Solvent and thermal stability, and pH kinetics, of proline-specific dipeptidyl peptidase IV-like enzyme from bovine serum

Proline-specific dipeptidyl peptidase-like (DPP IV; EC 3.4.14.5) activity in bovine serum has attracted little attention despite its ready availability and the paucity of useful proline-cleaving enzymes. Bovine serum DPP IV-like peptidase is very tolerant of organic solvents, particularly acetonitrile: upon incubation for 1 h at room temperature in 70% acetonitrile, 47% dimethylformamide, 54% DMSO and 33% tetrahydrofuran (v/v concentrations) followed by dilution into the standard assay mixture, the enzyme retained half of its aqueous activity. As for thermal performance in aqueous buffer, its relative activity increased up to 50 ◦C. Upon thermoinactivation at 71 ◦C, pH 8.0 (samples removed periodically, cooled on ice, then assayed under optimal conditions), residual activities over short times fit a first-order decay with a k-value of 0.071±0.0034 min−1. Over longer times, residual activities fit to a double exponential decay with k1 and k2 values of 0.218±0.025 min−1 (46±4% of overall decay) and 0.040±0.002 min−1 (54±4% of overall decay), respectively. The enzyme’s solvent and thermal tolerances suggest that it may have potential for use as a biocatalyst in industry. Kinetic analysis with the fluorogenic substrate Gly-Pro-7-aminomethylcoumarin over a range of pH values indicated two pK values at 6.18±0.07 and at 9.70±0.50. We ascribe the lower value to the active site histidine; the higher may be due to the active site serine or to a free amino group in the substrate

Elucidation of the RamA Regulon in Klebsiella pneumoniae Reveals a Role in LPS Regulation

Klebsiella pneumoniae is a significant human pathogen, in part due to high rates of multidrug resistance. RamA is an intrinsic regulator in K. pneumoniae established to be important for the bacterial response to antimicrobial challenge; however, little is known about its possible wider regulatory role in this organism during infection. In this work, we demonstrate that RamA is a global transcriptional regulator that significantly perturbs the transcriptional landscape of K. pneumoniae, resulting in altered microbe-drug or microbe-host response. This is largely due to the direct regulation of 68 genes associated with a myriad of cellular functions. Importantly, RamA directly binds and activates the lpxC, lpxL-2 and lpxO genes associated with lipid A biosynthesis, thus resulting in modifications within the lipid A moiety of the lipopolysaccharide. RamA-mediated alterations decrease susceptibility to colistin E, polymyxin B and human cationic antimicrobial peptide LL-37. Increased RamA levels reduce K. pneumoniae adhesion and uptake into macrophages, which is supported by in vivo infection studies, that demonstrate increased systemic dissemination of ramA overexpressing K. pneumoniae. These data establish that RamA-mediated regulation directly perturbs microbial surface properties, including lipid A biosynthesis, which facilitate evasion from the innate host response. This highlights RamA as a global regulator that confers pathoadaptive phenotypes with implications for our understanding of the pathogenesis of Enterobacter, Salmonella and Citrobacter spp. that express orthologous RamA proteins

The Victorian Newsletter (Spring 1962)

The Victorian Newsletter is edited for the English X Group of the Modern Language Association by William E. Buckler, 737 East Building, New York University, New York 3, New York.Some pages are missing from this record