Serine p-lactamase

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... 

Class C Serine p-lactamases AmpC enzymes of coti, Shigella spp., Enterobacterspp., C. freundii, M. morganii, Providencia spp. and Serratia spp. cephalos-porinases with wide spectrum of activity CMY, LAT, BIL, MOX, ACC, FOX and DHA types. All genes are ampC genes that have been mobilized by transfer to plasmid DNA. 

Class D Serine p-lactamases OXA enzymes (oxacillinases) of Acinetobac-ter spp. and some Aeromonas spp. Some OXA enzymes are carbapenemases Most OXA types are chromosomal... 

Pratt, R. F. Functional evolution of the serine P-lactamase active site. J. Chem. Soc. Perkin Trans. 2 2002, 851-861. 

Govardhan, C. P. Pratt, R. F. Kinetics and mechanism of the serine P-lactamase catalyzed hydrolysis of depsipeptides. Biochemistry 1987, 26, 3385-3395. 

JM Ghuysen. Serine P-lactamases and penicillin-binding proteins. Annu Rev Microbiol 45 37-67, 1991. 

The most common mechanism of bacterial resistance to P-lactam antibiotics such as the penicillins and cephalosporins is the synthesis of P-lactamases that cleave an amide bond in the antibiotics to generate inactive products (Wiedemann et al., 1989). Genes encoding P-lactamases can be found on the bacterial chromosome or on plasmids. The active site serine P-lactamases belong to a larger family of penicillin-recognizing enzymes that includes the penicillin binding proteins (Joris et al., 1988). All of these enzymes contain the active site serine as well as a conserved triad of K(S/T)G between the active site serine and the C-terminus (Joris et al, 1988). 

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. 

The asymmetric synthesis of c -3,4-disubstituted P-sultams 65, based on the oxidation of 1,2-aminothiols with H2O2 and ammonium heptamolybdate has been achieved <05S1807>. Different transition-state structures for the reactions of P-lactams and their sulfonyl analogues P-sultams with serine P-lactamases have been reported <05JA17556>. It has been discovered that the 3-oxo-P-sultam 66 is unusual in that it inhibits elastase by acylation resulting from substitution at the carbonyl center, C-N fission, and expulsion of the sulfonamide <05JA8946>. 

Known inhibitors of the class A, C, and D serine p-lactamases acylate the active site serine. The commercial inhibitors showed in Fig. 28 are selective for class A enzymes, since they stmcturally resemble penicillins more closely than they resemble cephalosporins. 

Because over half of aU the commercially available antibiotics are 3-lactams, and in recent years bacterial resistance to these molecules has become so prevalent (to the point that some infections can no longer be treated), metaUo-jJ-lactamases are not only important from the organometallic standpoint but they are enzymes central to human health [229]. Most of the p-lactamases identified to date belong to the A, C and D serine hydrolase classes [230-233]. Class B enzymes, on the other hand, require divalent (Zn, Co, Cd or Mn) metal ions for catalytic activity [234, 235]. Because only 10% of the 200-odd P-lactamases identified so far are metaUoenzymes little attention has been given to these proteins as potential threats to antibiotic therapy [236,237]. However, metallo-p-lactamases are able to hydrolyze compoimds that are resistant to serine-p-lactamases and they show little or no susceptibility to traditional p-lactamase inhibitors [238, 239]. This has resulted in ineffective use of the most available antibiotics in bacteria capable of producing metaUo-P-lactamases [240, 241]. 

P-Lactamases are enzymes that hydrolyze the P-lactam ring of P-lactamantibiotics (penicillins, cephalosporins, monobactams and carbapenems). They are the most common cause of P-lactam resistance. Most enzymes use a serine residue in the active site that attacks the P-lactam-amid carbonyl group. The covalently formed acylester is then hydrolyzed to reactivate the P-lacta-mase and liberates the inactivated antibiotic. Metallo P-lactamases use Zn(II) bound water for hydrolysis of the P-lactam bond. P-Lactamases constitute a heterogeneous group of enzymes with differences in molecular structures, in substrate preferences and in the genetic localizations of the encoding gene (Table 1). 

QUINONE METHIDES AND AZA-QUINONE METHIDES AS LATENT ALKYLATING SPECIES IN THE DESIGN OF MECHANISM-BASED INHIBITORS OF SERINE PROTEASES AND p-LACTAMASES... 

Buynak et al. [53] synthesized several 6-(mercaptomethyl) penicillanates (9r and 9s, Table 1) that include both C-6 stereoisomers as well as the sulfide and sulfone oxidation states of the penam thiazolidine sulfur. Selected mercaptomethyl penicillanates inactivated both metallo- and serine /5-lactamases, and displayed synergism with piperacillin against various //-lactamase-producing strains, including metallo-/5-lactamase-producing P. aeruginosa strain. Compound 9r would be capable of bidentate chelation of zinc subsequent to enzymatic hydrolysis of the /5-lactam (Scheme 3). 

J. Lamotte-Brasseur, G. Dive, O. Dideberg, P. Charlier, J. M. Frere, J. M. Ghuysen, Mechanism of Acyl Transfer by the Class A Serine /3-Lactamase of Streptomyces albus G , Biochem. J. 1991, 279, 213-221. 

Felici A, Perilli M, Segatore B, Franceschini N, Setacci D, Oratore A, Stefani S, Galleni M, Amicosante G (1995) Interactions of biapenem with active-site serine and metallo-P-lactamases. Antimicrob Agents Chemother 39 1300-1305. 

As a result, the penicillin occupies the active site of the enzyme, and becomes bound via the active-site serine residue. This binding causes irreversible enzyme inhibition, and stops cell-wall biosynthesis. Growing cells are killed due to rupture of the cell membrane and loss of cellular contents. The binding reaction between penicillinbinding proteins and penicillins is chemically analogous to the action of P-lactamases (see Boxes 7.20 and 13.5) however, in the latter case, penicilloic acid is subsequently released from the P-lactamase, and the enzyme can continue to function. Inhibitors of acetylcholinesterase (see Box 7.26) also bind irreversibly to the enzyme through a serine hydroxyl. 

Penicillin and related antibiotics are inactivated by P-lactamases (Box 20-G), some of which resemble serine proteases in forming acyl enzymes with active site serine side chains.656 657 Others are zinc metallogen-zymes.658/659 Amidohydrolases such as asparaginase and glutaminase,660/661 deacetylases,662 and many other hydrolases can also be described as acyltransferases. 

Figure 3 Example of a P-lactamase, which hydrolytically cleaves the p-lactam ring of penicillins and other P-lactam antibacterial agents. All proteins that specifically bind these agents have a similar folding motif even though homology in primary sequence of these proteins can be low. The active site serine and binding pocket are near the inner edge of the five-stranded 3-sheet.

Over 80 different (3-lactamases are now known. One classification is a system that divides the enzymes into three classes A, B, and C. Classes A and C are active-site serine enzymes. The serine residue in class A enzymes is at position 70. This class contains four major (3-lactamases 749/C (from B. licheniformis), PCI (from S. aureus), 569/H P-lactamase I (from B. cereus), and PBR322 and RTEM (from E. coli). As with other serine-type hydrolytic enzymes (acetylcholinesterase, trypsin), the mechanism of action requires initial formation of an acylated enzyme, in this case acylation of ser-70 followed by hydrolysis of the derivative to regenerate the enzyme ... 

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