Metallo-β-lactamases (MBLs) are a family of metalloenzymes that are capable of hydrolyzing β-lactam antibiotics and are an important means by which bacterial pathogens use to inactivate antibiotics. A database search of the available amino acid sequences from Serratia proteamaculans indicates the presence of an unusual MBL. A full length amino acid sequence alignment indicates overall homology to B3-type MBLs, but also suggests considerable variations in the active site, notably among residues that are relevant to metal ion binding. Steady-state kinetic measurements further indicate functional differences and identify two relevant pK a values for catalysis (3.8 for the enzyme-substrate complex and 7.8 for the free enzyme) and a preference for penams with modest reactivity towards some cephalosporins. An analysis of the metal ion content indicates the presence of only one zinc ion per active site in the resting enzyme. In contrast, kinetic data suggest that the enzyme may operate as a binuclear enzyme, and it is thus proposed that a catalytically active di-Zn(2+) center is formed only once the substrate is present.
Many bacteria secrete cellulose, which forms the structural basis for bacterial multicellular aggregates, termed biofilms. The cellulose synthase complex of Salmonella typhimurium consists of the catalytic subunits BcsA and BcsB and several auxiliary subunits that are encoded by two divergently transcribed operons, bcsRQABZC and bcsEFG. Expression of the bcsEFG operon is required for full-scale cellulose production, but the functions of its products are not fully understood. This work aimed to characterize the BcsG subunit of the cellulose synthase, which consists of an N-terminal transmembrane fragment and a C-terminal domain in the periplasm. Deletion of the bcsG gene substantially decreased the total amount of BcsA and cellulose production. BcsA levels were partially restored by the expression of the transmembrane segment, whereas restoration of cellulose production required the presence of the C-terminal periplasmic domain and its characteristic metal-binding residues. The high-resolution crystal structure of the periplasmic domain characterized BcsG as a member of the alkaline phosphatase/sulfatase superfamily of metalloenzymes, containing a conserved Zn-binding site. Sequence and structural comparisons showed that BcsG belongs to a specific family within alkaline phosphatase-like enzymes, which includes bacterial Zn-dependent lipopolysaccharide phosphoethanolamine transferases such as MCR-1 (colistin resistance protein), EptA, and EptC and the Mn-dependent lipoteichoic acid synthase (phosphoglycerol transferase) LtaS. These enzymes use the phospholipids phosphatidylethanolamine and phosphatidylglycerol, respectively, as substrates. These data are consistent with the recently discovered phosphoethanolamine modification of cellulose by BcsG and show that its membrane-bound and periplasmic parts play distinct roles in the assembly of the functional cellulose synthase and cellulose production.
There are currently no clinically available inhibitors of metallo-β-lactamases (MBLs). These enzymes confer resistance to bacteria against a broad range of commonly used β-lactam antibiotics, and are produced by an increasing number of bacterial pathogens. In this study, several thiol derivatives of l-amino acids were designed and synthesized, and their inhibitory effects against the metallo-β-lactamase IMP-1 (subclass B1) were investigated. The most potent compound, derived from l-tyrosine, exhibited competitive inhibition, with a Ki of 86 nM. The ability of this compound to render MBL-expressing bacteria susceptible to imipenem was examined. Reductions in MIC values up to 5.2-fold were observed.
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