Substrate-Induced Inactivation of the Escherichia coli AmiD N-Acetylmuramoyl-l-Alanine Amidase Highlights a New Strategy To Inhibit This Class of Enzyme

ABSTRACT In the eubacterial cell, the peptidoglycan is perpetually hydrolyzed throughout the cell cycle by different enzymes such as lytic transglycosylases, endopeptidases, and amidases. In Escherichia coli, four N-acetylmuramoyl-l-alanine amidases, AmiA, -B, -C, and -D, are present in the periplasm. AmiA, -B, and -C are soluble enzymes, whereas AmiD is a lipoprotein anchored in the outer membrane. To determine more precisely the specificity and the kinetic parameters of AmiD, we overproduced and purified the native His-tagged AmiD in the presence of detergent and a soluble truncated form of this enzyme by removing its signal peptide and the cysteine residue responsible for its lipidic anchorage. AmiD is a zinc metalloenzyme and is inactivated by a metal chelator such as EDTA. Native His-tagged and truncated AmiD hydrolyzes peptidoglycan fragments that have at least three amino acids in their peptide chains, and the presence of an anhydro function on the N-acetylmuramic acid is not essential for its activity. The soluble truncated AmiD exhibits a biphasic kinetic time course that can be explained by the inactivation of the enzyme by the substrate. This behavior highlights a new strategy to inhibit this class of enzymes.

Cloning and Characterization of the Glucooligosaccharide Catabolic Pathway β-Glucan Glucohydrolase and Cellobiose Phosphorylase in the Marine HyperthermophileThermotoga neapolitana

ABSTRACT Characterization in Thermotoga neapolitana of a catabolic gene cluster encoding two glycosyl hydrolases, 1,4-β-d-glucan glucohydrolase (GghA) and cellobiose phosphorylase (CbpA), and the apparent absence of a cellobiohydrolase (Cbh) suggest a nonconventional pathway for glucan utilization inThermotogales. GghA purified from T. neapolitana is a 52.5-kDa family 1 glycosyl hydrolase with optimal activity at pH 6.5 and 95°C. GghA releases glucose from soluble glucooligomers, with a preference for longer oligomers:k cat/Km values are 155.2, 76.0, and 9.9 mM−1 s−1 for cellotetraose, cellotriose, and cellobiose, respectively. GghA has broad substrate specificity, with specific activities of 236 U/mg towards cellobiose and 251 U/mg towards lactose. Withp-nitrophenyl-β-glucoside as the substrate, GghA exhibits biphasic kinetic behavior, involving both substrate- and end product-directed activation. Its capacity for transglycosylation is a factor in this activation. Cloning of gghA revealed a contiguous upstream gene (cbpA) encoding a 93.5-kDa cellobiose phosphorylase. Recombinant CbpA has optimal activity at pH 5.0 and 85°C. It has specific activity of 11.8 U/mg and aKm of 1.42 mM for cellobiose, but shows no activity towards other disaccharides or cellotriose. With its single substrate specificity and low Km for cellobiose (compared to GghA's Km of 28.6 mM), CbpA may be the primary enzyme for attacking cellobiose inThermotoga spp. By phosphorolysis of cellobiose, CbpA releases one activated glucosyl molecule while conserving one ATP molecule per disaccharide. CbpA is the first hyperthermophilic cellobiose phosphorylase to be characterized.

Pharmacokinetics of ciprofloxacin in layer chicks

Pharmacokinetics parameters of ciprofloxacin were calculated from constructed plasma disappearing curves (PDC) after oral or i.v. injection of 5mg / kg in two-week old hybird layer · The i.v. PDC indicated a first order biphasic kinetic. The distribution phase was too short to be considered. The parameters of the elimination phase phase indicated that the biological half – life ( t 12) was 3.7 hours and the volume of distribution (Vd) was 1.7 1/kg. The oral PDC indicated a first order kinetic with a peak plasma level (PPL) of 3.0 ug/ml achieved after 1.75 hours , a t 12 of 2.9 hours and a Vd of 1.7 1/kg . Bioavailability value was 84 +9.7% . Binding of ciprofloxacin to chicken plasma protein was estimated to be 25.4 61.3%.