Catalytic properties of an endogenous β-lactamase responsible for the resistance of Azospirillum lipoferum to β-lactam antibiotics

摘要

Azospirillum lipoferum RG20, a nitrogen-fixing bacterium found in all kind of soils, was found to be naturally resistant to penicillins and cephalosporins. 6-β-Bromopenicillanic acid, an irreversible inhibitor of serine-β-lactamases, completely abolished this resistance. A β-lactamase was purified 518-fold from a cell-free extract of A. lipoferum RG20. A single band on SDS-PAGE (apparent molecular mass 31?000 Da) and on isoelectric focussing (pI9·35) was observed with the purified protein. The enzyme hydrolysed benzylpenicillin, ampicillin, cephalothin and cephaloridine with comparable kcat values and catalytic efficiencies. However, carbenicillin and cefotaxime were hydrolysed with significantly lower kinetic parameters and oxacillin was hydrolysed at a rate 100 times slower. The purified β-lactamase was inhibited by clavulanic acid and sulbactam but not by EDTA or aztreonam. Its substrate and inhibitor profiles are consistent with those of the broad-spectrum β-lactamases inhibited by clavulanic acid (group 2b of the Bush–Jacoby–Medeiros scheme). The effect of pH on kcat and Km values for benzylpenicillin hydrolysis was studied. The dependence of kcat on pH suggests that the enzyme–substrate (ES) complex must be in at least three protonation states: two with kcat values equal to 2800 and 1450?s?1 and a third inactive one [pK1(ES) 4·7 and pK2(ES) 7·9]. Similarly, the dependence of kcat/Km on pH can be explained by postulating that the enzyme free form can be at least in three different protonation states: two of them with kcat/Km values equal to 2·7×106 and 3·7×108?M?1?s?1 and a third one unable to productively bind substrate. Interestingly, the dependence of kcat/Km on pH is consistent with positive cooperativity for proton binding to the enzyme free form [pK1(E) 8·5 and pK2(E) 7·2].