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A single amino acid substitution, Phe98 to Tyr98, in dihydrofolate reductase (DHFR) is the molecular origin of trimethoprim (TMP) resistance in Staphylococcus aureus. This active site amino acid substitution was found in all S. aureusTMP-resistant clinical isolates tested. In order to explore the structural role of Tyr98 in TMP-resistance the ternary complexes of the chromosomal S. aureusDHFR (SaDHFR) with methotrexate (MTX) and TMP in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) as well as that of mutant Phe98Tyr DHFR SaDHFR(F98Y) ternary folate-NADPH complex have been determined by X-ray crystallography. Critical evidence concerning the resistance mechanism has also been provided by NMR spectral analyses of 15N-labelled TMP in the ternary complexes of both wild-type and mutant enzyme. These studies show that the mutation results in loss of a hydrogen bond between the 4-amino group of TMP and the carbonyl oxygen of Leu5. This mechanism of resistance is predominant in both transferable plasmid-encoded and non-transferable chromosomally encoded resistance. Knowledge of the resistance mechanism at a molecular level could help in the design of antibacterials active against multi-resistant Staphylococcus aureus(MRSA), one of todays most serious problems in clinical infectology.

Senile plaques, a neuropathological hallmark of Alzheimer's disease, consist primarily of insoluble aggregates of ß-amyloid peptide (Aß). A 42-residue peptide (Aß1-42) appears to be the predominant form. In contrast to Aß1-40, Aß1-42is characterized by its extreme tendency to aggregate into fibers or precipitate. A tailored biotechnological method prevents aggregation of Aß1-42monomers during its production. The method is based on a protein tail fused to the amino terminus of Aß. This tail leads to a high expression in E. coli, and a histidine affinity tag facilitates purification. Selective cleavage of the fusion tail is performed with cyanogen bromide by immobilizing the fusion protein on a reversed phase chromatography column. Cleavage then occurs only at the methionine positioned at the designed site but not at the methionine contained in the membrane anchor sequence of Aß. Furthermore, immobilization prevents aggregation of cleaved Aß. Elution from the HPLC column and all succeeding purification steps are optimized to preserve Aß1-42as a monomer. Solutions of monomeric Aß1-42spontaneously aggregate into fibers within hours. This permits the investigation of the transition of monomers into fibers and the correlation of physico-chemical properties with biological activities. Mutations of Aß1-42at postion 35 influence the aggregation properties. Wild-type Aß1-42with methionine at position 35 has similar properties as Aß with a methionine sulfoxide residue. The fiber formation tendency, however, is reduced when position 35 is occupied by a glutamine, serine, leucine, or a glutamic acid residue.

Helicobacter pylori is an important human pathogen which causes both gastric and duodenal ulcers and is associated with gastric cancer and lymphoma. This microorganism synthesizes fucosylated oligosaccharides, predominantly the Galb-1,4GlcNAc (Type II) blood group antigens Lewis X and Y, whereas a small population also expresses the Galb-1,3GlcNAc (Type I) blood group antigens Lewis A and B. These carbohydrate structures are known to mimic host cell antigens and permit the bacteria to escape from the host immune response. Here, we report the cloning and characterization of a novel H. pylori α-1,4 fucosyltransferase (FucT). In contrast to the family members characterized to date, this enzyme shows exclusively Type I acceptor substrate specificity. The enzyme consisting of 432 amino acids (MW 50,502 Da) was cloned using a polymerase chain reaction (PCR)-based approach. It exhibits a high degree of identity (75–87%) and similar structural features, for example, in the heptamer repeat pattern, with other H. pylori FucTs. The kinetic characterization revealed a very efficient transferase (kcat/Km = 229 mM21s21) for the Type I acceptor substrate (Gal)-1,3 GlcNAc-Lem (1). Additionally, the enzyme possesses a broad tolerance toward nonnatural Type I acceptor substrate analogs and therefore represents a valuable tool for the chemoenzymatic synthesis of Lewis A, sialyl Lewis A as well as mimetics thereof.