Tuesday, December 3, 2024
12/3/2024
Metalloenzymes are capable of efficiently tuning the properties of a metal ion to catalyze very difficult chemical transformations. Yet the determinants of what guides the evolution of protein function still remain not fully understood. This lack of understanding of the subtle detail of interactions that determine enzymatic function limits our ability to rationally design catalysts. We will test why the same family of enzymes uses different metallocofactors. Metalloenzyme NrdF belongs to a class of ribonucleotide reductases (RNR), essential enzymes found in all organisms to catalyze the conversion of nucleotides to deoxynucleotides. RNRs rely on metals to oxidize a conserved cysteine in the active site into a thiyl radical, which then initiates nucleotide reduction. Several different classes of RNRs have been identified in various organisms and, interestingly, despite a remarkable conservation of the overall catalytic pathway, the enzyme can utilize different metals to achieve it. We have recently shown that dimanganese center in class Ib RNRs of pathogenic organisms performs the same task as the diiron center in humans. Moreover, other class I RNR enzymes utilize diiron and mixed iron- manganese centers for function. The molecular and biological determinants of this metal preference still remain unknown and present a major unanswered question in the field. To address this question, we devised three specific aims. Aim 1. Elucidation of the origins of specific metalation in RNR. Using class Ib RNR from Streptococcus sanguinis (Ss) we will address the fundamental question of how correct metalation of enzymes is controlled. Aim 2. Design of functional RNR models. We will use a stable and simple protein model of RNR (DFsc) to bind various metal ions and generate catalysts for practically useful reactions. This aim will give simple, inexpensive and biocompatible protein catalysts for redox transformations and pesticide/chemical weapons remediation in the environment, that could be easily handled in the applied setting. Aim 3. Structural characterization of metalloproteins. Here we will test how metal ions influence the structure of the protein. The work proposed in this aim will validate and correlate the studies on the natural enzymes.
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