Project Grant 2203472

COLLABORATIVE RESEARCH: ETHYLENE-FORMING ENZYME -WITH THE SUPPORT OF THE CHEMISTRY OF LIFE PROCESSES PROGRAM IN THE DIVISION OF CHEMISTRY, ROBERT HAUSINGER AND JIAN HU AT MICHIGAN STATE UNIVERSITY AND CHRISTO CHRISTOV AT MICHIGAN TECHNOLOGICAL UNIVERSITY ARE STUDYING THE CATALYTIC STRATEGY OF THE ETHYLENE-FORMING ENZYME (EFE). THIS BACTERIAL OR FUNGAL PROTEIN IS A MEMBER OF THE NON-HEME FE(II)- AND 2-OXOGLUTARATE (2OG)-DEPENDENT OXYGENASES AND IS DISTINCT FROM THE WELL-KNOWN PLANT ENZYME THAT ALSO FORMS ETHYLENE BUT BY A DISTINCT PROCESS. EFE CATALYZES TWO DISTINCT REACTIONS, WITH ONE PRODUCING ETHYLENE AND THE OTHER GENERATING THE NITROGEN-RICH COMPOUND GUANIDINE. ETHYLENE IS THE NATURAL PLANT-RIPENING HORMONE AND HAS BEEN ADVANCED AS A FUEL SOURCE ALTERNATIVE TO GASOLINE. GUANIDINE IS A POTENTIAL NITROGEN FERTILIZER. THUS, THE REACTIONS THAT PRODUCE ETHYLENE AND GUANIDINE ARE OF FUNDAMENTAL INTEREST AND HAVE POTENTIAL NATIONAL ECONOMIC BENEFITS AND SOCIETAL IMPACTS. INVESTIGATION OF THE EFE ENZYME WILL PROVIDE ADVANCED TRAINING IN BIOCHEMICAL, STRUCTURAL, AND COMPUTATIONAL APPROACHES TO TWO POSTDOCTORAL SCIENTISTS AND TO UNDERGRADUATE STUDENTS. SCIENTIFIC ADVANCES OBTAINED FROM THIS PROJECT WILL BE INCORPORATED INTO UNIVERSITY GRADUATE COURSES. IN ADDITION, THE FINDINGS FROM THESE STUDIES WILL BE COMMUNICATED TO LIFELONG LEARNERS IN THE GENERAL PUBLIC VIA PRESENTATIONS AT THE UNIVERSITY SCIENCE FESTIVAL. THE OVERARCHING GOAL OF THIS PROPOSAL IS TO ELUCIDATE THE CATALYTIC MECHANISM OF EFE BY A COMBINATION OF EXPERIMENTAL AND COMPUTATIONAL STUDIES. CRITICAL TO THIS PROCESS ARE STUDIES THAT EXAMINE THE ENZYME?S TWO REACTIONS: (1) DECOMPOSITION OF 2OG INTO THREE MOLECULES OF CARBON DIOXIDE/BICARBONATE AND ETHYLENE AND (2) THE CONVERSION OF 2OG TO SUCCINATE AND CARBON DIOXIDE AND TRANSFORMATION OF THE AMINO ACID L-ARGININE (L-ARG) INTO GUANIDINE AND L-?1-PYRROLINE-5-CARBOXYLATE. TO UNDERSTAND THE MOLECULAR DETERMINANTS THAT CONTROL THE RELATIVE ACTIVITIES FOR THE TWO REACTIONS, BIOCHEMICAL, STRUCTURAL, AND COMPUTATIONAL APPROACHES WILL BE APPLIED TO SITE-DIRECTED VARIANTS OF THE BEST STUDIED EFE FROM THE BACTERIUM PSEUDOMONAS SYRINGAE, AN EFE HOMOLOG FROM THE FUNGUS PENICILLIUM DIGITATUM, AND TO RECONSTRUCTED ANCESTRAL EFE PROTEINS. USING THIS INFORMATION ALONG WITH STRUCTURE-GUIDED PROTEIN ENGINEERING, EFE VARIANTS THAT ARE OPTIMIZED FOR PRODUCING THE BIOFUEL ETHYLENE OR THE PLANT FERTILIZER GUANIDINE WILL BE CREATED. THIS WORK SIGNIFICANTLY EXTENDS THE STRUCTURAL/MECHANISTIC INFORMATION AVAILABLE FOR OTHER IMPORTANT FE(II)- AND 2OG-DEPENDENT OXYGENASES THAT ARE UNABLE TO CATALYZE THESE REACTIONS. THIS AWARD REFLECTS NSF'S STATUTORY MISSION AND HAS BEEN DEEMED WORTHY OF SUPPORT THROUGH EVALUATION USING THE FOUNDATION'S INTELLECTUAL MERIT AND BROADER IMPACTS REVIEW CRITERIA.