Identification and characterization of an unusual metallo-β-lactamase from Serratia proteamaculans

Abstract

Metallo-β-lactamases (MBLs) are a family of metalloenzymes that are capable of hydrolyzing β-lactam antibiotics and are an important means by which bacterial pathogens use to inactivate antibiotics. A database search of the available amino acid sequences from Serratia proteamaculans indicates the presence of an unusual MBL. A full length amino acid sequence alignment indicates overall homology to B3-type MBLs, but also suggests considerable variations in the active site, notably among residues that are relevant to metal ion binding. Steady-state kinetic measurements further indicate functional differences and identify two relevant pK a values for catalysis (3.8 for the enzyme-substrate complex and 7.8 for the free enzyme) and a preference for penams with modest reactivity towards some cephalosporins. An analysis of the metal ion content indicates the presence of only one zinc ion per active site in the resting enzyme. In contrast, kinetic data suggest that the enzyme may operate as a binuclear enzyme, and it is thus proposed that a catalytically active di-Zn(2+) center is formed only once the substrate is present.

Metallo-β-Lactamases: A Major Threat to Human Health

“…Examples of the B2 and B3 subgroups are CphA from A. hydrophila [21] [22] [67]- [71], ImiS from A. veronii bv. Sobria and Sfh-I from S. fonticola [67] [71] [72], and L1 from S. maltophilia [24] [73]- [75], FEZ-1 from F. gormanii [76], BJP-1 from B. japonicum [77], MIM-1 from N. pentaromativorans [78], MIM-2 from S. agarivorans [78], SMB-1 from S. marcescens [79], CAR-1 from E carotovora [80] and THIN-B from J. lividum [81], while the recently proposed B4 subgroup is represented by SPR-1 from S. proteamaculans [25] and CSA-1 from C. sakazaki [15]. B1-type MBLs have two peptide loops, L3 and L8, in the vicinity of the metal ion-containing active site (Figure 3).These loops are believed to be crucial for the determination of the substrate specificity of these enzymes [2].…”

“…A preference for cephalosporins has been noted for this subgroup [2] [21]. Initially, SPR-1 from S. proteamaculans was also assigned to the B3 subgroup [25]. However, an analysis of its active site structure (see below), together with a homology sequence analysis has indicated that SPR-1 may represent the prototype of the B4 subgroup of MBLs [15] [25].…”

“…Initially, SPR-1 from S. proteamaculans was also assigned to the B3 subgroup [25]. However, an analysis of its active site structure (see below), together with a homology sequence analysis has indicated that SPR-1 may represent the prototype of the B4 subgroup of MBLs [15] [25]. Its substrate preference is similar to that of the B3-type MBL L1 from S. maltophilia [25]; no crystallographic data for SPR-1 is currently available.…”

“…However, an analysis of its active site structure (see below), together with a homology sequence analysis has indicated that SPR-1 may represent the prototype of the B4 subgroup of MBLs [15] [25]. Its substrate preference is similar to that of the B3-type MBL L1 from S. maltophilia [25]; no crystallographic data for SPR-1 is currently available. The active sites of MBLs generally accommodate space for two metal ions to bind in close proximity ( Figure 4).…”

“…The only characterised MBL that may belong to the recently proposed B4 subgroup, SPR-1 from Serratia proteamaculans, may also require two metal ions to be catalytically active. However, unlike other MBLs studied to date SPR-1 may be mononuclear in the absence of substrates; only upon the addition of an antibiotic reactant a binuclear centre is formed [25]. This behaviour may imply the presence of a regulatory mechanism whereby the enzyme is "switched on" only when needed, a mechanism reminiscent of that of the organophosphate-degrading enzyme GpdQ from Enterobacter aerogenes [26]- [33].…”