Crystal Structure of the Zinc-Dependent β-Lactamase from Bacillus cereus at 1.9 Å Resolution:  Binuclear Active Site with Features of a Mononuclear Enzyme^,

Abstract

The structure of the zinc-dependent beta-lactamase II from Bacillus cereus has been determined at 1.9 A resolution in a crystal form with two molecules in the asymmetric unit and 400 waters (space group P3121; Rcryst = 20.8%). The active site contains two zinc ions: Zn1 is tightly coordinated by His86, His88, and His149, while Zn2 is loosely coordinated by Asp90, Cys168, and His210. A water molecule (W1) lies between the two zinc ions but is significantly closer to Zn1 and at a distance of only 1.9 A is effectively a hydroxide moiety and a potential, preactivated nucleophile. In fact, Asp90 bridges W1 to Zn2, and its location is thus distinct from that of the bridging water molecules in the binuclear zinc peptidases or other binuclear zinc hydrolases. Modeling of penicillin, cephalosporin, and carbapenem binding shows that all are readily accommodated within the shallow active site cleft of the enzyme, and the Zn1-bound hydroxide is ideally located for nucleophilic attack at the beta-lactam carbonyl. This enzyme also functions with only one zinc ion present. The Zn1-Zn2 distances differ in the two independent molecules in the crystal (3.9 and 4.4 A), yet the Zn1-W1 distances are both 1.9 A, arguing against involvement of Zn2 in W1 activation. The role of Zn2 is unclear, but the B. cereus enzyme may be an evolutionary intermediate between the mono- and bizinc metallo-beta-lactamases. The broad specificity of this enzyme, together with the increasing prevalence of zinc-dependent metallo-beta-lactamases, poses a real clinical threat, and this structure provides a basis for understanding its mechanism and designing inhibitors.

Adaptive protein evolution grants organismal fitness by improving catalysis and flexibility

“…We solved the X-ray structure of M5 [supporting information (SI) Table S1], which showed that none of these mutations entails changes in the global fold (0.6 Å rmsd on all nonhydrogen atoms vs. wt BcII). The active site of BcII can be described as a shallow groove flanked by loops L3 and L10, hosting 2 Zn(II) ions located in its floor, which is defined by loops L7, L9, and L12 (20,21) (Fig. 1).…”

“…The structure of di-Zn(II) BcII from B. cereus [PDB ID code 1BC2 (20)] was selected as the reference starting structure for all our calculations, as reported in ref. 41.…”

“…Because there are no clinically useful inhibitors available for them, M␤Ls challenge the available therapeutics for bacterial infections (18). From the evolutionary point of view, several authors have considered them as young, still incompletely evolved enzymes, based on their lower catalytic efficiencies compared with S␤Ls (14,19,20). However, the broader substrate portfolio exhibited by M␤Ls may have been achieved at the expense of lower enzymatic activities.…”

Catalytic Role of the Metal Ion in the Metallo-β-lactamase GOB

“…Based on this heterogeneity, M␤Ls have been classified into three subclasses: B1, B2, and B3 (3). Subclass B1 includes several chromosomally encoded enzymes, such as BcII from Bacillus cereus (11)(12)(13), CcrA from Bacteroides fragilis (14 -17), BlaB from Elizabethkingia meningoseptica (18), and transferable VIM (19), IMP (20,21), SPM (22), and GIM (23) type enzymes. Subclass B2 includes the CphA (24,25) and ImiS (26) lactamases from Aeromonas species and Sfh-I from Serratia fonticola (27).…”

X-ray Absorption Spectroscopy of the Zinc-Binding Sites in the Class B2 Metallo-β-lactamase ImiS from Aeromonas veronii bv. sobria

“…Considerable information exists regarding the B1 and B3 enzymes, including X-ray diffraction (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29), spectroscopic (9,(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42), mechanistic (43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59), and computational studies (60)(61)(62)(63)(64)…”