A conserved tyrosyl-glutamyl catalytic dyad in evolutionarily linked enzymes: carbapenam synthetase and beta-lactam synthetase.
Beta-lactam-synthesizing enzymes carbapenam synthetase (CPS) and beta-lactam synthetase (beta-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, beta-LS) nitrogen source. Unlike AS-B, crystal structures of CPS and beta-LS revealed a putative Tyr-Glu dyad (CPS, Y345/E380; beta-LS, Y348/E382) proposed to deprotonate the respective internal nucleophile. CPS and beta-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 beta-lactam product. However, [(32)P]PP(i)-ATP exchange assays demonstrated Y345A and E380A undergo the first half-reaction, with the remaining active mutants showing decreased forward commitment to beta-lactam cyclization. pH-rate profiles of CPS and beta-LS supported the importance of a Tyr-Glu dyad in beta-lactam formation and suggested its reverse protonation in beta-LS. The kinetics of CPS double-site mutants reinforced the synergism of Tyr-Glu in catalysis. Furthermore, significant solvent isotope effects on k(cat) ((D)k(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 beta-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 beta-LS acting as a carbapenam synthetase.
Department of Chemistry, The Johns Hopkins University, Baltimore, Maryland 21218, USA.
Mary L
ML
Raber
Samantha O
SO
Arnett
Craig A
CA
Townsend
D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001217:Aspartate-Ammonia Ligase / Q000737:chemistry; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D017124:Conserved Sequence; D019143:Evolution, Molecular; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016973:Pectobacterium carotovorum / Q000201:enzymology* / Q000235:genetics; D011522:Protons; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics; D014783:Viscosity
D011522:Protons; D018698:Glutamic Acid; D014443:Tyrosine; D000581:Amidohydrolases; C114020:beta-lactam synthetase; D019731:Carbon-Nitrogen Ligases; D001217:Aspartate-Ammonia Ligase; C479139:carbapenam synthetase