Prolyl-tRNA synthetases (ProRSs) catalyze the covalent attachment of proline onto cognate transfer ribonucleic acids (tRNAs), an indispensable step for protein synthesis in all living organisms. ProRSs are modular enzymes and the "prokaryotic-like" ProRSs are distinguished from "eukaryotic-like" ProRSs by the presence of an editing insertion domain (INS) inserted between motifs 2 and 3 of the main catalytic domain. Earlier studies suggested that the presence of coupled-domain dynamics could contribute to catalysis; however, the role that the distal and highly mobile INS domain plays in catalysis at the synthetic active site is not completely understood. In the present study, a combination of theoretical and experimental approaches has been used to elucidate the precise role of INS domain dynamics. Quantum mechanical/molecular mechanical simulations were carried out to model catalytic prolyl-adenylate formation by Enterococcus faecalis ProRS. The energetics of the adenylate formation by the wild-type enzyme was computed and contrasted with variants containing active site mutations as well as a deletion mutant lacking the INS domain. The combined results revealed that two distinct types of dynamics contribute to the enzyme's catalytic power. One set of motions is intrinsic to the INS domain and leads to conformational preorganization that is essential for catalysis. A second type of motion, stemming from the electrostatic reorganization of active site residues, impacts the height and width of the energy profile and has a critical role in fine-tuning the substrate orientation to facilitate reactive collisions. Thus, motions in a distal domain can preorganize the active site of an enzyme to optimize catalysis.

Enzymes play important roles in many biological processes. Amino acid residues in the active site pocket of an enzyme, which are in direct contact with the substrate(s), are generally believed to be critical for substrate recognition and catalysis. Identifying and understanding how these "catalytic" residues help enzymes achieve enormous rate enhancement has been the focus of many structural and biochemical studies over the past several decades. Recent studies have shown that enzymes are intrinsically dynamic and dynamic coupling between distant structural elements is essential for effective catalysis in modular enzymes. Therefore, distal residues are expected to have impacts on enzyme function. However, few studies have investigated the role of distal residues on enzymatic catalysis. In the present study, effects of distal residue mutations on the catalytic function of an aminoacyl-tRNA synthetase, namely, prolyl-tRNA synthase, were investigated. The present study demonstrates that distal residues significantly contribute to catalysis of the modular Escherichia coli prolyl-tRNA synthetase by maintaining intrinsic protein flexibility.

No abstract

Metals enable numerous physiological processes ranging from respiration to nitrogen fixation. However, the role of metals in biology and biocatalysis is not appreciated by the general public. This lack of knowledge around biological metals can lead to misinformation, especially regarding vaccines and health products.Here, we present a series of easy-to-implement experiments and demonstrations that can be incorporated in the high school curriculum to introduce students to the role of metals in biology. Our results from running these experiments/demonstrations in virtual (N = 6−10) and in-person (N = 22; N = 9−12) formats reveal that only 9−30% of high school students are aware of the presence of metals in humans. These statistics can be changed to 48−100% by incorporating proposed experiments and content in the curriculum.

Metals enable numerous physiological processes ranging from respiration to nitrogen fixation. However, the role of metals in biology and biocatalysis is not appreciated by the general public. This lack of knowledge around biological metals can lead to misinformation, especially regarding vaccines and health products. Here, we present a series of easy-to-implement experiments and demonstrations that can be incorporated in high school and undergraduate curricula to introduce students to the role of metals in biology. Our results from running these experiments/demonstrations in virtual (N = 6-10) and in-person (N = 22) formats reveal that only 9-30% of high school students are aware of the presence of metals in humans. These statistics can be changed to 48-100% by incorporating proposed experiments and content in the curriculum.