Importance of the beta 5-beta 6 Loop for the Structure, Catalytic Efficiency, and Stability of Carbapenem-Hydrolyzing Class D beta-Lactamase Subfamily OXA-143

Author(s)

Antunes, Victor U. ^[1] ; Llontop, Edgar E. ^[2] ; Vasconcelos, Fernanda N. da Costa ^[3] ; de los Santos, Yossef Lopez ^[4] ; Oliveira, Ronaldo J. ^[5] ; Lincopan, Nilton ^[6] ; Farah, Chuck S. ^[2] ; Doucet, Nicolas ^{[7, 4]} ; Mittermaier, Anthony ^{[8, 7]} ; Favaro, Denize C. ^{[1, 8]}

Affiliation

^[1] Univ Estadual Campinas, Dept Organ Chem, BR-13083970 Sao Paulo, SP - Brazil

^[2] Univ Sao Paulo, Inst Chem, Dept Biochem, Av Prof Lineu Prestes 748, BR-05508000 Sao Paulo, SP - Brazil

^[3] Univ Florida, Citrus Res & Educ Ctr, Lake Alfred, FL 33850 - USA

^[4] Univ Quebec, INRS, Ctr Armand Frappier Sante Biotechnol, Laval, PQ H7V 1B7 - Canada

^[5] Univ Fed Triangulo Mineiro, Inst Ciancias Exatas Nat & Educ, Dept Fis, Lab Biofis Teor, BR-38064200 Uberaba, MG - Brazil

^[6] Univ Sao Paulo, Inst Biomed Sci, Dept Microbiol, BR-05508900 Sao Paulo, SP - Brazil

^[7] Univ Laval, PROTEO, Quebec City, PQ G1V 0A6 - Canada

^[8] McGill Univ, Dept Chem, Montreal, PQ H3A 0G4 - Canada

• Document type: Journal article
• Source: BIOCHEMISTRY; v. 58, n. 34, p. 3604-3616, AUG 27 2019.
• Web of Science Citations: 0

Abstract

The class D beta-lactamase OXA-143 has been described as an efficient penicillinase, oxacillinase, and carbapenemase. The D224A variant, known as OXA-231, was described in 2012 as exhibiting less activity toward imipenem and increased oxacillinase activity. Additionally, the P227S mutation was reported as a case of convergent evolution for homologous enzymes. To investigate the impact of both mutations (D224A and P227S), we describe in this paper a deep investigation of the enzymatic activities of these three homologues. OXA-143(P227S) presented enhanced catalytic activity against ampicillin, oxacillins, aztreonam, and carbapenems. In addition, OXA-143(P227S) was the only member capable of hydrolyzing ceftazidime. These enhanced activities were due to a combination of a higher affinity (lower K-m) and a higher turnover number (higher k(cat)). We also determined the crystal structure of apo OXA-231. As expected, the structure of this variant is very similar to the published OXA-143 structure, except for the two M223 conformations and the absence of electron density for three solvent-exposed loop segments. Molecular dynamics calculations showed that both mutants experience higher flexibility compared to that of the wild-type form. Therefore, our results illustrate that D224A and P227S act as deleterious and positive mutations, respectively, within the evolutionary path of the OXA-143 subfamily toward a more efficient carbapenemase. (AU)

• FAPESP's process: 17/17303-7 - Structure and function of bacterial secretion systems
• Grantee: Shaker Chuck Farah
• Support type: Research Projects - Thematic Grants
• FAPESP's process: 17/22822-3 - Structural and dynamical origins of catalytic differences between OXA-143 and OXA-231
• Grantee: Denize Cristina Favaro
• Support type: Scholarships abroad - Research