Structure and Mechanism of Copper- and Nickel-Substituted Analogues of Metallo-β-lactamase L1

Abstract

In an effort to further probe metal binding to metallo-β-lactamase L1 (mβl L1), Cu-(Cu-L1) and Nisubstituted (Ni-L1) L1 were prepared and characterized by kinetic and spectroscopic studies. Cu-L1 bound 1.7 equivalents of Cu and small amounts of Zn(II) and Fe. The EPR spectrum of Cu-L1 exhibited two overlapping, axial signals, indicative of type 2 sites with distinct affinities for Cu(II). Both signals indicated multiple nitrogen ligands. Despite the expected proximity of the Cu(II) ions, however, only indirect evidence was found for spin-spin coupling. Cu-L1 exhibited higher k cat (96 s −1 ) and K m (224 μM) values, as compared to the values of dinuclear Zn(II)-containing L1, when nitrocefin was used as substrate. The Ni-L1 bound 1 equivalent of Ni and 0.3 equivalents of Zn(II). Ni-L1 was EPR-silent, suggesting that the oxidation state of nickel was +2; this suggestion was confirmed by 1 H NMR spectra, which showed relatively sharp proton resonances. Stopped-flow kinetic studies showed that ZnNi-L1 stabilized significant amounts of the nitrocefin-derived intermediate and that the decay of intermediate is rate-limiting. 1 H NMR spectra demonstrate that Ni(II) binds in the Zn 2 site and that the ring-opened product coordinates Ni(II). Both Cu-L1 and ZnNi-L1 hydrolyze cephalosporins and carbapenems, but not penicillins, suggesting that the Zn 2 site modulates substrate preference in mβ1 L1. These studies demonstrate that the Zn 2 site in L1 is very flexible and can accommodate a number of different transition metal ions; this flexibility could possibly offer an organism that produces L1 an evolutionary advantage when challenged with β-lactam containing antibiotics.β-Lactams are inexpensive and widely-used antibiotics against microbes since the 1940's(1). There are three different major classes of β-lactams, penicillins, cephalosporins, and carbapenems, that have been used clinically. However, most microorganisms have obtained the ability to either pump the β-lactams out of the cell via transporter proteins (2) or to hydrolyze these compounds by secreting β-lactamases into the periplasm or milieu (3). Four distinct classes of β-lactamases have been identified (4). Unlike class A, C, and D β-lactamases, which utilize an active site serine as a nucleophile, class B β-lactamases, metallo-β-lactamases or Mβl's, are a group of enzymes that require Zn(II) to hydrolyze β-lactams (5). There have been >50 Mβl's identified and categorized into three subgroups, according to amino acid sequence homology, the requirement of Zn(II) (1 or 2) for maximal activity, the identity of the metal

β-Lactam antibiotic-degrading enzymes from non-pathogenic marine organisms: a potential threat to human health

“…The exception is Cu(II), which renders MIM-2 significantly more reactive Table 4 indicate that with respect to the β-lactamase activity of MIM-1 and MIM-2, Zn(II) is the preferred metal ion, they also demonstrate that substrate preference is affected by the metal ion composition, and thus alternative in vivo functions may be possible for these enzymes. A similar behavior was observed previously for the B3-type MBL L1 from S. maltophilia, where the substrate specificity for the Ni(II) and Cu(II) derivatives of that enzyme varies distinctly from that of the Zn(II) derivative [53]. Here, a particularly intriguing observation was that Ca(II) was rather proficient in reconstituting ampicillinase activity in MIM-1 (Table 4).…”

Controlling Dicopper Protein Functions

“…68 Crowder et al reported that two cupric ions can be incorporated into the same site, when the apo-form crystals of metallo-β-lactamase is soaked with Cu 2+ , because of metal-binding promiscuity (Figure 9a). 69 Thus, we tried to develop an artificial dinuclear copper oxidase by using metallo-β-lactamase as a platform for dinuclear copper reaction center. 70 We have succeeded in the preparation of copper-substituted metallo-β-lactamase in the periplasm of E. coli cells during the cultivation by using the copper-containing medium.…”

Identification of Hypertension Susceptibility Loci on Rat Chromosome 12

“…In the line C region we identified 20 nonsynonomous amino acid changes in conserved validated genes between SS and BN, of which Mblac1, Il3ra, Spry3, Elfn1 , and Azgp1 were predicted by PolyPhen to be damaging variants (Table 1). Mblac1 is a poorly characterized metal-binding enzyme 35 with no reported role in BP or renal function. Il3ra and Spr3 are involved in hematopoiesis 36 and neurogenesis, 37 respectively, but also have no reported role in BP or renal function.…”