Folding strategy to prepare Co(II)-substituted metallo-β-lactamase L1

Abstract

In an effort to overcome previous problems with the preparation of Co(II)-substituted metallo-beta-lactamase L1, two strategies were undertaken. Attempts to prepare Co(II)-substituted L1 using biological incorporation resulted in an enzyme that contained only 1 Eq of cobalt and exhibited no catalytic activity. Co(II)-substituted L1 could be prepared by refolding metal-free L1 in the presence of Co(II), and the resulting enzyme contained 1.8 Eq of cobalt, yielded a UV-Vis spectrum consistent with 5-coordinate Co(II), and exhibited a k(cat) of 63 s(-1) and K(m) of 20 microM when using nitrocefin as the substrate. Pre-steady-state fluorescence and UV-Vis studies demonstrated that refolded, Co(II)-substituted L1 uses the same kinetic mechanism as Zn(II)-containing L1, in which a reaction intermediate is formed when using nitrocefin as substrate. The described refolding strategy can be used to prepare other Co(II)-substituted Zn(II)-metalloenzymes, particularly those that contain a solvent-exposable disulfide, which often causes oxidation of Co(II) to Co(III).

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].…”

Metal-dependent amyloid β-degrading catalytic antibody construct

“…, Zn 2+ accelerates proteolytic auto-activation of Factor XII, but the rate is reduced by excess Zn 2+ (Bernardo et al 1993). Co 2+ can serve as the replacement metal ion at protein Zn 2+ -binding sites (Auld 1995; Hu et al 2008), but differing concentrations of the two metals are required for activation and inhibition of metal-dependent enzymes displaying multiple metal-binding sites (Hashida and Inouye 2007; Sabel et al 2011). Removal of bound metal did not decrease the IgV nucleophilic reactivity, suggesting that the metal is not involved in initiating IgV nucleophilic attack on Aβ.…”

Dilution of dipolar interactions in a spin-labeled, multimeric metalloenzyme for DEER studies

“…[26]. Metal-free (apo) L1 samples, both wild-type and mutants, were generated as described by Hu and coworkers [28]. The metal-free, double mutant of L1 (1 mL, 200 µM) was mixed with 1 mL of 800 µM apo-wild-type L1, and the mixture was unfolded in 18 mL of 7 M Gdn-HCl containing 100 µM Zn(II).…”

“…The enzymes were made metal-free according to previously reported procedures [28]. The apo-enzymes were unfolded with Gdn-HCl, and the spin-diluted sample was made by refolding 4 molar equivalents of unfolded wild-type L1 with 1 molar equivalent of the unfolded T163C/K286C in the presence of Zn(II).…”