Abstract
SummarySynthesis and scission of phosphodiester bonds in DNA and RNA regulate vital processes within the cell. Enzymes that catalyze these reactions operate mostly via the recognized two-metal-ion mechanism. Our analysis reveals that basic amino acids and monovalent cations occupy structurally conserved positions nearby the active site of many two-metal-ion enzymes for which high-resolution (<3 Å) structures are known, including DNA and RNA polymerases, nucleases such as Cas9, and splicing ribozymes. Integrating multiple-sequence and structural alignments with molecular dynamics simulations, electrostatic potential maps, and mutational data, we found that these elements always interact with the substrates, suggesting that they may play an active role for catalysis, in addition to their electrostatic contribution. We discuss possible mechanistic implications of this expanded two-metal-ion architecture, including inferences on medium-resolution cryoelectron microscopy structures. Ultimately, our analysis may inspire future experiments and strategies for enzyme engineering or drug design to modulate nucleic acid processing.
Highlights
Enzymatic cleavage and formation of phosphodiester bonds in DNA and RNA is central to life and health
K1 and K2 produce two discrete regions of positive electrostatic potential (+7.4 kT/e) at 4–8 Afrom the active site, which interrupts the negative potential created by first-shell coordinators of MA-MB (Figure 1A)
Stiffening of the active site, modulation of its electrostatic environment, and orientation of the reactants relative to the MA-MB center are needed by two-metal-ion enzymes to ensure specificity in substrate recognition and to augment the fidelity of the reaction (Hanoian et al, 2015; Jeltsch et al, 1993; Kurpiewski et al, 2004; Ramachandrakurup et al, 2016; Warshel et al, 2006)
Summary
Enzymatic cleavage and formation of phosphodiester bonds in DNA and RNA is central to life and health These reactions allow processing nucleic acids during DNA replication, DNA recombination, DNA repair, transcription, splicing, and defense from pathogens (Yang et al, 2006). All these key chemical processes are controlled by vital cellular machineries including both protein and RNA enzymes (Strater et al, 1996), such as endo- and exonucleases, DNA and RNA polymerases, and ribozymes (i.e., group II intron and spliceosome) (Table 1). A first-shell structural architecture centered on two conserved and positively charged elements located in the catalytic site is crucial for efficient DNA and RNA processing (Palermo et al, 2015)
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