Metal Content of Metallo-β-lactamase L1 Is Determined by the Bioavailability of Metal Ions

Abstract

In an effort to probe whether the metal content of metallo-β-lactamase L1 is affected by metal ion bioavailability, L1 was over-expressed as mature protein (M-L1) and full-length (FL-L1) analogs, and the analogs were characterized with metal analyses, kinetics, and EPR spectroscopy. FL-L1, containing the putative leader sequence, was localized in the periplasm of E. coli and shown to bind Zn(II) preferentially. The metal content of FL-L1 could be altered if the enzyme was over-expressed in minimal medium containing Fe and Mn, and surprisingly, an Fe-binding analog was obtained. On the other hand, M-L1, lacking the putative leader sequence, was localized in the cytoplasm of E. coli and shown to bind various amounts of Fe and Zn(II), and like FL-L1, the metal content of the resulting enzyme could be affected by the amount of metal ions in the growth medium. L1 was refolded in the presence of Fe, and a dinuclear Fe-containing analog of L1 was obtained, although this analog is catalytically-inactive. EPR spectra demonstrate the presence of an antiferromagnetically-coupled Fe(III)Fe(II) center in Fe-containing L1 and suggests the presence of a Fe(III)Zn(II) center in M-L1. Metal analyses on the cytoplasmic and periplasmic fractions of E. coli showed that the concentration of metal ions in the periplasm is not tightly controlled and increases as the concentration of metal ions in the growth medium increases. In contrast, the concentration of Zn(II) in the cytoplasm is tightly-controlled while that of Fe is less so.Bacterial resistance to β-lactam containing antibiotics such as penicillins, cephalosporins, and carbapenems is most often accomplished by expression of β-lactamases, which hydrolyze the C-N bond of these antibiotics ( 1-4 ). A majority of these β-lactamases utilize an active site serine group for the nucleophilic attack on the β-lactam carbonyl, and the serine β-lactamases have been studied extensively for many years ( 4 ). On the other hand, one class (Class B) of β-lactamases utilizes a metal-assisted hydrolysis pathway to inactivate β-lactam containing antibiotics, and these enzymes are called metallo-β-lactamases (mβl's) ( 1, 2, 5-7 ). The mβl's have been further divided into subgroups based on sequence identity, Zn(II) content, substrate preference, and biochemical properties. Subgroup B1 enzymes require 2 Zn(II) ions for full catalytic activity, exhibit kinetic preference for penicillins as substrates, exhibit >23% sequence identity toward other subgroup B1 members, and are represented by mβl's CcrA from Bacteroides fragilis, BcII from Bacillus cereus, and IMP-1 from various sources ( 1, 5 ). Subgroup B2 enzymes require only 1 Zn(II) ion for full catalytic activity, preferentially hydrolyze carbapenems, exhibit 11% sequence identity with the subgroup B1 enzymes, and

Secretion of GOB Metallo-β-Lactamase in Escherichia coli Depends Strictly on the Cooperation between the Cytoplasmic DnaK Chaperone System and the Sec Machinery: Completion of Folding and Zn(II) Ion Acquisition Occur in the Bacterial Periplasm

“…Remarkably, the GOB form secreted to the E. coli periplasm contained only Zn(II) ions (33). Similar observations concerning the lack of selectivity between Zn(II) and Fe(II) ions were made recently by other authors using an L1 M␤L form produced in the E. coli cytoplasm (20). Therefore, secretion of M␤Ls by the Sec rather than by the Tat pathway, as shown in this work, supports the proposal that a given secretory machinery may serve to overcome low intrinsic specificities of a given apoenzyme for the desired metal ion (45).…”

Low-Molecular-Mass Penicillin Binding Protein 6b (DacD) Is Required for Efficient GOB-18 Metallo-β-Lactamase Biogenesis in Salmonella enterica and Escherichia coli

“…Individual folding/assembly landscapes of secreted macromolecules are certainly affected in this complex and highly dynamic scenario, and it is therefore not surprising to find a number of periplasmic proteins with the ability to assist the folding, assembly, transit, and degradation of secreted proteins (6-8, 26, 27). Yet, whether the final biogenesis steps of MBLs also require assistance by periplasmic factors represented an open question, although evidence from a number of experiments (9,11,12) pointed in this direction. The results presented here indicate that the final steps of GOB-18 MBL biogenesis can be significantly affected by periplasmic factors.…”

“…Second, production of different apo MBL forms in the E. coli cytoplasm by means of expression vectors lacking transit sequences directing secretion resulted in the accumulation in this compartment of mostly Fe-containing inactive MBLs (11,12). By contrast, Zn-containing native MBLs are predominantly recovered when the same apoproteins are directed to the periplasm (11,12), suggesting the existence of factors in this compartment mediating Zn 2ϩ supply or assisting folding (or refolding) until the native conformation is reached.By using different genetic approaches we searched here for the existence of factors assisting biogenesis of the GOB-18 MBL in the periplasm of Escherichia coli and Salmonella enterica. For this purpose the preapoprotein was produced from expression plasmids (5) and the resulting GOB-18-mediated cefotaxime (CTX) resistance of the cells was analyzed.…”

“…Due to the high protease content of the periplasm, some means of protection of these nonnative forms from degradation must exist (10), thus suggesting the existence of periplasmic factors performing this role, which is typical of folding assistants (8). Second, production of different apo MBL forms in the E. coli cytoplasm by means of expression vectors lacking transit sequences directing secretion resulted in the accumulation in this compartment of mostly Fe-containing inactive MBLs (11,12). By contrast, Zn-containing native MBLs are predominantly recovered when the same apoproteins are directed to the periplasm (11,12), suggesting the existence of factors in this compartment mediating Zn 2ϩ supply or assisting folding (or refolding) until the native conformation is reached.…”

Structure of PhnP, a Phosphodiesterase of the Carbon-Phosphorus Lyase Pathway for Phosphonate Degradation

“…It is certainly possible that in vivo Zn 2ϩ may serve as the active metal ion in PhnP, since cytoplasmic Zn 2ϩ concentrations are maintained at 45 M in E. coli (35), which would afford a low level of activity (Table 3) sufficient for cell growth. However, E. coli also has a dedicated Mn 2ϩ transport system (36) and can achieve cytoplasmic levels of this metal ion well into the 10 Ϫ4 M range (37,38).…”