| http://purl.uniprot.org/citations/19371088 | http://www.w3.org/1999/02/22-rdf-syntax-ns#type | http://purl.uniprot.org/core/Journal_Citation |
| http://purl.uniprot.org/citations/19371088 | http://www.w3.org/1999/02/22-rdf-syntax-ns#type | http://purl.uniprot.org/core/Journal_Citation |
| http://purl.uniprot.org/citations/19371088 | http://www.w3.org/1999/02/22-rdf-syntax-ns#type | http://purl.uniprot.org/core/Citation |
| http://purl.uniprot.org/citations/19371088 | http://www.w3.org/2000/01/rdf-schema#comment | "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."xsd:string |
| http://purl.uniprot.org/citations/19371088 | http://purl.org/dc/terms/identifier | "doi:10.1021/bi900432n"xsd:string |
| http://purl.uniprot.org/citations/19371088 | http://purl.org/dc/terms/identifier | "doi:10.1021/bi900432n"xsd:string |
| http://purl.uniprot.org/citations/19371088 | http://purl.uniprot.org/core/author | "Townsend C.A."xsd:string |
| http://purl.uniprot.org/citations/19371088 | http://purl.uniprot.org/core/author | "Townsend C.A."xsd:string |
| http://purl.uniprot.org/citations/19371088 | http://purl.uniprot.org/core/author | "Arnett S.O."xsd:string |
| http://purl.uniprot.org/citations/19371088 | http://purl.uniprot.org/core/author | "Arnett S.O."xsd:string |
| http://purl.uniprot.org/citations/19371088 | http://purl.uniprot.org/core/author | "Raber M.L."xsd:string |
| http://purl.uniprot.org/citations/19371088 | http://purl.uniprot.org/core/author | "Raber M.L."xsd:string |
| http://purl.uniprot.org/citations/19371088 | http://purl.uniprot.org/core/date | "2009"xsd:gYear |
| http://purl.uniprot.org/citations/19371088 | http://purl.uniprot.org/core/date | "2009"xsd:gYear |
| http://purl.uniprot.org/citations/19371088 | http://purl.uniprot.org/core/name | "Biochemistry"xsd:string |
| http://purl.uniprot.org/citations/19371088 | http://purl.uniprot.org/core/name | "Biochemistry"xsd:string |
| http://purl.uniprot.org/citations/19371088 | http://purl.uniprot.org/core/pages | "4959-4971"xsd:string |
| http://purl.uniprot.org/citations/19371088 | http://purl.uniprot.org/core/pages | "4959-4971"xsd:string |
| http://purl.uniprot.org/citations/19371088 | http://purl.uniprot.org/core/title | "A conserved tyrosyl-glutamyl catalytic dyad in evolutionarily linked enzymes: carbapenam synthetase and beta-lactam synthetase."xsd:string |
| http://purl.uniprot.org/citations/19371088 | http://purl.uniprot.org/core/title | "A conserved tyrosyl-glutamyl catalytic dyad in evolutionarily linked enzymes: carbapenam synthetase and beta-lactam synthetase."xsd:string |
| http://purl.uniprot.org/citations/19371088 | http://purl.uniprot.org/core/volume | "48"xsd:string |
| http://purl.uniprot.org/citations/19371088 | http://purl.uniprot.org/core/volume | "48"xsd:string |
| http://purl.uniprot.org/citations/19371088 | http://www.w3.org/2004/02/skos/core#exactMatch | http://purl.uniprot.org/pubmed/19371088 |
| http://purl.uniprot.org/citations/19371088 | http://www.w3.org/2004/02/skos/core#exactMatch | http://purl.uniprot.org/pubmed/19371088 |
| http://purl.uniprot.org/citations/19371088 | http://www.w3.org/2004/02/skos/core#exactMatch | http://purl.uniprot.org/pubmed/19371088 |