Mechanism of inhibition of the class C .beta.-lactamase of Enterobacter cloacae P99 by phosphonate monoesters

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: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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

Kumar

Journal of Biological Chemistry

et al. 2004

“…The classical mechanism-based inhibitors of class A ␤-lactamases are not generally effective against class D (16). A variety of phosph(on)ates have been found to be covalent inhibitors of class A and class C ␤-lactamases (4,5,9,(17)(18)(19). This paper describes the screening of a panel of these compounds, 1 to 9, against the class D OXA-1 ␤-lactamase.…”

mentioning

confidence: 99%

“…Second-order rate constants of inactivation were obtained from measurements of the loss of enzyme activity as a function of time (18,19). All kinetics studies were performed at 25°C in a buffer at pH 7.5 containing 20 mM MOPS (3-morpholinopropanesulfonic acid) and 50 mM sodium bicarbonate (3).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Inhibition of Class D β-Lactamases by Acyl Phosphates and Phosphonates

Adediran

Antimicrob Agents Chemother

Baurin

et al. 2005

Self Cite

The susceptibility of typical class D ␤-lactamases to inhibition by acyl phosph(on)ates has been determined. To a large degree, these class D enzymes behaved very similarly to the class A TEM ␤-lactamase towards these reagents. Dibenzoyl phosphate stood out in both cases as a lead compound towards a new class of effective inhibitors.The resistance of bacteria to ␤-lactam antibiotics is to a large extent due to ␤-lactamases (14). There is a wide range of ␤-lactamases, although from a structural standpoint, they can be divided into four classes, A, B, C, and D (11). Each class contains many variants of widely differing substrate specificities and thus clinical importance. Of the canonical four classes, it is perhaps class D that at present is least well studied at the molecular level.The class D ␤-lactamases, also generally known as oxacillinases because of their general specificity for oxacillin and its derivatives, represent a diverse class of enzymes (1) that hydrolyze a broad spectrum of substrates (2). Like class A and C ␤-lactamases, the class D enzymes are serine hydrolases, catalyzing substrate hydrolysis by way of a covalent acyl-enzyme (acyl-serine) intermediate and thus by a double-displacement mechanism. Structural studies indicate that the class D ␤-lactamases are more closely related to class A than to class C, although there are distinct differences in active-site structures and thus, presumably, in chemical mechanisms (20). Although class D ␤-lactamases are clinically significant, there are no specific inhibitors known for them other than certain inhibitory ␤-lactams (8, 12) and a series of anthraquinone dyes (15). The classical mechanism-based inhibitors of class A ␤-lactamases are not generally effective against class D (16). A variety of phosph(on)ates have been found to be covalent inhibitors of class A and class C ␤-lactamases (4,5,9,(17)(18)(19). This paper describes the screening of a panel of these compounds, 1 to 9, against the class D OXA-1 ␤-lactamase. This enzyme is representative of one subclass of the D enzymes (1) and is clinically important in its own right; a crystal structure is also available (20). The more effective compounds of 1 to 9 (Fig. 1) were also tested against the OXA-10 enzyme, a representative of another major class D subgroup (1).The OXA-1 and OXA-10 ␤-lactamases were prepared and purified as described previously (8,20). The various phosph(on)ates were available from previous studies (4, 5, 9, 17-19). The phosphates 2 and 4 to 8 and the phosphonates 1, 3, and 9 were all irreversible or slowly reversible inhibitors of the OXA ␤-lactamases, and the inactivation step could be described simply by scheme 1 as follows:where E is the free enzyme and I is the inhibitor. Second-order rate constants of inactivation were obtained from measurements of the loss of enzyme activity as a function of time (18,19). All kinetics studies were performed at 25°C in a buffer at pH 7.5 containing 20 mM MOPS (3-morpholinopropanesulfonic acid) and 50 mM sodium bicarbonate (3). The enzyme conc...

Patai's Chemistry of Functional Groups

Biological Activity of Phosphonic and Phosphinic Acids

Kalir

2009