Beta-lactamase: lessons in the art of survival.

1. A beta-lactamase molecule is a marvelous device enabling its marker to thrive in a hostile and potentially lethal environment. What is more, the protective effect is extended to otherwise vulnerable organisms, securing their survival in the sheltered neighborhood. Curiously, such environmental implications had no discernible impact on the strategy of antibacterial chemotherapy. Moreover, traditional diagnostic routines are largely responsible for the sheltered survival of susceptible pathogens. 2. For half a century, since their existence was discovered by Abraham and Chain, beta-lactamases have never ceased to amaze us with an ever-widening spectrum of structural and functional variants, appearing in increasingly rapid succession in an ever broader range of bacteria. Whereas the phenomenon itself was the predicted outcome of unguarded expansion of beta-lactam therapy, its scope is a testimony to the unique advantage of an otherwise irrelevant, and thus freely modifiable and transmissible, enzyme as a key to survival. 3. Perhaps the least expected lesson is to be had from the adaptive behaviour of the beta-lactamase molecule itself. Faced with virtually countless structural variations on the beta-lactam theme, nearly all created so as to frustrate its function, the beta-lactamase molecule proves to be so versatile in overcoming so many of its potential inhibitors that it makes us reflect on the evolutionary significance of coping and survival at the molecular level.

Inducible oxacillin-hydrolyzing beta-lactamase in a methylotrophic bacterium.

A novel beta-lactamase (beta-lactam-hydrolase, EC 3.5.2.6) was detected in a culture of Pseudomonas C, an obligatory methylotroph. This is the first beta-lactamase discovered in a methylotrophic organism. The inducible cell-bound enzyme with broad-spectrum activity against penicillins, was purified 77-fold from cell extracts of the methanol-grown bacterium, and its molecular weight was estimated to be 30,000. As a group, the isoxazolyl penicillins are the favored substrates, while cephalosporins are resistant to hydrolysis and act as mild competitive inhibitors. The activity of this M-OXA beta-lactamase focused as a single band at an acidic pI value (5.5) similar to that of PSE- and TEM-type enzymes, but can be clearly distinguished from other OXA-type beta-lactamases, all of which focus in the alkaline region. The enzyme is coded by a non-transferable gene. Based on the sum of its physical and biochemical properties, the M-OXA beta-lactamase is distinguishable from all previously described beta-lactamases, although immunological studies revealed some cross reactivity with the plasmid mediated OXA-2 enzyme.

In situ detection of beta-lactamase activity in sodium dodecyl sulfate-polyacrylamide gels.

After beta-lactamase had been denatured by boiling in the presence of sodium dodecyl sulfate (SDS) and then electrophoresed in SDS-polyacrylamide gels, activity could be restored and could be detected in situ as specific molecular species. Renaturation was simple and facilitated by the presence of a carrier protein. The assay was sensitive, detecting 0.8 ng beta-lactamase activity in the gel.

Conformational adaptation of RTEM beta-lactamase to cefoxitin.

Cefoxitin, a poor substrate of the RTEM beta-lactamase (penicillin amido-beta-lactam hydrolase, EC 3.5.2.6), induces a reversible change in the conformation of the enzyme. The change is manifested in gradual loss of catalytic activity and increased susceptibility to proteolytic inactivation. It is prevented by antibodies, which stabilize the native conformation. By contrast, divalent cations, which have no effect on the native enzyme, delay recovery from the cefoxitin-induced state, presumably by reacting with sites made accessible in the partly unfolded enzyme. Prolonged exposure to excess of cefoxitin causes a similar delay. The kinetic evidence, namely, the initial burst of consumption of cefoxitin and the subsequent gradual recovery of activity with better substrates, appears to be consistent with acylation of the active site by cefoxitin followed by a slower deacylation step [Fisher et al. (1980) Biochemistry 19, 2895-2901]. However, additional evidence leads us to conclude that the kinetics observed reflect deformation of the active site, rather than its blockage, by cefoxitin. Of most significance is the transient change in specificity, i. e. a preferential interaction of the recovering enzyme with substrates which are closest in structure to cefoxitin.

Interaction of the pBR 322-coded RTEM beta-lactamase with substrates. Evidence for specific conformational transitions.

The rate of inactivation of RTEM-1 beta-lactamase by Pronase is accelerated by class A ('resistant') penicillins. Other substrates (class S penicillin and cephalosporins) protect against the inactivation. Cefoxitin, a semi-synthetic cephamycin, induces a more extensive, hysteretic response. In its presence the enzyme is inactivated by trypsin as well as by Pronase.

Cross-linking preserves conformational changes induced in penicillinase by its substrates.

Exopenicillinase of Bacillus cereus 569/H was cross-linked with toluene 2,4-diisocyanate in the presence of cephalothin, cloxacillin or no substrate. The derivatives show significant differences in susceptibility to inactivation by heat, urea, iodination or proteolysis. Such differences can be predicted from the contrasting effects of these substrates on the conformation of the enzyme.

Preferential suppression of normal exoenzyme formation by membrane-modifying agents.

Cerulenin, phenethyl alcohol, benzyl alcohol, procaine, and a series of aliphatic alcohols selectively suppressed production of active exoenzymes by various bacterial strains.

Preparation and properties of an immobilized derivative of penicillinase.

Penicillinase (beta-lactamase I, EC 3.5.2.6) secreted by Bacillus cereus, strain 569/H, was covalently attached to aminoethyl cellulose via glutaraldehyde. The immobilized derivative shows increased thermostability and decreased susceptibility to conformational changes induced by certain substrates of penicillinase. The decline in the rate of hydrolysis of such substrates was consequently suppressed by immobilization. A marked increase in Km was observed with all substrates except for the unsubstituted 6-aminopenicillanic acid. The altered properties of the new derivative are attributed to the constraint imposed by immobilization on the conformational flexibility of the enzyme molecule. Thus, apart from obvious technological interest, immobilized penicillinase provides a useful model for the study of the role of flexibility in the function of an enzyme.

Evidence linking penicillinase formation and secretion to lipid metabolism in Bacillus licheniformis.

The formation of penicillinase by cultures of Bacillus licheniformis was preferentially suppressed by cerulenin, an antibiotic known to specifically inhibit fatty acid synthesis in microorganisms. The effect was studied at cerulenin concentrations that had almost no effect on the rate of cell growth and overall protein synthesis, but that reduced the rate of [14C]acetate incorporation (by 50 to 70%), indicating partial inhibition of lipid synthesis. The levels of both the released enzyme (exopenicillinase) and its cell-bound precursor were reduced to the same extent (70% to 80%). Enzyme formation was gradually resumed after the removal of cerulenin or the addition of a mixture of fatty acids prepared from lipids extracted from B. licheniformis. Reversal was less effective as the time interval between treatment with cerulenin and addition of fatty acids increased. We conclude that de novo synthesis of fatty acids is required for the formation of both the membrane-bound and extracellular penicillinase. Suppression of the membrane-bound enzyme is a likely consequence of the altered membrane (decreased lipid-to-lipid ratio and increased density) seen in cerulenin-treated preparations. The corresponding suppression of exopenicillinase is consistent with the view that it is derived from the membrane-bound form. A mechanism linking the general class of exportable proteins to specific aspects of lipid synthesis is discussed.

Acquisition of substrate-specific parameters during the catalytic reaction of penicillinase.

The progress of the catalytic reaction of penicillinase (EC 3.5.2.6; penicillin amido-beta-lactamhydrolase) depends on the structure of the side-chain in derivatives of 6-aminopenicillanic acid (the parent substrate). Side-chains of one class promote the rate of the reaction and cause no deviation from the linear kinetics observed with the parent compound. By contrast, side-chains of the other class induce a time-dependent, reversible change in the parameters of the catalytic reaction. The rate decelerates considerably and then becomes constant; the decrease in kcat is accompanied by a corresponding decrease in Km. The initial parameters of the biphasic reaction, determined by stopped-flow spectrophotometry, approach those of the unsubstituted 6-aminopenicillanic acid. The final parameters, which are specific for each derivative, are not acquired when the native conformation of the enzyme is stabilized by homologous antibodies.