The β-lactamase cycle: a tale of selective pressure and bacterial ingenuity

Kumar

Journal of Biological Chemistry

et al. 2004

Bacterial resistance to the third-generation cephalosporins is an issue of great concern in current antibiotic therapeutics. An important source of this resistance is from production of extended-spectrum (ES) ␤-lactamases by bacteria. The Enterobacter cloacae GC1 enzyme is an example of a class C ES ␤-lactamase. Unlike wild-type (WT) forms, such as the E. cloacae P99 and Citrobacter freundii enzymes, the ES GC1 ␤-lactamase is able to rapidly hydrolyze third-generation cephalosporins such as cefotaxime and ceftazidime. To understand the basis for this ES activity, m-nitrophenyl 2-(2-aminothiazol-4-yl)-2-[(Z)-methoxyimino]acetylaminomethyl phosphonate has been synthesized and characterized. This phosphonate was designed to generate a transition state analog for turnover of cefotaxime. The crystal structures of complexes of the phosphonate with both ES GC1 and WT C. freundii GN346 ␤-lactamases have been determined to high resolution (1.4 -1.5 Å). The serine-bound analog of the tetrahedral transition state for deacylation exhibits a very different binding geometry in each enzyme. In the WT ␤-lactamase the cefotaxime-like side chain is crowded against the ⍀ loop and must protrude from the binding site with its methyloxime branch exposed. In the ES enzyme, a mutated ⍀ loop adopts an alternate conformation allowing the side chain to be much more buried. During the binding and turnover of the cefotaxime substrate by this ES enzyme, it is proposed that ligand-protein contacts and intra-ligand contacts are considerably relieved relative to WT, facilitating positioning and activation of the hydrolytic water molecule. The ES ␤-lactamase is thus able to efficiently inactivate third-generation cephalosporins.␤-Lactam antibiotics are widely used because of their effectiveness and safety. However, ␤-lactam-resistant bacteria have become widespread. The main cause of this resistance is the production of ␤-lactamases, which hydrolyze the amide bond in the ␤-lactam ring. ␤-Lactamases are grouped into four classes, A-D, on the basis of amino acid sequence. Although class B ␤-lactamases are metallo-␤-lactamases, class A, C, and D ␤-lactamases are serine-reactive hydrolases that function by acylation and deacylation steps employing tetrahedral intermediates (1, 2) (see Reaction 1).To overcome ␤-lactam resistance, third-generation cephalosporins such as cefotaxime (1), ceftazidime (2), and ceftriaxon have been employed since the 1980s because of their relative stability to serine ␤-lactamases and their broad antibacterial spectrum. Third-generation cephalosporins typically contain a bulky group such as a 2-(2-aminothiazole-4-yl)-2-oxyimino substituent (R 1 ) at position C7 of the cephalosporin nucleus.From the kinetic perspective, third-generation cephalosporins show high K m and low k cat values for class A and D ␤-lactamases, and low K m (K i ) and low k cat values for class C enzymes. This behavior suggests that these cephalosporins cannot effectively bind to the active site of wild-type (WT) 1 class A ␤-lactamases and acylate them...

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Hydrolysis of Third-generation Cephalosporins by Class C β-Lactamases

Kumar

Journal of Biological Chemistry

et al. 2004

pKa calculations for class C ?-lactamases: The role of tyr-150

2000

Self Cite

The Poisson-Boltzmann method was used to compute the pK(a) values of titratable residues in a set of class C beta-lactamases. In these calculations, the pK(a) of the phenolic group of residue Tyr150 is the only one to stand out with an abnormally low value of 8.3, more than one pK(a) unit lower than the measured reference value for tyrosine in solution. Other important residues of the catalytic pocket, such as the conserved Lys67, Lys315, His314, and Glu272 (hydrogen-bonded to the ammonium group of Lys315), display normal protonation states at neutral pH. pK(a) values were also computed in catalytically impaired beta-lactamase mutants. Comparisons between the relative k(cat) values and the Tyr150 pK(a) value in these mutants revealed a striking correlation. In active enzymes, this pK(a) value is always lower than the solution reference value while it is close to normal in inactive enzymes. These results thus support the hypothesis that the phenolate form of Tyr150 is responsible for the activation of the nucleophilic serine. The possible roles of Lys67 and Lys315 during catalysis are also discussed.

The structure of Toho1 β‐lactamase in complex with penicillin reveals the role of Tyr105 in substrate recognition

et al. 2016

The role of the conserved residue Tyr105 in class A β‐lactamases has been the subject of investigation using both structural studies and saturation mutagenesis. Both have shown that while it does not need to be strictly conserved for activity, it is important for substrate recognition. With this in mind we determined the crystal structure of Toho1 β‐lactamase at 15 K to 1.10 Å resolution in complex with penicillin. As expected a ring‐opened penicillin molecule bound to Ser70 the catalytic nucleophile, can clearly be seen in electron density in the active site. In addition to the trapped penicillin, however, are two additional intact ring‐closed penicillin molecules, captured by the enzyme through noncovalent interactions at the edge of the active site.