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

Silvana B. Boggio

Departamento de Química Biológica, Area Biofísica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, (S2002LRK) Rosario, Argentina

Correspondence
Oscar A. Roveri
oroveri{at}fbioyf.unr.edu.ar

Azospirillum lipoferum RG20, a nitrogen-fixing bacterium found in all kind of soils, was found to be naturally resistant to penicillins and cephalosporins. 6--Bromopenicillanic acid, an irreversible inhibitor of serine--lactamases, completely abolished this resistance. A -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 k_cat 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 -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 -lactamases inhibited by clavulanic acid (group 2b of the Bush–Jacoby–Medeiros scheme). The effect of pH on k_cat and K_m values for benzylpenicillin hydrolysis was studied. The dependence of k_cat on pH suggests that the enzyme–substrate (ES) complex must be in at least three protonation states: two with k_cat values equal to 2800 and 1450 s^-1 and a third inactive one [pK_1(ES) 4·7 and pK_2(ES) 7·9]. Similarly, the dependence of k_cat/K_m 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 k_cat/K_m values equal to 2·7x10⁶ and 3·7x10⁸ M^-1 s^-1 and a third one unable to productively bind substrate. Interestingly, the dependence of k_cat/K_m on pH is consistent with positive cooperativity for proton binding to the enzyme free form [pK_1(E) 8·5 and pK_2(E) 7·2].