Evolution and Adaptation of Enzymesʹ Active Sites: Enzymes Use Functionally Similar Active Site for Same Type of Reactions with Structurally Similar Substrates Irrespective of the Organism

Enzymes have played a crucial role in the evolution of life on the planet Earth, as they could accelerate thermodynamically favourable reactions approximately a million times faster. The evolution of enzymes with their unique 3D structures and specific active sites for each reaction is still a mystery. Interestingly, now it is found that two different transferases from two different metabolic pathways use the same active site amino acids, suggesting that active sites of enzymes are designed based on the specific molecular interactions between the active site amino acids and their substrate molecules. The present data further reveal that not only the active site is designed for a particular biological reaction during evolution, but also adapted and conserved in all life forms, from viruses to humans. This is based on the fact that just two-point mutations, viz. S356 and Q363, instead of the conserved Phe356 and a positively charged residue K363, respectively, rendered the enzyme, fructosyl transferase from Geobacillus stearothermophilus inactive. The importance of these two amino acids in fructosyl transferases was further confirmed from analysis of hundreds of fructosyl transferase sequences from levan- and inulosucrases, and other glycoside hydrolase GH68 family of enzymes from a large number of organisms. The fructosyl transferases are bifunctional enzymes and function both as a hydrolase and as a transferase. The hydrolase uses a catalytic triad in its active site, viz. 2 Asp residues where one acts as the nucleophile and the other acts as the transition-state stabilizer and a Glu residue that acts as a general acid-base catalyst. Similarly, the transferase also uses a catalytic triad, viz. a positively charged amino acid residue, K/R/H, as the proton abstractor and an R at -3 from the proton abstractor as the donor selection amino acid and an Y or a branched chain amino acid with a G as the template-binding pair. It is interesting to note that the same catalytic triad is observed in the active sites of nucleotidyl transferases like DNA/RNA polymerases [1-3]. For example, the nucleotidyl transferase (E. coli DNA polymerase I) and the fructosyl transferase (Bacillus subtilis levansucrase) possess more or less the same active site structure, (-R-4RSAK758AINFGLIY766GM- and -D358SR-3GSK363MTIDGITSNDIYML377GY-, respectively) where K758 in the E. coli enzyme is  shown to be the proton abstractor, the R at -4 as the nucleotide selection amino acid and the –YG- as the template–binding pair. Furthermore, extensive analysis of different DNA/RNA polymerases and fructosyl transferases from a large number organisms, from viruses to humans, revealed the same active site structure [1-3]. These data confirm that during evolution, not only similar active site is designed and used for the same types of reactions, but also conserved and adapted to all forms of life, from viruses to humans.