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Alanine Ligase (alanine + ligase)
Selected AbstractsA six amino acid deletion, partially overlapping the VanSB G2 ATP-binding motif, leads to constitutive glycopeptide resistance in VanB-type Enterococcus faeciumMOLECULAR MICROBIOLOGY, Issue 3 2003Florence Depardieu Summary Enterococcus faecium clinical isolate BM4524, resistant to vancomycin and susceptible to teicoplanin, harboured a chromosomal vanB cluster, including the vanSB / vanRB two-component system regulatory genes. Enterococcus faecium strain BM4525, isolated two weeks later from the same patient, was resistant to high levels of both glycopeptides. The ddl gene of BM4525 had a 2 bp insertion leading to an impaired d -alanine: d -alanine ligase. Sequencing of the vanB operon in BM4525 also revealed an 18 bp deletion in the vanSB gene designated vanSB, . The resulting six amino acid deletion partially overlapped the G2 ATP-binding domain of the VanS B, histidine kinase leading to constitutive expression of the resistance genes. Sequence analysis indicated that the deletion occurred between two tandemly arranged heptanucleotide direct repeats, separated by 11 base-pairs. The VanS B , VanS B, and VanR B proteins were overproduced in Escherichia coli and purified. In vitro autophosphorylation of the VanS B and VanS B, histidine kinases and phosphotransfer to the VanR B response regulator did not differ significantly. However, VanS B, was deficient in VanR B phosphatase activity leading to accumulation of phosphorylated VanR B . Increased glycopeptide resistance in E. faecium BM4525 was therefore a result of the lack of production of d -alanyl- d -alanine ending pentapeptide and to constitutive synthesis of d -alanyl- d -lactate terminating peptidoglycan precursors, following loss of d -alanine: d -alanine ligase and of VanS B phosphatase activity respectively. We suggest that the heptanucleotide direct repeat in vanSB may favour the appearance of high level constitutively expressed vancomycin resistance through a ,slippage' type of genetic rearrangement in VanB-type strains. [source] Structure of d -alanine- d -alanine ligase from Thermus thermophilus HB8: cumulative conformational change and enzyme,ligand interactionsACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2009Yoshiaki Kitamura d -Alanine- d -alanine ligase (Ddl) is one of the key enzymes in peptidoglycan biosynthesis and is an important target for drug discovery. The enzyme catalyzes the condensation of two d -Ala molecules using ATP to produce d -Ala- d -Ala, which is the terminal peptide of a peptidoglycan monomer. The structures of five forms of the enzyme from Thermus thermophilus HB8 (TtDdl) were determined: unliganded TtDdl (2.3,Å resolution), TtDdl,adenylyl imidodiphosphate (2.6,Å), TtDdl,ADP (2.2,Å), TtDdl,ADP,d -Ala (1.9,Å) and TtDdl,ATP,d -Ala- d -Ala (2.3,Å). The central domain rotates as a rigid body towards the active site in a cumulative manner in concert with the local conformational change of three flexible loops depending upon substrate or product binding, resulting in an overall structural change from the open to the closed form through semi-open and semi-closed forms. Reaction-intermediate models were simulated using TtDdl-complex structures and other Ddl structures previously determined by X-ray methods. The catalytic process accompanied by the cumulative conformational change has been elucidated based on the intermediate models in order to provide new insights regarding the details of the catalytic mechanism. [source] Crystallization and preliminary X-ray analysis of a d -alanyl- d -alanine ligase (EcDdlB) from Escherichia coliACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2010Sarah Batson A recombinant form of Escherichia coli DdlB (EcDdlB) has been prepared and cocrystallized with ADP and d -alanyl- d -alanine to represent the ternary complex of EcDdlB. Furthermore, EcDdlB has been cocrystallized under the same conditions with the ligands ATP and d -alanyl- d -alanine, representing the product-inhibited complex. The crystals belonged to space group P212121, with unit-cell parameters a = 53.0, b = 97.6, c = 109.5,Å and a = 51.2, b = 97.8, c = 110.1,Å, respectively, and both contained two molecules in the asymmetric unit. Complete data sets were collected to 1.5 and 1.4,Å resolution, respectively, from single crystals under cryogenic conditions using synchrotron radiation. [source] |