Class A p-lactamase

The class A P-lactamases are a subset of the active-site serine P-lactamases. TEM-1 P-lactamase is a class A enzyme encoded by the ft/ajEM-l gene that is present on the transposons Tn2 and Tn3 (Datta et al, 1965). Epidemiological studies have shown that TEM-1 is the most common plasmid-mediated P-lactamase and is therefore a major determinant of bacterial resistance to P-lactam antibiotics (Wiedemann et al, 1989). Compounding the problem of resistance is the discovery that TEM-1 mutant variants with altered substrate specificity have been identified in natural isolates (Jacoby and Medieros, 1991). These variant enzymes contain from one to three amino acid substitutions that enable the enzyme to hydrolyze the newer extended-spectrum cephalosporin antibiotics such as cefotaxime and ceftazidime (Jacoby and Medieros, 1991). Thus, the selective pressure of antibiotic therapy le s to die creation of new enzymes with expanded hydrolytic capabilities. 

A large number of class A P-lactamases have now been identified and sequenced and an alignment of 20 class A P-lactamases has been published (Ambler et al., 1991). These aligned sequences permit a comparison between the conserved amino acid residue positions among class A p-lactamases and the conserved positions among the functional random mutants in TEM P-lactamase (Fig. 2). In general, there is agreement between the tolerance of a residue in TEM P-lactamase to amino acid substitutions and the amount a position is substituted in... 

Figure 2. Comparison of sequence variability among functional TEM-1 p-lactamase mutants and twenty aligned class A P-lactamases. The wild type TEM-1 -lactamase primary sequence is shown. Above the sequence are the different amino acids that were identified at that sequence position among functional random mutants. Below the TEM-1 primary sequence are the different amino acids that appear at these positions in an alignment of 20 class A P-lactamases (Figure adapted from Huang et al., 1996).

Continuing use of the third-generation cephalosporins and the introduction of p-lactamase inhibitor combinations (clavulanate with amoxycillin or ticarcillin, sulbactam with ampicillin, and tazobactam with piperacillin see section 4.2) resulted in the appearance of plasmids encoding class C P-lactamases. After several unconfirmed reports, the first proof that a class C P-lactamase had been captured on a plasmid came in 1990 when transmissible resistance to a-methoxy and oxyimino-P-lactams was shown to be mediated by an enzyme whose gene was 90% identical to the ampC gene of E. cloacae. They have subsequently been found worldwide. Strains with plasmid-mediated AmpC enzymes are typically resistant to aminopenicillins (ampicillin or amoxycillin), carboxypenicillins (carbenicillin or ticarcillin) and ureidopenicillins (piperacillin). The enzymes also provide resistance to the oxyimino cephalosporins (ceftazidime, cefo-... 

The 3-chloromethyl cephalosporin 41 has been used for the preparation of nitrocefin 42 via Finkelstein and Wittig reactions <05JOC367>. The bicyclic P-lactam 43, a simple C3 homologue of sulbactam, has been prepared and evaluated as an improved inhibitor of class C P-lactamases <05JOC4510>. 

Purine-9-ylalkyl derivatives (15) containing both the phosphonic and phosphinic substituent inhibit purine nucleoside phosphorylase. Compound 15 [R = NH2, R = OH, R r4 = h, = N, X = H, X = CH2)3] had K, = 0.0026 pmol (ref. 39). The phosphonate monoesters 16 (R = Me, R = o-, m- or P-NO2) inhibit the class A j5-lactamase, probably by phosphorylation of the active site of this enzyme" . When R = Ph they also inhibit class C j5-lactamase of Enterobacter cloacae P99 and may led to new antibiotics. They should be useful as active titrants of the enzyme ... 

The presence of a phenyl group at the C-4 position of the azetidinone ring favored a specific hydrophobic interaction with the active site of class A p-lacta-mases. Instead, the stereochemistry of the C-4 position appeared to be not important for the inhibition [310]. Studies recently reported for the structure-function analyses of the sulfonate moiety have argued for the requirement of a hydrophobic functionality, but its size did not appear to be restrictive. The absence of any hydrophobic functionality at this position lowered the ability of the molecules to inhibit (3-lactamases [314]. 

The examples discussed above constitute a selection of recent applications of the acid and basic hydrolysis of (3-lactams in synthesis. Hydrolysis and alcoholysis of (3-lactams can also be effected under roughly neutral reaction conditions when enzymes are the promoters [47]. The (3-lactamases catalyzed hydrolysis of (3-lactams is an efficient process for a broad spectrum of substrates, including those (3-lactams with base or acid sensitive groups [12 14]. This process proceeds through an acyl enzyme intermediate to give ring opened p-amino acids. The class C p-lactamases in particular, in Scheme 9, have the ability to catalyze the alcoholysis reaction and hence p-amino esters are the products formed. 

The predicted polypeptide was similar to carboxypeptidase DD from Streptomyces R61, penicillin-binding proteins from S. lactamdurans and B. subtilis, and class C p-lactamases. Thus, the enzyme was categorized as a new "penicillin-recognizing enzyme"... 

The proposed biosynthetic pathway of A-503083B (39) is reported in the Figure 7.12. Cap W, a putative class C p-lactamase, was thought to be involved in the formation of the amide bond between L-amino-caprolactam and the methyl ester moiety as the ultimate step of the biosynthesis of 39. Cap S and Cap K are two putative methyltransferases (carboxyl-methyltransferse and O-methylttansferase, respectively), whereas Cap B is a carbamoyltransferase. Cap U and likely Cap V are required for L-amino-caprolactam formation [24]. 

Kaur et al. presented the inhibition of class D p-lactamases by different acyl-phosphonates (01JACS10436). These compounds were synthetized starting with ethyl o-toluate 109. Benzylic bromination with N-bromosuccinimide followed by a subsequent Arbuzov reaction with triethyl phosphite and hydrolysis under acidic conditions afforded the phosphonic acid 110. The final dehydration of diacid 110 in refluxing xylene produced the cycHc phospho-nolactone 111 (Scheme 26). 

Biological assays have shown that class A and class C P-lactamases can be effectively inhibited by compound 116a. The phosphate analog 116b (Figure 15), also synthetized, is more effective than the phosphonolactone and represents a new lead for further development of P-lactamase inhibitors. 

These recent data indicate that MALDI-TOF MS has the potential to directly detect the most clinically important AmpC P-lactamases, such as the CMY-2-like, ACC, and DHA types, in clinical isolates of Enterobacteriaceae. In agreement with other MALDI-TOF MS applications (Hrabak 2013), the described protocol is quick and economical. In addition, detection of p-lactamases by MALDI-TOF MS in a proteomic approach allowing the study of the behavior of the tested strains can complement the already used techniques for characterization of P-lactamases, such as PCR and isoelectric focusing (lEF). MALDI-TOF MS can directly detect the class A (Camara and Hays 2007) and class C p-lactamases, as well as other mechanisms such as methylation of rRNA and cell wall components (Cai et al. 2012 Hrabak et al. 2013). We conclude that establishing a MALDI-TOF supplementary database of resistance mechanisms would promote further research in this field. 

Several bacterial penicillin-binding proteins have been shown to be serine enzymes (Section 2). P-Lactamases are efficient and clinically important enzymes which play an important part in bacterial resistance to the normally lethal action of P-lactam antibiotics. A major class of P-lactamases are also serine enzymes that function by covalent catalysis with the intermediate formation of an acyl-enzyme (Knott-Hunziker et al., 1982 Cohen and Pratt, 1980 Fisher et al., 1980 Anderson and Pratt, 1983 Cartwright and Fink, 1982, Joris et al., 1984). The P-lactamase catalysed hydrolysis of a penicillin thus proceeds by formation of an acyl-enzyme which is an a-penicilloyl ester of a serine residue. The mechanism of reaction of penicillin with alcohols is therefore of obvious relevance, but in addition acyl transfer from nitrogen to oxygen nucleophiles is of current interest. 

Boronic acid transition-state inhibitors (BAT-Sis) represent one of the most promising classes of P-lactamase inhibitors. A small library of l-amido-2-triazolyl-ethaneboronic acids as new class of BAT-Sis have been synthesized through click chemistry and screened against two clinically relevant class C P-lactamases from Enterobacter spp. and Pseudomonas aeruginosa [102]. Among these compounds, compound 65 showed a good activity (4nM) with a significant reduction of bacterial resistance to the combination with cefotaxime (Scheme 2.6 and Table 2.13). 

In essence, the cephalosporin acts as a carrier (63) for the quinolone. The quinolone is replaced in the bacterial ceU after the action of P-lactamase on the cephalosporin portion of the molecule. This codmg combination represents a relatively new class of antibacterial agents which appear to offer advantages over the separated components (64). A good introductory discussion of these exciting agents can be found (65) (see also Antibiotics P-lactams ... 

One approach to combating antibiotic resistance caused by P-lactamase is to inhibit the enzyme (see Enzyme inhibition). Effective combinations of enzyme inhibitors with P-lactam antibiotics such as penicillins or cephalosporins, result in a synergistic response, lowering the minimal inhibitory concentration (MIC) by a factor of four or more for each component. However, inhibition of P-lactamases alone is not sufficient. Pharmacokinetics, stability, ability to penetrate bacteria, cost, and other factors are also important in determining whether an inhibitor is suitable for therapeutic use. Almost any class of P-lactam is capable of producing P-lactamase inhibitors. Several reviews have been pubUshed on P-lactamase inhibitors, detection, and properties (8—15). 

Class A Serine p-lactamases SHV-1 penicillinase in K. pneumoniae, and Koxy with activity against certain third generation cephalosporins in K. oxytoca BlaZ staphylococcal penicillinase TEM, SHV, VEB, PER and CTX-M penicillinases and ESBLs (P-lactamases with activity against third generation cephalosporins and aztreo-nam) KPC, IMI/NMC and SME carbapenemases... 

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