A Conserved Tyrosyl−Glutamyl Catalytic Dyad in Evolutionarily Linked Enzymes: Carbapenam Synthetase and β-Lactam Synthetase

β-Lactam-synthesizing enzymes carbapenam synthetase (CPS) and β-lactam synthetase (β-LS) are evolutionarily linked to a common ancestor, asparagine synthetase B (AS-B). These three relatives catalyze substrate acyl-adenylation and nucleophilic acyl substitution by either an external (AS-B) or internal (CPS, β-LS) nitrogen source. Unlike AS-B, crystal structures of CPS and β-LS revealed a putative Tyr-Glu dyad (CPS, Y345/E380; β-LS, Y348/E382) proposed to deprotonate the respective internal nucleophile. CPS and β-LS site-directed mutagenesis (Y345/8A, Y345/8F, E380/2D, E380/2Q, E380A) resulted in the reduction of their catalytic efficiency, with Y345A, E380A, and E382Q producing undetectable amounts of β-lactam product. However, [³²P]PP_i−ATP exchange assays demonstrated Y345A and E380A undergo the first half-reaction, with the remaining active mutants showing decreased forward commitment to β-lactam cyclization. pH−rate profiles of CPS and β-LS supported the importance of a Tyr-Glu dyad in β-lactam formation and suggested its reverse protonation in β-LS. The kinetics of CPS double-site mutants reinforced the synergism of Tyr-Glu in catalysis. Furthermore, significant solvent isotope effects on k_cat (^Dk_cat) for Y345F (1.9) and Y348F (1.7) maintained the assignment of Y345/8 in proton transfer. A proton inventory on Y348F determined its ^D(k_cat/K_m) = 0.2 to arise from multiple reactant-state fractionation factors, presumably from water molecule(s) replacing the missing Tyr hydroxyl. The role of a CPS and β-LS Tyr-Glu catalytic dyad was solidified by a significant decrease in mutant k_cat viscosity dependence with respect to the wild-type enzymes. The evolutionary relation and potential for engineered biosynthesis were demonstrated by β-LS acting as a carbapenam synthetase.