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PubMed2001

Sublethal effects of ultraviolet A radiation on Enterobacter cloacae.

We report the sublethal effects of ultraviolet A (UVA) on Enterobacter cloacae in comparison with those produced in Escherichia coli. UVA-induced sublethal effects were investigated in either bacterial membrane and at tRNA level. Limited dependence on oxygen concentration for photoinduced inhibition of biochemical membrane functions and low levels of oxidative damage during the irradiation period were found in En. cloacae. On the other hand, ultraviolet spectroscopy and reversed-phase HPLC analysis of hydrolysed tRNA showed that radio induced damage to tRNA is similar in En. cloacae and E. coli. Nevertheless, growth delay induced by UVA in En. cloacae was shorter than that found in E. coli submitted to the same experimental conditions. A limited post-irradiation ppGpp accumulation and the absence of any influence of the membrane damage on the growth delay extent seem to be responsible for the shortness of this effect in En. cloacae. Most of the differences between En. cloacae and E. coli could be attributed to an increased ability of En. cloacae to overcome oxidative stress during UVA exposure.

Cell Membrane↗
PubMed2002

Transient reduction in the tRNA 4-thiouridine content induced by ultraviolet A during post-irradiation growth in Enterobacter cloacae.

The influence of previous exposure to ultraviolet-A radiation (UVA) was studied on the susceptibility of Enterobacter cloacae to undergo the growth delay effect. Comparison of growth curves corresponding to irradiated and control cells showed that a previous treatment with UVA almost abolished the growth delay effect. UV absorption spectra of tRNA, and reverse phase HPLC analysis of hydrolysed tRNA, demonstrated a low content of 4-thiouridine in E. cloacae cells grown after UVA exposure at low doses. Since 4-thiouridine is the UVA target responsible for initiation of growth delay, this observation explained the influence of previous exposure to UVA on the susceptibility of this organism to undergo growth delay. A similar but weaker alteration was found when Escherichia coli was assayed. The results suggest that, in addition to cross-linking with cytidine residues, the content of 4-thiouridine in tRNA may be modified by UVA by an unknown mechanism.

Dose-Response Relationship, Radiation↗
PubMed1997

DNA-dependent RNA polymerase from Enterobacter cloacae is closely related to Escherichia coli.

The RNA polymerase holoenzyme (RNAP) of Enterobacter cloacae was purified by gel filtration and heparin affinity chromatography and shown to consist of four subunits (beta', beta, alpha and sigma) of 156, 151, 45 and 82 kDa, as measured by SDS-PAGE. The 82 kDa protein was shown to be related to the Escherichia coli primary sigma factor by western blot analysis with polyclonal antisera raised against purified E. coli sigma 70. Functional reconstitution of E. cloacae core enzyme with purified E. coli sigma 70 showed that E. cloacae and E. coli sigma factors are closely related. The RNAP of E. cloacae initiated transcription from the tac promoter with an efficiency similar to that of E. coli. Measuring promoter-specific transcription, the dependence of holoenzyme activity on salt, divalent cation and temperature was also similar to that of the E. coli RNAP. We also showed that the transcriptional inhibitor, rifampicin, inhibits the enzyme activity of the purified RNAP of E. cloacae and E. coli at similar concentrations. We conclude, based on these data, that the RNAP of E. cloacae and E. coli, both enterobacteria, are closely related.

Chromatography, Affinity↗
PubMed2003

Overexpression and purification of the three components of the Enterobacter aerogenes AcrA-AcrB-TolC multidrug efflux pump.

The tripartite AcrA-AcrB-TolC system is the major efflux pump of the nosocomial pathogen Enterobacter aerogenes. AcrA is a trimeric periplasmic lipoprotein anchored in the inner membrane, AcrB is an inner membrane transporter and TolC is a trimeric outer membrane channel. In order to reconstitute the AcrA-AcrB-TolC system of E. aerogenes in artificial membranes, we overexpressed and purified the three proteins. The E. aerogenes acrA, acrB and tolC open reading frames were individually inserted in the expression vector pET24a(+), in frame with a sequence coding a C-terminal hexahistidine tag to allow purification by INAC (Immobilized Nickel Affinity Chromatography). The mature AcrA-6His was overproduced in a soluble form in the cytoplasm of Escherichia coli BL21(DE3). AcrA-6His was purified under native conditions in two steps using INAC and gel permeation chromatography. We obtained about 25 mg of 97% pure AcrA-6His per liter of culture. AcrB-6His was solubilized from the membrane fraction of E. coli C43(DE3) in 300 mM NaCl, 5% Triton X-100 and purified in one step by INAC. The AcrB-6His enriched fraction was eluted with 100 mM imidazole. The final yield was 1-2 mg of 95% pure AcrB-6His per liter of culture. The membrane fraction of E. coli BL21(DE3)pLysS containing TolC-6His was first treated with 2% Triton X-100, 30 mM MgCl(2) to solubilize the inner membrane proteins. After ultracentrifugation, the pellet was treated with 5% Triton X-100, 5 mM EDTA to solubilize the outer membrane proteins. Approximately 5 mg of 95% pure TolC-6His trimers per liter of culture was purified by INAC.

Bacterial Proteins↗
PubMedDate not supplied

An outbreak of gentamicin-resistant Enterobacter cloacae infections in a pediatric intensive care unit.

The intensive care unit at Children's Medical Center in Dallas is a medical-surgical unit that cares for pediatric patients of all ages. In 1978 an outbreak of infections occurred that was caused by a gentamicin-resistant strain of Enterobacter cloacae. Thirty of the 34 patients involved in the outbreak were neonates. Six patients developed bacteremia, five of them neonates. The neonates who became infected were significantly smaller (less 1500 g) and more premature (less than 35 weeks) than control patients. Neonates with bacteremia had a significantly higher incidence of congenital anomalies. In a multi-specialty pediatric intensive care unit newborn infants were the group of patients at high risk for nosocomial infection.

Disease Outbreaks↗
PubMed1989

Electrophoretic typing of Enterobacter cloacae with a limited set of enzyme stains.

Hospital isolates of Enterobacter cloacae were analysed by polyacrylamide gel electrophoresis for enzyme polymorphism and the results were compared with established serotyping, phage typing and biotyping techniques. Initially, the diversity of electromorphs of 13 enzymes was determined on a representative set of 62 distinct strains. Two broad clusters of strains were found in the species, and analysis by serotype suggested a limited diversity within the most frequent O serotypes. A subset of three enzymes, lactate dehydrogenase, 6-phosphogluconate dehydrogenase, glutamate dehydrogenase and an unidentified marker, were selected and used to type groups of hospital isolates. There was good general agreement between the two systems, although the enzyme method failed to distinguish between some strains with the same serotype. This method provided useful epidemiological information and, in the absence of established typing systems, it is a practical approach to subdividing the species.

Bacterial Typing Techniques↗
PubMed1991

The use of the PhP-KE biochemical fingerprinting system in epidemiological studies of faecal Enterobacter cloacae strains from infants in Swedish neonatal wards.

The PhenePlate (PhP) biochemical fingerprinting system is an automated method for typing of bacteria, based on the evaluation of the kinetics of biochemical reactions, performed in microtitre plates. In the present study the PhP-Klebsiella/Enterobacter (KE) system was evaluated for typing of Enterobacter cloacae and employed to study the epidemiology of faecal E. cloacae strains isolated from infants in 22 Swedish neonatal wards. The PhP-KE system showed a high reproducibility and discrimination for E. cloacae isolates. Among 64 epidemiologically unrelated E. cloacae strains, 49 distinct phenotypes were found, and the diversity index was 0.985. E. cloacae was found as a part of the dominating Gram-negative aerobic bacterial flora in 83 out of 953 infants studied. The incidences of E. cloacae colonization varied between 0 and 35% in different wards, but in contrast to previous data for Klebsiella spp. and Escherichia coli, there was little evidence of spread of particular strains in the wards. We also discuss two different measures of nosocomial transmission of bacterial strains: transmissible strains and epidemic index.

Bacterial Typing Techniques↗
PubMed1995

Steady-state kinetics of the binding of beta-lactams and penicilloates to the second binding site of the Enterobacter cloacae P99 beta-lactamase.

Previous research has shown that the class C beta-lactamase of Enterobacter cloacae P99 is able to catalyze the hydrolysis and aminolysis of acyclic depsipeptides. The steady kinetics of these reactions are complicated by the presence of an additional (depsi)peptide binding site in addition to the active site [Pazhanisamy, S., & Pratt, R. F. (1989) Biochemistry 28, 6875-6882]. The present paper presents a steady-state kinetic analysis of the inhibition of depsipeptide hydrolysis by sodium benzylpenicilloate, methyl benzylpenicilloate, 6-aminopenicillanic acid, and 7-aminocephalosporanic acid. The two beta-lactams are considerably poorer substrates than the depsipeptide employed, m-[[(phenylacetyl)glycyl]oxy]benzoic acid. The aim was to determine the relative affinity of these ligands for the active site and the second site. Three types of experiments were employed: (i) measurements of direct inhibition of depsipeptide hydrolysis, (ii) measurements of the effect of an active-site-directed inhibitor, m-(dansylamidophenyl)-boronic acid, on the effectiveness of the ligands as inhibitors, and (iii) measurements of the effect of a preferential second site ligand, N-(phenylacetyl)glycyl-D-phenylalanine, on the effectiveness of the ligands as inhibitors. The results suggest that all four ligands preferentially bind to the active site, with weaker binding at the second site. The necessarily weaker binding of a ligand to the second site when the active site is occupied by a transition-state analog inhibitor was analyzed. Perhaps surprisingly, the intact beta-lactams appeared to bind more firmly to the alternative site than do the flexible penicilloates.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1995

Inactivation of the Enterobacter cloacae P99 beta-lactamase by a fluorescent phosphonate: direct detection of ligand binding at the second site.

The synthesis of a fluorescent beta-lactamase inhibitor, p-nitrophenyl [(dansylamido)methyl]-phosphonate is described. The compound inactivated the class C beta-lactamase of Enterobacter cloacae P99 with stoichiometric release of p-nitrophenol, presumably, as with other phosphonate inhibitors, by phosphonylation of the active site serine. The inhibited enzyme exhibited typical dansyl fluorescence emission at 533 nm with excitation maxima at 345 and 283 nm; the latter excitation peak probably arises from radiationless energy transfer to the dansyl group from aromatic chromophores on the protein-inspection of the crystal structure shows that the closest are tyrosines. The fluorescence of the p-nitrophenyl phosphonate and the inhibited enzyme varied with pH in a very similar fashion, reflecting dissociation of the dimethylammonium ion in the ground state at low pH and of the sulfonamide in the excited state above pH 6. No perturbation of the fluorescence of the inhibited enzyme due to active site functional groups was observed. This may reflect the distance between the dansyl fluorophore and the phosphonyl group and/or the high pKa's of the protonated active site functional groups in the presence of the phosphonate. The addition of certain small molecular weight N-acyl amino acids, of preferred structure D-RCONHCHR'CO2-, to the inhibited enzyme led to an enhancement of dansyl fluorescence intensity and a blue shift in the emission maximum. This suggested that these molecules bind to the beta-lactamase at a site other than the active site and supports previous kinetic data to this effect [Dryjanski, M., & Pratt, R. F., (1995) Biochemistry 34, preceding paper in this issue].(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed2000

Determination of the pKa value of C115 in MurA (UDP-N-acetylglucosamine enolpyruvyltransferase) from Enterobacter cloacae.

The enzyme UDP-N-acetylglucosamine (UDP-NAG) enolpyruvyltransferase (MurA) catalyzes the formation of enolpyruvyl-UDP-NAG, a precursor in peptidoglycan biosynthesis. The residue at position 115 in MurA has been proposed to act as a general acid in the enzymatic reaction. This is also the primary site of action of the antibiotic fosfomycin. In this paper, the pK(a) of Cys-115 has been determined to be 8.3, by titration of Enterobacter cloacae MurA with the alkylating agent iodoacetamide as a function of pH. Use of site-directed mutagenesis has established that only C115 is essential for catalysis, and the three other cysteine residues (C251, C354, and C381) are nonessential. Mass spectrometric analysis demonstrated that C115 is not alkylated at pH <7, but is alkylated significantly at pH >7. Measurement of the enzymatic inhibition by iodoacetamide as a function of pH showed maximum inhibition at pH >9, with a second-order rate constant of inhibition of 44 M(-)(1) s(-)(1) at pH 10. The presence of either one of the substrates did not influence the inactivation behavior, while the presence of both substrates resulted in a 5-fold reduction in the extent of alkylation. The covalent species that results from PEP bound to C115 of MurA exhibited 50-100-fold increased resistance against alkylation by iodoacetamide. These results imply that C115 is appreciably protonated at physiological pH and, therefore, is capable of acting as a proton donor in the enzyme-catalyzed reaction. However, it also implies that C115 is appreciably deprotonated at physiological pH also, whereupon the resultant thiolate nucleophile may play an important role in the formation of the covalent O-phosphothioketal species, whose role in catalysis is yet to be established.

Alkyl and Aryl Transferases↗
PubMed1992

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

The class C serine beta-lactamase of Enterobacter cloacae P99 was inhibited by a series of aryl methylphosphonate monoester monoanions. The effectiveness of these inhibitors was promoted by an acylamido substituent on the methyl group and a good leaving group at phosphorus. The former preference suggests that noncovalent interaction of these inhibitors with the enzyme resembles that of substrates, while the latter suggests that nucleophilic displacement at phosphorus occurs as part of the inhibition mechanism. The truth of the latter proposition was confirmed by observation of release of 1 equiv of phenol concomitant with inhibition and of the presence of an equivalent amount of 14C-label on the enzyme after inhibition by a 14C-labeled phosphonate. The hydrolytically inert nature of the enzyme-inhibitor adduct, and its 31P chemical shift, suggested that O-phosphonylation of the enzyme had occurred. Although, by analogy with substrates, one might expect that the hydroxyl of the active site serine residue would be covalently modified by these inhibitors, successive alkali and acid treatment of the enzyme-inhibitor adduct generated no pyruvate. Instead, 1 equiv of lysinoalanine was found. This product was rationalized to arise through intramolecular capture by an adjacent lysine amine group of the dehydroalanine residue produced by alkali treatment of an O-phosphonylated serine residue. One equivalent of lysinoalanine was also produced by alkali treatment of the enzyme that had been inhibited by 6 beta-bromopenicillanic acid, a mechanism-based inhibitor known to acylate the hydroxyl group of the active site serine residue. It is therefore likely that the aryl phosphonates phosphonylate this residue. These compounds should be useful as beta-lactamase active site titrants and as sources of fresh insight into the chemical properties of the active site. The significant mechanistic features of the inhibition, in particular its strong leaving group dependence and the distinctive ability of the beta-lactamase active site to stabilize a dianionic transition state containing a pentacoordinated phosphorus, are discussed with respect to the active site structure. The comparison with phosph(or/on)yl inhibitors of serine proteinases is made, and the mechanism-based features of inhibition of serine hydrolases by phosph(on)ates are noted.

Enterobacter cloacae↗
PubMed1994

Role of residue Lys315 in the mechanism of action of the Enterobacter cloacae 908R beta-lactamase.

The role of the highly conserved Lys315 residue in the catalytic mechanism of a class C beta-lactamase has been probed by site-directed mutagenesis. Lys315 has been replaced by a histidine in the Enterobacter cloacae 908R beta-lactamase, thus introducing a tritratable group to probe the role of the positive charge, and by a glutamine. The effects of these mutations have been studied on the kinetics of penicillin G and cephalothin turnover and on the pre-steady-state kinetics with carbenicillin at different pH. Results showed that substrate binding was not impaired by the mutations, so that an interaction with the substrate-free carboxylate in the Henri-Michaelis complex could be ruled out. Lys315 must have a catalytic role as shown by the decreased acylation and deacylation rates observed with the mutant enzymes. The mutants exhibited a lower activity at acidic pH, and this observation could be correlated with a decreased affinity for (3-aminophenyl)boronate, a compound devoid of free carboxylate which binds to the active site and forms an adduct mimicking the tetrahedral intermediate. This suggested that Lys315 was somehow involved in accelerating the nucleophilic substitutions along the reaction pathway. The study was extended to modified substrates where the free carboxylate had been esterified. Neither acylation nor deacylation seemed severely impaired with these compounds, showing that the interaction between the enzyme and the substrate-free carboxylate did not play a major role in catalysis.

Amino Acid Sequence↗
PubMed1994

Crystallographic structure of a phosphonate derivative of the Enterobacter cloacae P99 cephalosporinase: mechanistic interpretation of a beta-lactamase transition-state analog.

The crystal structure of a complex formed on reaction of the Enterobacter cloacae P99 cephalosporinase (beta-lactamase) with a phosphonate monoester inhibitor, m-carboxyphenyl [[N-[(p-iodophenyl)acetyl]amino]methyl]phosphonate, has been obtained at 2.3-A resolution. The structure shows that the inhibitor has phosphonylated the active site serine (Ser64) with loss of the m-carboxyphenol leaving group. The inhibitor is positioned in the active site in a way that can be interpreted in terms of a transition-state analog. The arylacetamido side chain is placed as anticipated from analogous beta-lactamoyl complexes of penicillin-recognizing enzymes, with the amino group hydrogen-bonded to the backbone carbonyl of Ser318 (of the B3 beta-strand) and to the amides of Gln120 and Asn152. There is support in the asymmetry of the hydrogen bonding of this side chain to the protein and in the 2-fold disorder of the benzyl group for the considerable breadth in substrate specificity exhibited by class C beta-lactamases. One phosphonyl oxygen atom is in the oxyanion hole, hydrogen-bonded to main-chain NH groups of Ser318 and Ser64, while the other oxygen is solvated, not within hydrogen-bonding distance of any amino acid side chain. The closest active site functional group to the solvated oxygen atom is the Tyr150 hydroxyl group (3.4A); Lys67 and Lys315 are quite distant (4.3 and 5.7 A, respectively). Rather, Tyr150 and Lys67 are more closely associated with Ser64O gamma (2.9 and 3.3 A). This arrangement is interpreted in terms of the transition state for breakdown of the tetrahedral intermediate in the deacylation step of catalysis, where the Tyr150 phenol seems the most likely general acid. Thus, Tyr150, as the phenoxide anion, would be the general base catalyst in acylation, as proposed by Oefner et al. [Nature (1990) 343, 284-288]. The structure is compared with that of a similar phosphonate derivative of a class A beta-lactamase [Chen et al. (1993) J. Mol. Biol. 234, 165-178], and mechanistic comparisons are made. The sensitivity of serine beta-lactamases, as opposed to serine proteinases, toward inhibition by phosphonate monoanions is supported by electrostatic calculations showing a net positive potential only in the catalytic sites of the beta-lactamases.

Binding Sites↗
PubMed2001

Mechanism of reaction of acyl phosph(on)ates with the beta-lactamase of Enterobacter cloacae P99.

A series of acyl phosph(on)ates has been prepared to more closely examine the details of the interactions of this class of molecule with beta-lactamases. In general, they were found to react with the class C beta-lactamase of Enterobacter cloacae P99 in two ways, by acylation and by phosphylation. The acyl-enzymes generated by the former reaction were transiently stable with half-lives of between 3 and 45 s, of comparable lifetime therefore to those generated by the inhibitory beta-lactams cefotaxime, cefuroxime, and cefoxitin. On the other hand, phosphylation led to a completely inactive enzyme. In general, the second-order rate constants for acylation (k(cat)/K(m)) were larger than for phosphylation (k(i)). As expected on chemical grounds, phosphylation was found to be relatively more effective for the phosphonates than the phosphates. The acyl phosphates were much more effective acylating agents however. The acylation reaction was found to be enhanced by hydrophobic substituents in both the acyl and leaving group moieties. Thus, the most reactive compound in this series was benzo[b]thiophene-2-carbonyl 2'-naphthyl phosphate with a K(m) value of 0.15 microM and a k(cat) of 0.2 s(-1); k(cat)/K(m) is therefore 1.3 x 10(6) s(-1) M(-1), making this compound the most specific acyclic acylation reagent for this beta-lactamase yet described. Significant substrate inhibition by this compound suggested that further binding regions may be available for exploitation in inhibitor design. A linear free energy analysis showed that the transition states for acylation of the beta-lactamase by aroyl phosphates are analogues of the corresponding aryl boronic acid adducts. Molecular modeling suggested that the aroyl phosphates react with the P99 beta-lactamase with the aroyl group in the side chain/acyl group site of normal substrates and the phosphate in the leaving group site. In this orientation, the phosphate leaving group interacts strongly with Lys 315.

Acylation↗
PubMed1979

Stereochemistry of the hydrolysis of the endo isomer of uridine 2',3'-cyclic phosphorothioate catalyzed by the nonspecific phosphohydrolase from Enterobacter aerogenes.

The nonspecific phosphohydrolase from Enterobacter aerogenes (ATCC 13048) requires divalent metal ions for activity, since zinc present in the isolated enzyme can be removed by extensive dialysis against 8-hydroxyquinoline-5-sulfonate at pH 7.5 to yield an inactive enzyme which can be reactivated by addition of Zn2+, Cd2+, Co2+, Mn2+, or Ni2+; six ions of either zinc or cadmium can be incorporated into the inactive enzyme, and this incorporation of metal ion can be correlated with the regaining of activity (J. A. Gerlt, R. Dhesi, and H. C. Hemmings, unpublished experiments). The cadmium-reactivated phosphohydrolase catalyzes the hydrolysis of the endo isomer of uridine 2',3'-cyclic phosphorothioate (U greater than pS) to yield uridine 3'-monophosphorothioate as the major product. After enzymatic hydrolysis of the cyclic phosphorothioate in 19.8% H218O and chemical recyclization of the 18O-labeled acyclic phosphorothioates to yield a mixture of the endo and exo isomers of U greater than pS, 18O is found primarily in the exo isomer, as judged by examination of the 145.7-MHz phosphorus-31 nuclear magnetic resonance spectrum of the mixture. This observation indicates that the cadmium phosphosphohydrolase catalyzes hydrolysis of endo-U greater than pS with inversion of configuration, implying that the hydrolysis reaction proceeds by an in-line attack of water on the phosphorus.

Enterobacter↗
PubMed2002

Mechanism of inhibition of the beta-lactamase of Enterobacter cloacae P99 by 1:1 complexes of vanadate with hydroxamic acids.

The class C beta-lactamase of Enterobacter cloacae P99 is competitively inhibited by low concentrations of 1:1 complexes of vanadate and hydroxamic acids. Structure-activity studies indicated that the hydroxamic acid functional group was essential to this inhibition. Both aryl and alkyl hydroxamic acids form inhibitory ternary complexes with vanadate and the enzyme, although, in certain cases of the latter, the inhibition may not be seen because of the low formation constants of the vanadate-hydroxamic acid complex. After all of the vanadate species present in solution had been taken into account, "real" K(i) values for the vanadate complexes could be determined. The K(i) value of the best of the inhibitors that were investigated, the 1:1 complex of vanadate with 4-nitrobenzohydroxamic acid, was 0.48 microM. Kinetics studies showed that the association and dissociation rate constants of this complex with the enzyme were 1.48 x 10(6) s(-1) M(-1) and 0.73 s(-1), respectively; the magnitude of the latter indicates covalent interaction of the complex with the enzyme. (51)V NMR and UV-vis spectra suggest that the structure of the vanadate complex bound to the enzyme may be very similar to that in solution. A (13)C NMR spectrum of the enzyme complex with 4-nitrobenzo[(13)C]hydroxamic acid and vanadate yields a coordination-induced shift (CIS) of 7.74 ppm. This is significantly larger than that of the vanadate complex in free solution (3.62 ppm), suggesting either, somewhat contrary to the (51)V and UV-vis spectra, greater interaction between vanadium and the hydroxamate carbonyl oxygen in the enzyme complex than in free solution or, more likely, polarization of the hydroxamate by interaction, e.g., hydrogen bonding, with the enzyme. Molecular modeling indicates that a pentacoordinated vanadate complex may well be able to snugly occupy the enzyme active site; Asn 152 is suitably placed to hydrogen bond to the hydroxamic acid oxygen atom. The experimental results are in accord with a model whereby the vanadate-hydroxamate-enzyme complex is a moderately good analogue of the transition state of the reaction of the beta-lactamase with phosphonate inhibitors.

Dose-Response Relationship, Drug↗
PubMed2004

Kinetics of turnover of cefotaxime by the Enterobacter cloacae P99 and GCl beta-lactamases: two free enzyme forms of the P99 beta-lactamase detected by a combination of pre- and post-steady state kinetics.

Third-generation cephalosporins bearing oximino side chains are resistant to hydrolysis by class C beta-lactamases such as that from Enterobacter cloacae P99. For example, steady state parameters for hydrolysis of cefotaxime by this enzyme are as follows: k(cat) = 0.41 s(-1), K(m) = 17.2 microM, and k(cat)/K(m) = 2.3 x 10(4) s(-1) M(-1). On the other hand, however, the K(i) value for cefotaxime as an inhibitor of cephalothin hydrolysis is 27 nM. The discrepancy between K(m) and K(i) indicated that a real steady state had not been achieved in at least one of these experiments. Analysis indicated that only two to three cefotaxime turnovers occurred during the K(i) determination. This suggested that the first few turnovers of cefotaxime by the P99 beta-lactamase may be different from those in the subsequent steady state. A direct pre-steady state experiment confirmed this hypothesis. The simplest reaction scheme that fitted the data involved replacement of the initial enzyme form, E, which bound cefotaxime tightly, with a second more weakly binding form, E', by partitioning of the acyl-enzyme intermediate during the first few turnovers. Steady state turnover of cefotaxime then largely involved E' as the free enzyme form. E' slowly reverted to E in the post-steady state regime. Further evidence for this scheme included quantitative analysis of the post-steady state and observation of a difference in the catalytic activity of E and E' in 2 M ammonium sulfate. The kinetics of P99 beta-lactamase-catalyzed hydrolysis of an acyclic depsipeptide substrate bearing a third-generation cephalosporin side chain showed that the side chain is necessary but not sufficient for production of resistance to beta-lactamase; a combination of the side chain and the dihydrothiazine ring of a cephalosporin is required. The beta-lactamase of E. cloacae GC1, an extended spectrum mutant of the P99 enzyme, rapidly hydrolyzes third-generation cephalosporins, without the structural transition described above. The flexibility of the extended Omega loop of the GC1 enzyme probably leads to this situation. Conformational restriction of the loop in the P99 enzyme is probably responsible for the long-lived acyl-enzyme intermediate and the transition to E' induced by cefotaxime.

Acylation↗
PubMed2005

The D-methyl group in beta-lactamase evolution: evidence from the Y221G and GC1 mutants of the class C beta-lactamase of Enterobacter cloacae P99.

The beta-lactam antibiotics act through their inhibition of D-alanyl-D-alanine transpeptidases (DD-peptidases) that catalyze the last step of bacterial cell wall synthesis. Bacteria resist beta-lactams by a number of mechanisms, one of the more important of which is the production of beta-lactamases, enzymes that catalyze the hydrolysis of these antibiotics. The serine beta-lactamases are evolutionary descendants of DD-peptidases and retain much of their structure, particularly at the active site. Functionally, beta-lactamases differ from DD-peptidases in being able to catalyze hydrolysis of acyl-enzyme intermediates derived from beta-lactams and being unable to efficiently catalyze acyl transfer reactions of D-alanyl-D-alanine terminating peptides. The class C beta-lactamase of Enterobacter cloacae P99 is closely similar in structure to the DD-peptidase of Streptomyces R61. Previous studies have demonstrated that the evolution of the beta-lactamase, presumably from an ancestral DD-peptidase similar to the R61 enzyme, included structural changes leading to rejection of the D-methyl substituent of the penultimate D-alanine residue of the DD-peptidase substrate. This seems to have been achieved by suitable placement of the side chain of Tyr 221 in the beta-lactamase. We show in this paper that mutation of this residue to Gly 221 produces an enzyme that more readily hydrolyzes and aminolyzes acyclic D-alanyl substrates than glycyl analogues, in contrast to the wild-type beta-lactamase; the mutant is therefore a more efficient DD-peptidase. Molecular modeling showed that the D-alanyl methyl group fits snugly into the space originally occupied by the Tyr 221 side chain and, in doing so, allows the bound substrate to assume a conformation similar to that on the R61 DD-peptidase, which has a hydrophobic pocket for this substituent. Another mutant of the P99 beta-lactamase, the extended spectrum GC1 enzyme, also has space available for a D-alanyl methyl group because of an extended omega loop. In this case, however, no enhancement of activity against D-alanyl substrates with respect to glycyl was observed. Accommodation of the penultimate D-alanyl methyl group is therefore necessary for efficient DD-peptidase activity, but not sufficient.

Binding Sites↗