Catalytic Mechanism of Metallo beta-Lactamases: Insights from Calculations and Experiments

DAL PERARO, M; A.J. VILA; CARLONI, P

Quantum Biochemistry

Wiley VCH

Lugar: Washington; Año: 2010; p. 605 - 618

Beta-Lactam antibiotics are the most widespread antibiotics on the market nowadays.They have exerted great evolutionary pressure on bacteria, triggering sophisticatedresistance mechanisms. Among them, the most widespread is the expression of blactamases.This is a family of hydrolases that uses different protein scaffolds andcatalytic architectures to inactivate b-lactam drugs (Scheme 21.1) [1].b-Lactamases from classes A, C and D are serine hydrolases, whereas class B blactamasesare characterized by the presence of Zn ions bound to their active sites [2].Metallo b-lactamases (MbLs), despite not being as ubiquitous as serine b-lactamases,represent the largest group of carbapenemases, that is, they hydrolyze a veryimportant class of antibiotics, the carbapenems. MbLs are increasingly spreadingamong pathogenic bacteria in the clinical setting and are resistant to all currentclinical inhibitors on the market [3–6]. Thus, understanding their function at themolecular level is of paramount importance for designing effective drugs (eitherMbLinhibitors or antibiotics refractory to hydrolysis by MbLs).MbLs are classified by sequence homology in three subclasses: B1, B2 and B3[4–7]. The B1 subclass includes several chromosomally-encoded enzymes, such asthe ones from Bacillus cereus (BcII) [8–13], Bacteroides fragilis (CcrA) [14–16],Elizabethkingia meningoseptica (BlaB) [17, 18], as well as the transferable VIM,IMP, SPM and GIM-type enzymes [19–25]. Subclass B2 includes the CphA andImiS lactamases from Aeromonas species, and Sfh-I from Serratia fonticola [26–28].Subclass B3, along with the extensively characterized enzyme L1 from Stenotrophomonasmaltophilia [29–32], includes enzymes from environmental bacteria,such as CAU-1 from Caulobacter crescentus [33], and THIN-B from Janthinobacteriumlividum [34], and from opportunistic pathogens like FEZ-1 from Legionellagormanii [35–37], and GOB from E. meningoseptica [38]. B1 and B3 enzymes displaya broad substrate spectrum, being able to actively hydrolyze b-lactams of all three b-lactam families (e.g., penicillins, cephalosporins and carbapenems), whilst B2MbLs are selective carbapenemases.The metal ions are essential for hydrolysis: Fast mixing techniques coupled to Trpfluorescence have shown that apo-BcII, despite being properly folded, is unable tobind any substrate [13]. Unfortunately, characterization of the catalytic mechanismin Zn-enzymes by means of experimental techniques is difficult: they are silent tomost spectroscopic techniques, in contrast to transition metal-ion based enzymes.Scheme 21.1 Metallo b-lactamase hydrolysis of a general b-lactam, and b-lactam substratesrepresentative of major antibiotic families.606j 21 Catalytic Mechanism of Metallo b-Lactamases: Insights from Calculations and ExperimentsComputational quantum mechanical methods may provide a full structural andenergetics description of the reactive mechanism, along with the metal polyhedronreorganization, providing a valuable instrument to dissect enzymatic catalysis.Here we discuss the enzymatic reaction mechanism of MbLs from both acomputational and experimental point of view. We mostly focus on subclass B1,which represents to date the best structurally and functionally characterized MbLspecies [6, 8, 10–12, 14–16, 28, 32, 39–43], based on its clinical relevance [5].