Structure of the Extended-Spectrum Class C β-Lactamase of <i>Enterobacter cloacae</i> GC1, a Natural Mutant with a Tandem Tripeptide Insertion<sup>,</sup>

Kumar

et al. 2004

Self Cite

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: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 98%

See 1 more Smart Citation

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

Kumar

et al. 2004

Self Cite

“…In NMC-A, the unique disulfide bond between Cys 69 and Cys 238 induces distortion in the ␤-strand B3, which increases the space between residue 170 and this strand (10). GC1 ␤-lactamase has a three-residue insertion in the ⍀-loop, which produces the wider binding cavity (11). The complex structure of class C ␤-lactamase AmpC with ceftazidime (21) proposes the importance of this insertion for the improved activity of GC1.…”

Section: Comparison With the Acyl-intermediate Structures Of Non-mentioning

confidence: 99%

Acyl-intermediate Structures of the Extended-spectrum Class A β-Lactamase, Toho-1, in Complex with Cefotaxime, Cephalothin, and Benzylpenicillin

Shimamura

Shimizu‐Ibuka

Fushinobu

et al. 2002

103

154

Bacterial resistance to ␤-lactam antibiotics is a serious problem limiting current clinical therapy. The most common form of resistance is the production of ␤-lactamases that inactivate ␤-lactam antibiotics. Toho-1 is an extended-spectrum ␤-lactamase that has acquired efficient activity not only to penicillins but also to cephalosporins including the expanded-spectrum cephalosporins that were developed to be stable in former ␤-lactamases. We present the acyl-intermediate structures of Toho-1 in complex with cefotaxime (expanded-spectrum cephalosporin), cephalothin (non-expanded-spectrum cephalosporin), and benzylpenicillin at 1.8-, 2.0-, and 2.1-Å resolutions, respectively. These structures reveal distinct features that can explain the ability of Toho-1 to hydrolyze expanded-spectrum cephalosporins. First, the ⍀-loop of Toho-1 is displaced to avoid the steric contacts with the bulky side chain of cefotaxime. Second, the conserved residues Asn 104 and Asp 240 form unique interactions with the bulky side chain of cefotaxime to fix it tightly. Finally, the unique interaction between the conserved Ser 237 and cephalosporins probably helps to bring the ␤-lactam carbonyl group to the suitable position in the oxyanion hole, thus increasing the cephalosporinase activity.

“…This suggests that positively charged Lys 67 and Lys 315 in the "KTG box" are involved in the reduction of the pK a of Tyr 150 (35 (Fig. 5C) (20), and class C ␤-lactamase (Lys 67 /Tyr 150 ) (33). In addition, Lys 315 in "KTG box" of class C ␤-lactamase was not conserved in Hyb-24 (Fig.…”

mentioning

confidence: 99%

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

Negoro

Ohki

Shibata

et al. 2005

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...