Newly Identified Genetic Variations in Common Escherichia coli MG1655 Stock Cultures

We have recently identified seven mutations in commonly used stocks of the sequenced Escherichia coli strain MG1655 which do not appear in the reference sequence. The mutations are likely to cause loss of function of the glpR and crl genes, which may have serious implications for physiological experiments using the affected strains

Kinetic and structural characterization of bacterial glutaminyl cyclases from Zymomonas mobilis and Myxococcus xanthus

AbstractAlthough enzymes responsible for the cyclization of amino-terminal glutamine residues are present in both plant and mammal species, none have yet been characterized in bacteria. Based on low sequence homologies to plant glutaminyl cyclases (QCs), we cloned the coding sequences of putative microbial QCs fromZymomonas mobilis(ZmQC) andMyxococcus xanthus(MxQC). The two recombinant enzymes exhibited distinct QC activity, with specificity constantskcat/Kmof 1.47±0.33 mm-1s-1(ZmQC) and 142±32.7 mm-1s-1(MxQC) towards the fluorescent substrate glutamine-7-amino-4-methyl-coumarine. The measured pH-rate profile of the second order rate constant displayed an interesting deviation towards the acidic limb of the pH chart in the case of ZmQC, whereas MxQC showed maximum activity in the mild alkaline pH range. Analysis of the enzyme variants ZmQCGlu46Gln and MxQCGln46Glu show that the exchanged residues play a significant role in the pH behaviour of the respective enzymes. In addition, we determined the three dimensional crystal structures of both enzymes. The tertiary structure is defined by a five-bladed β-propeller anchored by a core cation. The structures corroborate the putative location of the active site and confirm the proposed relation between bacterial and plant glutaminyl cyclases.</jats:p

Structural and functional analyses of pyroglutamate amyloid beta specific antibodies as a basis for Alzheimer immunotherapy

Alzheimer disease is associated with deposition of the amyloidogenic peptide A in the brain. Passive immunization using A-specific antibodies has been demonstrated to reduce amyloid deposition both in vitro and in vivo. Because N-terminally truncated pyroglutamate (pE)-modified A species (A(pE3)) exhibit enhanced aggregation potential and propensity to form toxic oligomers, they represent particularly attractive targets for antibody therapy. Here we present three separate monoclonal antibodies that specifically recognize A(pE3) with affinities of 1-10 nm and inhibit A(pE3) fibril formation in vitro. In vivo application of one of these resulted in improved memory in A(pE3) oligomer-treated mice. Crystal structures of F-ab-A(pE3) complexes revealed two distinct binding modes for the peptide. Juxtaposition of pyroglutamate pE3 and the F4 side chain (the pEF head) confers a pronounced bulky hydrophobic nature to the A(pE3) N terminus that might explain the enhanced aggregation properties of the modified peptide. The deep burial of the pEF head by two of the antibodies explains their high target specificity and low cross-reactivity, making them promising candidates for the development of clinical antibodies

The protein-free IANUS peptide array uncovers interaction sites between Escherichia coli parvulin 10 and alkyl hydroperoxide reductase

The reliable identification of interacting structural elements without prior isolation of interacting proteins can be achieved by using the novel fluorescence resonance energy transfer-coupled IANUS (Induced orgANization of strUcture by matrix-assisted togethernesS) peptide array. Here we report that parvulin 10 (Par10), an abundant Escherichia coli peptidyl prolyl cis/trans isomerase (PPIase), physically interacts with the alkyl hydroperoxide reductase subunit C (AhpC) in bacterial cell extracts, as determined by affinity chromatography and chemical cross-linking experiments. A Par10-negative E. coli strain showed increased sensitivity toward hydrogen peroxide compared to the wild-type strain. The IANUS experiment revealed three segments of the peroxiredoxin AhpC chain as potential Par10 binding partners. Inhibition of the Par10 PPIase activity by the corresponding AhpC-derived peptides as well as NMR data of (15)N-labeled Par10 in the presence of the AhpC(115-132) peptide or full-length AhpC confirmed that the putative Par10 active site is involved in the Par10-AhpC interaction. Moreover, NMR-based docking calculations as well as NOESY exchange peaks between the proline cis and trans isomers revealed the Asp125-Pro126 moiety of the AhpC segment G115-A132 as a substrate for Par10 enzymatic action. On the basis of these data, we conclude that Par10 catalytic activity is involved in the cellular protection against oxidative stress