOCHROME P450 REDUCTASES, AND THE NATURAL CYP51-FERREDOXIN FUSION PROTEIN FROM THE CYTOPLASM OF A STEROL- MAKING BACTERIUM. ENZYMES FROM PLANTS THAT CONTAIN MULTIPLE CYP51 GENES WILL BE ADDED AS THE RESEARCH PROGRAM PROGRESSES.