Site-saturation mutagenesis and three-dimensional modelling of ROB-1 define a substrate binding role of Ser 130 in class A <i>β</i>-lactamases

Juteau, Jean-Marc; Billings, Eric M.; Knox, James R.; Levesque, Roger C.

doi:10.1093/protein/5.7.693

Cited by 57 publications

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“…The major effect of the S130G mutation on apparent K m and K I emphasizes the precise topological role of this amino acid (17,(22)(23)(24)(25)(26)(27). A direct consequence of the Ser 3 Gly substitution is reflected by the amount of S130G versus SHV-1 inhibited in 20 min, the time required for E. coli cell division.…”

Section: Discussionmentioning

confidence: 99%

Understanding Resistance to β-Lactams and β-Lactamase Inhibitors in the SHV β-Lactamase

Helfand

Bethel

Hujer

et al. 2003

Journal of Biological Chemistry

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Bacterial resistance to ␤-lactam/␤-lactamase inhibitor combinations by single amino acid mutations in class A ␤-lactamases threatens our most potent clinical antibiotics. In TEM-1 and SHV-1, the common class A ␤-lactamases, alterations at Ser-130 confer resistance to inactivation by the ␤-lactamase inhibitors, clavulanic acid, and tazobactam. By using site-saturation mutagenesis, we sought to determine the amino acid substitutions at Ser-130 in SHV-1 ␤-lactamase that result in resistance to these inhibitors. Antibiotic susceptibility testing revealed that ampicillin and ampicillin/clavulanic acid resistance was observed only for the S130G ␤-lactamase expressed in Escherichia coli. Kinetic analysis of the S130G ␤-lactamase demonstrated a significant elevation in apparent K m and a reduction in k cat /K m for ampicillin. Marked increases in the dissociation constant for the preacylation complex, K I , of clavulanic acid (SHV-1, 0.14 M; S130G, 46.5 M) and tazobactam (SHV-1, 0.07 M; S130G, 4.2 M) were observed. In contrast, the k inact s of S130G and SHV-1 differed by only 17% for clavulanic acid and 40% for tazobactam. Progressive inactivation studies showed that the inhibitor to enzyme ratios required to inactivate SHV-1 and S130G were similar. Our observations demonstrate that enzymatic activity is preserved despite amino acid substitutions that significantly alter the apparent affinity of the active site for ␤-lactams and ␤-lactamase inhibitors. These results underscore the mechanistic versatility of class A ␤-lactamases and have implications for the design of novel ␤-lactamase inhibitors.

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Section: Discussionmentioning

confidence: 99%

Understanding Resistance to β-Lactams and β-Lactamase Inhibitors in the SHV β-Lactamase

Helfand

Bethel

Hujer

et al. 2003

Journal of Biological Chemistry

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“…Mutational studies have demonstrated that substitutions at residues Ser-130 and Ser-235 result in a significant decrease in cephalosporinase activity, and yet do not greatly alter penicillinase activity (25,26). Each study proposed that the loss of a hydrogen bond to the substrate carboxylate from the serine side chain was detrimental only for cephalosporin hydrolysis.…”

Section: Discussionmentioning

confidence: 99%

“…The conclusion from these studies was that the hydrogen bond network in the active site is developed to such an extent for penicillinase activity that the enzyme can afford the mutational loss of one or two hydrogen bonds and retain function. For example, it was proposed that the S130G mutation was compensated for in penicillin hydrolysis by a hydrogen bond to the substrate carboxylate from Arg-244 that is shorter (stronger) for penicillins than for cephalosporins (26). Therefore, because the TEM enzyme has more interactions with penicillins than with cephalosporins, the loss of any specific interaction due to a residue substitution is critical to cephalosporin hydrolysis and therefore is not tolerated.…”

Section: Discussionmentioning

confidence: 99%

Cephalosporin Substrate Specificity Determinants of TEM-1 β-Lactamase

Cantu

Huang

Palzkill

1997

Journal of Biological Chemistry

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␤-lactamase is a bacterial enzyme that catalyzes the hydrolysis of ␤-lactam antibiotics such as penicillins and cephalosporins. TEM-1 ␤-lactamase is a prevalent ␤-lactamase found in Gram-negative bacteria and is capable of hydrolyzing both penicillins and cephalosporins, except for the extended-spectrum cephalosporins. To identify the sequence determinants in the active site for a given antibiotic substrate, random libraries were constructed that each contain all possible amino acid combinations for the designated region of TEM-1 ␤-lactamase. To establish the determinants of substrate specificity for cephalosporins versus those for penicillins, these active site libraries have been screened for mutants with high levels of activity for the second generation cephalosporin cephaloridine. Based on the sequence results, substitutions of W165S, A237T, and E240C were identified as cephalosporin-specific. Kinetic analysis of these mutants was done to determine whether each is capable of distinguishing between the two classes of antibiotics. Both the A237T and E240C substitutions, alone or in combination, exhibited increased cephalosporinase activity and decreased penicillinase activity relative to the wild-type enzyme. A sequence comparison between functional mutants selected for cephaloridine hydrolytic activity and functional mutants previously selected for ampicillin hydrolytic activity suggests that TEM-1 ␤-lactamase has greater restrictions in maintaining cephalosporinase activity versus maintaining penicillinase activity.␤-lactam antibiotics, such as the penicillins and cephalosporins, are among the most commonly used antimicrobial agents. The production of ␤-lactamases, which catalyze ␤-lactam hydrolysis, is the predominant mechanism of bacterial resistance to these antibiotics (1). ␤-lactamases are grouped into 4 classes (A, B, C, and D) based on primary sequence homology (2, 3). Classes A, C, and D are serine hydrolases (3) while class B consists of zinc metalloenzymes (4). TEM-1 ␤-lactamase is a plasmid encoded, class A enzyme that can efficiently cleave the penicillins and some cephalosporins (5). The third generation or extended-spectrum cephalosporins, such as ceftazidime and cefotaxime, however, are poor substrates for the TEM-1 enzyme (6).Changes in the substrate specificity of TEM-1 ␤-lactamase due to amino acid substitutions in the enzyme have been observed among resistant clinical isolates in response to selective pressure applied by extended-spectrum antibiotics (6). The most common amino acid substitutions occur at residues 104, 164, and 237-240, both as individual and as combinatorial substitutions (numbering of amino acids according to Ambler et al. (7)). The substitutions are thought to provide more favorable interactions between the enzyme and the bulkier side chains of the extended-spectrum cephalosporins, thus increasing the catalytic efficiency of the enzyme (6).Because of the importance of mutation-meditated changes in TEM-1 ␤-lactamase substrate specificity during the development of antibiot...

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“…It has been reported that Ser130 mutants have less substrate affinity. 39,40) Several structural studies on the acylenzyme intermediate analogues indicated that Ser130 made a hydrogen bond with the carboxyl group. 7,[41][42][43] From these experimental findings, the function of Ser130 was thought to be binding a substrate.…”

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

confidence: 99%

Substrate Deacylation Mechanisms of Serine-.BETA.-lactamases

Hata

Fujii

Tanaka

et al. 2006

Biological & Pharmaceutical Bulletin

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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...

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Site-saturation mutagenesis and three-dimensional modelling of ROB-1 define a substrate binding role of Ser 130 in class A β-lactamases

Cited by 57 publications

References 0 publications

Understanding Resistance to β-Lactams and β-Lactamase Inhibitors in the SHV β-Lactamase

Understanding Resistance to β-Lactams and β-Lactamase Inhibitors in the SHV β-Lactamase

Cephalosporin Substrate Specificity Determinants of TEM-1 β-Lactamase

Substrate Deacylation Mechanisms of Serine-.BETA.-lactamases

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