[14] Removal and replacement of metal ions in metallopeptidases

Class B metallo-β-lactamases (MBLs) are Zn²⁺-dependent enzymes that catalyze the hydrolysis of β-lactam antibiotics to confer resistance in bacteria. Several problematic groups of MBLs belong to subclass B1, including the binuclear New Delhi MBL (NDM), Verona integrin-encoded MBL, and imipenemase-type enzymes, which are responsible for widespread antibiotic resistance. Aspergillomarasmine A (AMA) is a natural aminopolycarboxylic acid that functions as an effective inhibitor of class B1 MBLs. The precise mechanism of action of AMA is not thoroughly understood, but it is known to inactivate MBLs by removing one catalytic Zn²⁺ cofactor. We investigated the kinetics of MBL inactivation in detail and report that AMA is a selective Zn²⁺ scavenger that indirectly inactivates NDM-1 by encouraging the dissociation of a metal cofactor. To further investigate the mechanism in living bacteria, we used an active site probe and showed that AMA causes the loss of a Zn²⁺ ion from a low-affinity binding site of NDM-1. Zn²⁺-depleted NDM-1 is rapidly degraded, contributing to the efficacy of AMA as a β-lactam potentiator. However, MBLs with higher metal affinity and stability such as NDM-6 and imipenemase-7 exhibit greater tolerance to AMA. These results indicate that the mechanism of AMA is broadly applicable to diverse Zn²⁺ chelators and highlight that leveraging Zn²⁺ availability can influence the survival of MBL-producing bacteria when they are exposed to β-lactam antibiotics.

The open reading frame of an α-amylase coding gene, amy19, was obtained from a fosmid genomic library of hot spring bacterium Bacillus BI-19 by a plate-based assay of carrageenase activity. After heterologous expression of the gene, the recombinant Amy19 was found to possess α-amylase, agarase, carrageenase, and cellulase activities, and could degrade soluble starch, agarose, carrageen, and sodium cellulose into oligosaccharides with low degrees of polymerization. To explore the multifunctional mechanism of Amy19, three continuous glycosyl hydrolase 70 (GH70) motifs in the Amy19 encoding sequence were deleted one by one, then in pairs, then all at once. The GH70 motifs may play an important role in the multifunctionality of Amy19, but the multifunctionality was not determined by the GH70 motifs alone. To our knowledge, this is the first report of an α-amylase from a hot spring bacterium with additional agarase, carrageenase, and cellulase activities.

Putative PFI1625c was cloned, over-expressed and purified to homogeneity. It is a 56.2 kDa monomeric protease which preferentially catalyzes the degradation of gelatin with a K_m = 30μM. It is a slow acting enzyme with optimal pH 8.5 and temperature 37 °C, and activity is sensitive to metalloprotease inhibitor 1,10-phenanthroline. PFI1625c active site was probed with a series of heterocyclic compounds and three molecules namely, BNPC-Inh2, DDBM-Inh1 and BHPM-Inh1 from the series were inhibiting PFI1625c protease activity. These heterocyclic compounds were found to irreversible inhibiting PFI1625c protease activity. Parasite culture was treated with these inhibitors and PFI1625c isolated from culture was found to be inactive without affecting other gelatinases present in the parasite. These inhibitors were used to generate chemically knockout PFI1625c in the parasite. PFI1625c knockout parasite remained at ring stage and was unable to complete its erythrocytic schizogony. Also, these knockout parasites were incapable to multiply. More careful analysis indicate these parasites develop oxidative stress as evident by the increase in lipid peroxidation, protein-carbonyl and a decrease of GSH level. In summary, the current study has employed biochemical, computational and pharmacological approaches to explore the role of PFI1625c in the parasite, its utility as a potential drug target to develop anti-malarials.

Anthrax lethal factor (LF) is a zinc-dependent endopeptidase involved in the cleavage of proteins critical to the maintenance of host signaling pathways during anthrax infections. Although zinc is typically regarded as the native metal ion in vivo, LF is highly tolerant to metal substitution, with its replacement by copper yielding an enzyme (CuLF) 4.5-fold more active than the native zinc protein (at pH 7). The current study demonstrates that by careful choice of the buffer, ionic strength, pH and substrate, the activity ratio of CuLF and native LF can be increased to >40-fold. Using a fluorogenic LF substrate, such optimized assay conditions can be exploited to detect LF concentrations as low as 2 pM. In contrast to the zinc form, CuLF was found to be inhibited by bromide and iodide ions, to be resistant to metal loss under acidic conditions, and to display a sharp pH dependence with significantly shifted alkaline limb towards more acidic conditions. The alkaline limb in the enzyme's pH profile is suggested to originate from changes in the protonation state of the metal-bound water molecule which serves as the nucleophile in the catalytic mechanism. Based on these observations and studies on other zinc proteases, a minimal mechanism for LF is proposed.

A large part of the more than 11 million tons of nanomaterials produced every year are metal and metal oxide nanoparticles (NPs), which ultimately end up in soil. NPs could enter into microbial cells either through endocytosis or by penetrating cells, and many of them, especially silver, copper, and zinc, have antimicrobial properties. Thus after entering the soil, NPs could adversely affect microorganisms present and disrupt essential functions of the soil. The effect of NPs on microorganisms and the mechanisms of their toxicity are comprehensively reviewed and discussed in this chapter. Both physical characteristics and chemical interactions of NPs at the nano–bio interface could elicit toxic effects, depending on their physical structure and chemical composition. Physical interactions, depending on size and surface properties of NPs at the nano–bio interface, could cause membrane disintegration and interference in transport processes. NPs, which are the same size as a protein molecule, interact with proteins and change their configuration, thus obstructing signaling processes in cells. Their shape can also affect toxicity by influencing uptake efficiency (rod-shaped NPs show maximum uptake and cubes minimum uptake), subsequently causing physical disintegration of membranes (truncated triangular silver nanoplates display the strongest biocidal action against Escherichia coli). Once inside the cells, NPs affect the catalytic activity of enzymes and stability of protein structures. Release of metal ions and the generation of reactive oxygen species restrict normal physiological redox-regulated functions of cells and lead to oxidative modification of proteins, which causes mortality of cells. Carbon-based NPs are less toxic than metal or metal oxide NPs but generation of reactive oxygen remains a major mechanism behind their toxicity.

A novel endo-type β-agarase, AgaB, was cloned from an agar-degrading bacterium, Flammeovirga sp. SJP92. The gene agaB consists of 2, 550 bp and encodes a protein of 849 amino acids including a 19 amino acids signal peptide. Based on the amino acid sequence similarity, AgaB belongs to the glycoside hydrolase family GH16. The recombinant AgaB was expressed in Escherichia coli and exhibited maximal activity at around 45 °C and pH 8.0, with a specific activity of 254.2 U/mg, a K_m of 3.99 mg/ml and a V_max of 700 U/mg for agarose. The agarase was stable at neutral to mildly alkaline condition, and remained 85%–90% of activity after treatment for 1 h, a characteristic much more different from other agarases reported. The recombinant enzyme was sensitive to some metal ions (Cu²⁺, Co²⁺ and Zn²⁺), but resistant to some denaturants (urea and SDS). It can hydrolyze the β-1, 4-glycosidic linkages of agarose, yielding neoagarotetraose and neoagarohexaose as the main products. These properties could make AgaB has a potential application in the food, cosmetic and medical industries.