Enzymes, the protein catalysts for biological reactions, are able to efficiently perform difficult chemical transformations with a high degree of specificity. The evolution of enzymatic function is believed to begin with a dynamic promiscuous enzyme, which then evolves to be more rigid and specific. By contrast, the protein design community has historically targeted highly stable and inflexible protein scaffolds. While the field has achieved the ability to design proteins with atomic-level precision, the creation of biologically and industrially relevant levels of enzymatic function has proven more elusive. CDM13 is simple, dynamic self-assembling ?pre-evolved? binuclear metalloprotein capable of performing a number of widely differing catalytic reactions. This promiscuous primordial enzyme provides a unique model system to study in detail what early enzymes were like and to examine the pathways in functional sequence space that connects these primordial enzymes to present day enzymes. This project will not only generate an improved understanding of natural binuclear enzyme evolution and function, but also in a palette of robust binuclear enzymes, which may prove useful in future microbial biofuel projects and as environmentally friendly hydroxylation catalysts. As part of the project undergraduates, graduate students and New York City Public High School teachers will be trained in the interdisciplinary science of biophysics at an institution with a student body which is composed of >55% underrepresented minorities. An outreach plan is presented which includes the development of a new planetarium demonstration of protein structure and dynamics.
CDM13 is a simple, dynamic self-assembling ?pre-evolved? binuclear metalloprotein capable of performing a number of widely differing catalytic tasks in solution and in vivo at rates that approach those observed in the natural binuclear enzymes that catalyze these reactions, including methane hydroxylation and arylamine oxidation as a diiron enzyme and hydrogen peroxide oxidation as a di-manganese enzyme. The binuclear enzyme family members each have two metal ions bound within a D2-symmetric antiparallel four alpha helical bundle. This class of proteins is ideal for because the natural binuclear enzymes have almost superimposable backbone structures, and thus aspects of the evolved catalytic functions can be easily transplanted. This project centers on the use of CDM13 to look at the evolution of specificity, dynamics, and bioenergetic coupling to protons on several different forms of multielectron catalysis in a single scaffold, positively identifying critical features necessary for enzyme function and specificity and giving us a new window on how modern enzymes have evolved their substrate and structural specificity.
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.
