Site-Directed Mutagenesis of Glutamate-166 in .beta.-Lactamase Leads to a Branched Path Mechanism

Escobar, Walter A.; Tan, Anthony K.; Lewis, Evan R.; Fink, Anthony L.

doi:10.1021/bi00190a015

Cited by 54 publications

(68 citation statements)

References 30 publications

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“…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: 49%

“…26) Another mechanism that involves proton migration from Glu166O e to Ser70O g via a water molecule located between Glu166O e and Ser70O g has been also proposed. [28][29][30][31] However, no water molecule was observed between Glu166O e and Ser70O g during the MD calculation. 26) Judging from the facts above, Glu166 is not thought to be a candidate for the catalytic residue.…”

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

confidence: 98%

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

confidence: 49%

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

confidence: 98%

Substrate Deacylation Mechanisms of Serine-.BETA.-lactamases

Hata

Fujii

Tanaka

et al. 2006

Biological & Pharmaceutical Bulletin

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“…The question is which residue acts as a general base. For class A ␤-lactamases, Lys 73 in the "Ser-X-X-Lys motif" (24) or Glu 166 in the ⍀-loop (21,23,25,28,29) are candidates. To function as a general base, the pK a of Lys 73 should be decreased.…”

mentioning

confidence: 99%

“…5D). Moreover, this network is believed to be responsible for the ␤-lactam hydrolysis (23,25,28). In Hyb-24, however, the distance between C ␣ of Gly 181 and the O ␥ of Ser 112 is 11.84 Å (Fig.…”

mentioning

confidence: 99%

X-ray Crystallographic Analysis of 6-Aminohexanoate-Dimer Hydrolase

Negoro

Ohki

Shibata

et al. 2005

Journal of Biological Chemistry

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6-Aminohexanoate-dimer hydrolase (EII), responsible for the degradation of nylon-6 industry by-products, and its analogous enzyme (EII) that has only ϳ0.5% of the specific activity toward the 6-aminohexanoate-linear dimer, are encoded on plasmid pOAD2 of Arthrobacter sp. (formerly Flavobacterium sp.) KI72. Here, we report the three-dimensional structure of Hyb-24 (a hybrid between the EII and EII proteins; EII-level activity) by x-ray crystallography at 1.8 Å resolution and refined to an R-factor and R-free of 18.5 and 20.3%, respectively. The fold adopted by the 392-amino acid polypeptide generated a two-domain structure that is similar to the folds of the penicillin-recognizing family of serine-reactive hydrolases, especially to those of D-alanyl-D-alanine-carboxypeptidase from Streptomyces and carboxylesterase from Burkholderia. Enzyme assay using purified enzymes revealed that EII and Hyb-24 possess hydrolytic activity for carboxyl esters with short acyl chains but no detectable activity for D-alanyl-D-alanine. In addition, on the basis of the spatial location and role of amino acid residues constituting the active sites of the nylon oligomer hydrolase, carboxylesterase, D-alanyl-D-alanine-peptidase, and ␤-lactamases, we conclude that the nylon oligomer hydrolase utilizes nucleophilic Ser 112 as a common active site both for nylon oligomer-hydrolytic and esterolytic activities. However, it requires at least two additional amino acid residues (Asp 181 and Asn 266 ) specific for nylon oligomer-hydrolytic activity. Here, we propose that amino acid replacements in the catalytic cleft of a preexisting esterase with the ␤-lactamase fold resulted in the evolution of the nylon oligomer hydrolase.Microorganisms are believed to be highly adaptable toward environmental conditions. This can be elucidated from the observations that microorganisms capable of degrading unnatural synthetic compounds can be isolated relatively easily. Unnatural synthetic compounds include various chemicals such as endocrine disrupters and toxic compounds, which have unfavorable effects on living cells. A suitable system to enhance the biodegradability of these compounds is important from an environmental point of view. We have been studying the degradation of a by-product of nylon-6 manufacture (i.e. 6-aminohexanoate oligomers (namely nylon oligomers)) (Fig. 1), by Flavobacterium sp. KI72 as a model for studying the adaptation of microorganisms toward unnatural compounds (1, 2).2 Three enzymes, 6-aminohexanoate-cyclic dimer hydrolase (3), 6-aminohexanoate-dimer hydrolase (EII) 3 (4), and endotype 6-aminohexanoate-oligomer hydrolase (5), encoded on the plasmid pOAD2 (45,519 bp) (6) in strain KI72, were found to be responsible for the degradation of the nylon oligomers. It was also established that the EII-analogous protein (EIIЈ) is located on a different part of the pOAD2 (7,8). EIIЈ has 88% homology to EII (7) but has very low catalytic activity ( 1 ⁄ 200 of EII activity) toward the 6-aminohexanoate-linear dimer (Ald), suggesting that EII h...

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“…Mutation of this residue to any amino acids that cannot activate the hydrolytic water molecule generates a protein that still would experience acylation by ␤-lactam antibiotics but is unable to undergo deacylation rapidly (9,34). We replaced Glu-166 with Asn in both the wild-type (construct pTZ-TEM Mut ) and anchored TEM ␤-lactamase (construct pTZ-ATEM Mut ).…”

Section: Resultsmentioning

confidence: 99%

Cytoplasmic-Membrane Anchoring of a Class A β-Lactamase and Its Capacity in Manifesting Antibiotic Resistance

Suvorov

Vakulenko

Mobashery

2007

Antimicrob Agents Chemother

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Bacterial ␤-lactamases are the major causes of resistance to ␤-lactam antibiotics. Three classes of these enzymes are believed to have evolved from ancestral penicillin-binding proteins (PBPs), enzymes responsible for bacterial cell wall biosynthesis. Both ␤-lactamases and PBPs are able to efficiently form acyl-enzyme species with ␤-lactam antibiotics. In contrast to ␤-lactamases, PBPs are unable to efficiently turn over antibiotics and therefore are susceptible to inhibition by ␤-lactam compounds. Although both PBPs and gram-negative ␤-lactamases operate in the periplasm, PBPs are anchored to the cytoplasmic membrane, but ␤-lactamases are not. It is believed that ␤-lactamases shed the membrane anchor in the course of evolution. The significance of this event remains unclear. In an attempt to demonstrate any potential influence of the membrane anchor on the overall biological consequences of ␤-lactamases, we fused the TEM-1 ␤-lactamase to the C-terminal membrane-anchor of penicillin-binding protein 5 (PBP5) of Escherichia coli. The enzyme was shown to express well in E. coli and was anchored to the cytoplasmic membrane. Expression of the anchored enzyme did not result in any changes in antibiotic resistance pattern of bacteria or growth rates. However, in the process of longer coincubation, the organism that harbored the plasmid for the anchored TEM-1 ␤-lactamase lost out to the organism transformed by the plasmid for the nonanchored enzyme over a period of 8 days of continuous growth. The effect would appear to be selection of a variant that eliminates the problematic protein through elimination of the plasmid that encodes it and not structural or catalytic effects at the protein level. It is conceivable that an evolutionary outcome could be the shedding of the sequence for the membrane anchor or alternatively evolution of these enzymes from nonanchored progenitors.

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Site-Directed Mutagenesis of Glutamate-166 in .beta.-Lactamase Leads to a Branched Path Mechanism

Cited by 54 publications

References 30 publications

Substrate Deacylation Mechanisms of Serine-.BETA.-lactamases

Substrate Deacylation Mechanisms of Serine-.BETA.-lactamases

X-ray Crystallographic Analysis of 6-Aminohexanoate-Dimer Hydrolase

Cytoplasmic-Membrane Anchoring of a Class A β-Lactamase and Its Capacity in Manifesting Antibiotic Resistance

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