Difference in the Inhibitory Effect of Thiol Compounds and Demetallation Rates from the Zn(II) Active Site of Metallo-ß-lactamases (IMP-1 and IMP-6) Associated with a Single Amino Acid Substitution.

Gram-negative bacteria producing metallo-ß-lactamases (MBLs) have become a considerable threat to public health. MBLs including the IMP, VIM, and NDM types are Zn(II) enzymes that hydrolyze the ß-lactam ring present in a broad range of antibiotics, such as N-benzylpenicillin, meropenem, and imipenem. Among IMPs, IMP-1 and IMP-6 differ in a single amino acid substitution at position 262, where serine in IMP-1 is replaced by glycine in IMP-6, conferring a change in substrate specificity. To investigate how this mutation influences enzyme function, we examined lactamase inhibition by thiol compounds. Ethyl 3-mercaptopropionate acted as a competitive inhibitor of IMP-1, but a noncompetitive inhibitor of IMP-6. A comparison of the crystal structures previously reported for IMP-1 (PDB code: 5EV6) and IMP-6 (PDB code: 6LVJ) revealed a hydrogen bond between the side chain of Ser262 and Cys221 in IMP-1 but the absence of hydrogen bond in IMP-6, which affects the Zn2 coordination sphere in its active site. We investigated the demetallation rates of IMP-1 and IMP-6 in the presence of chelating agent ethylenediaminetetraacetic acid (EDTA) and found that the demetallation reactions had fast and slow phases with a first-order rate constant (kfast = 1.76 h-1, kslow = 0.108 h-1 for IMP-1, and kfast = 14.0 h-1 and kslow = 1.66 h-1 for IMP-6). The difference in the flexibility of the Zn2 coordination sphere between IMP-1 and IMP-6 may influence the demetallation rate, the catalytic efficiency against ß-lactam antibiotics, and the inhibitory effect of thiol compounds.

Recovery of platinum(0) through the reduction of platinum ions by hydrogenase-displaying yeast.

Biological technologies for recycling rare metals, which are essential for high-tech products, have attracted much attention because they could prove to be more environmentally friendly and energy-saving than other methods. We have developed biological recycling technologies by cell surface engineering for the selective recovery of toxic heavy metal ions and rare metal ions from aqueous wastes. In this study, we aimed to construct a unique biological technique to recover rare metals 'in solid' form by reducing rare metal ions, leading to a practical next-generation recovery system. Sulfate-reducing bacteria (SRB) can reduce Pt(II) to Pt(0), and hydrogenases of SRB contribute to the reduction. Therefore, we constructed yeasts displaying their hydrogenases on the 'cell membrane', and reduction experiments were performed under anaerobic conditions without any electron donors. As a result, hydrogenase-displaying yeasts produced black precipitates in PtCl42- solution. Based on X-ray photoelectron spectroscopy (XPS) and transmission electron microscopy (TEM) observations, the constructed yeasts were found to successfully produce the precipitates of Pt(0) through the reduction of Pt(II). Interestingly, the precipitates of Pt(0) were formed as nanoparticles, suitable for industrial usage.