Site-directed mutagenesis of .beta.-lactamase leading to accumulation of a catalytic intermediate

Escobar, Walter A.; Tan, Anthony K.; Fink, Anthony L.

doi:10.1021/bi00108a025

Cited by 81 publications

(82 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The TI formation mechanism obtained in this work is consistent with results of several experimental studies showing that the deacylation ability of the Glu166 mutant was greatly impaired compared to that of the wildtype enzyme. 6,23,27,29,44,45) In our study, the activation energy for the TI formation in pathway A (Fig. 2) was 24.6 kcal/mol, and the reaction appeared to be the rate-determining step of the deacylation step.…”

Section: Deacylation Mechanism Of Class a B-lacta-masementioning

confidence: 51%

Substrate Deacylation Mechanisms of Serine-.BETA.-lactamases

Hata

Fujii

Tanaka

et al. 2006

Biological & Pharmaceutical Bulletin

View full text Add to dashboard Cite

INTRODUCTIONb-Lactamases are produced by pathogenic bacteria and are enzymes that cause resistance to b-lactam antibiotics by hydrolyzing their b-lactam rings.1-3) b-Lactamases are classified into two major types on the basis of the main component of the active site: serine-b-lactamases and metallo-b-lactamases. 4,5) Serine-b-lactamases are further classified into three classes: classes A, C, and D; i.e., metallo-b-lactamase is classified into class B.As is well known, the catalytic mechanism of serine-b-lactamases involves acylation ( Fig. 1 (a) to (c) via (b)) and deacylation ( Fig. 1 (c) to (d)). The acylation mechanism has been examined in many experimental [6][7][8][9][10][11][12][13][14] and theoretical works, 11,[15][16][17][18][19][20][21][22] and acylation is common to serine-b-lactamase and penicillin-binding protein (PBP). Deacylation, on the other hand, is only seen in serine-b-lactamase. This means that the essence of antibiotic resistance by serine-blactamase arises from the deacylation reaction. Hence, an understanding of the reaction mechanism of deacylation is important for developing novel b-lactam antibiotics and potent b-lactamase inhibitors. We have investigated the deacylation mechanisms of serine-b-lactamases by using theoretical calculation. In this paper, the results of our recent computational analyses for all classes of serine-b-lactamases are presented. DEACYLATION MECHANISM OF CLASS A b-LACTA-MASEClass A b-lactamase is more frequently detected in a clinical setting than are other classes of b-lactamase. Since the study by Knox et al. suggested that the E166A mutant of blactamase from Bacillus licheniformis induced the accumulation of an acyl-enzyme intermediate, 23) the catalytic residue involved in the deacylation reaction has been presumed to be only Glu166. However, it is difficult to conclude that the deacylation reaction occurs only due to Glu166 because the distance between Ser70O g and Glu166O e is too far to interact (ca. 3.74 Å) according to the results of our molecular dynamics (MD) simulation of the structure of b-lactam antibioticsb-lactamase complex. 24) We have speculated that not only Glu166 but also Lys73 is involved in the deacylation reaction because Lys73N z is located between Ser70 and Glu166 and interacts with Ser70O g and Glu166O e , nearly forming hydrogen bonds (2.77 and 3.21 Å, respectively).24) Furthermore, a hydrogen bond network among Lys73N z , Ser130O g and the carboxyl group of the substrate (b-lactam antibiotics) should be taken into account to clearly explain the reaction mechanism. Ishiguro et al. also discussed the importance of the hydrogen bond network based on the results of their molecular mechanics calculations.15) Accordingly, we investigated the whole mechanism of the deacylation reaction by theoretical The substrate deacylation mechanisms of serine-b b-lactamases (classes A, C and D) were investigated by theoretical calculations. The deacylation of class A proceeds via four elementary reactions. The rate-determining process is the tetrahedra...

show abstract

Section: Deacylation Mechanism Of Class a B-lacta-masementioning

confidence: 51%

Substrate Deacylation Mechanisms of Serine-.BETA.-lactamases

Hata

Fujii

Tanaka

et al. 2006

Biological & Pharmaceutical Bulletin

View full text Add to dashboard Cite

show abstract

“…This marked decrease in activity demonstrates that Glu 166 is important for the catalytic mechanism of the N170G enzyme, as it is for the wild-type enzyme (20,24,26,27,55). A puzzling observation, however, is the increase in activity of the E166A/N170G double mutant in comparison with the single mutation of E166A.…”

Section: Discussionmentioning

confidence: 93%

Structural and Biochemical Evidence That a TEM-1 β-Lactamase N170G Active Site Mutant Acts via Substrate-assisted Catalysis

Brown

Shanker

Prasad

et al. 2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

TEM-1 ␤-lactamase is the most common plasmid-encoded166 and the hydrolytic water. The goal of this study was to examine the importance of Asn 170 for catalysis and substrate specificity of ␤-lactam antibiotic hydrolysis. The codon for position 170 was randomized to create a library containing all 20 possible amino acids. The random library was introduced into Escherichia coli, and functional clones were selected on agar plates containing ampicillin. DNA sequencing of the functional clones revealed that only asparagine (wild type) and glycine at this position are consistent with wild-type function. The determination of kinetic parameters for several substrates revealed that the N170G mutant is very efficient at hydrolyzing substrates that contain a primary amine in the antibiotic R-group that would be close to the Asn 170 side chain in the acyl-intermediate. In addition, the x-ray structure of the N170G enzyme indicated that the position of an active site water important for deacylation is altered compared with the wildtype enzyme. Taken together, the results suggest the N170G TEM-1 enzyme hydrolyzes ampicillin efficiently because of substrate-assisted catalysis where the primary amine of the ampicillin R-group positions the hydrolytic water and allows for efficient deacylation.

show abstract

“…The reactive Ser-64 is at the N terminus of the H2 helix and is adjacent to invariant Lys-67. A second conserved lysine, Lys-315, lies on 1-strand B3 and is part of a signature K(H)T(S)G tripeptide seen in all penicillin-recognizing enzymes (21,22 in class A enzymes (4,25,26), Glu-166, is without a counterpart in this and other (14) class C ,B-lactamases. The nearest acid residue, Asp-217, is 10 A away from Ser-64, and its carboxylate group is directed away from Ser-64 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The primary cause of this resistance is the constitutive or ,B-lactam-inducible overproduction of 83-lactamase (EC 3.5.2.6), which catalyzes, via a serine-bound acyl intermediate, the hydrolysis of the ,B-lactam to an inactive acid: The 3-lactamases have been grouped into classes A through D (2) or groups 1 through 4 (3). Most studied have been the class A /-lactamases (group 2), whose members are commonly called penicillinases and use a non-classical serine mechanism (4). The body of data on the class C enzymes, the so-called cephalosporinases or group 1 /3-lactamases, is considerably smaller than that for class A.…”

mentioning

confidence: 99%

Evolution of an enzyme activity: crystallographic structure at 2-A resolution of cephalosporinase from the ampC gene of Enterobacter cloacae P99 and comparison with a class A penicillinase.

Lobkovsky

Moews

Liu

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

208

272

View full text Add to dashboard Cite

The structure of the class C ampC 3lactam-ase (cephalosporinase) from Enterobacter cloacae strain P99 has been established by x-ray crystallography to 2-resolution and compared to a class A ,B-lactamase (penicillinase) struc- comparison of overall tertary folding shows that the cephalosporinase, more than the penicillinase, is broadly similar to the ancestral 3lactam-inhibited enzymes of bacterial cell wall synthesis. On this basis, it is proposed that the cephalosporinase is the older of the two 3lactamases, and, therefore, that a local refolding in the active site, rather than a simple point mutation, was required for the primordial class C &3-lactamase to evolve to the dass A -lactamase having an improved ability to catalyze the deacylation step of 3lactam hydrolysis.

show abstract

Site-directed mutagenesis of .beta.-lactamase leading to accumulation of a catalytic intermediate

Cited by 81 publications

References 23 publications

Substrate Deacylation Mechanisms of Serine-.BETA.-lactamases

Substrate Deacylation Mechanisms of Serine-.BETA.-lactamases

Structural and Biochemical Evidence That a TEM-1 β-Lactamase N170G Active Site Mutant Acts via Substrate-assisted Catalysis

Evolution of an enzyme activity: crystallographic structure at 2-A resolution of cephalosporinase from the ampC gene of Enterobacter cloacae P99 and comparison with a class A penicillinase.

Contact Info

Product

Resources

About