Site - Specific Mutations of Conserved C - Terminal Residues in Aminoglycoside 3'-Phosphotransferase II [APH(3')-II]: Phenotypic and Structural Analysis of the Mutant Enzymes

In order to test the biological importance of amino acids in the C-terminal quarter of aminoglycoside 3′-phosphotransferase II enzyme, seven of the highly conserved residues in this region, His-188, Asp-190, Asp-208, Gly-210, Arg-211, Asp-216 and Asp-220, were changed via site-directed mutagenesis. The phenotype of each mutant was compared to wildtype in terms of antibiotic susceptibilities and enzymatic activities. All of the substitutions either abolished or significantly reduced the resistance of the resulting strains to kanamycin, neomycin, butirosin, ribostamycin, paromomycin, gentamicin A, and G-418. Similarly, enzyme activities in crude extracts were substantially reduced for the mutant strains. Affinity of the enzyme for Mg+2-ATP decreased with His-188, Asp-190, Asp-216 and Asp-220 substitutions as revealed by Km measurements. Secondary structure analysis predicted that substitutions at the conserved residues caused severe conformational distortions at the corresponding regions of the protein

Altered Substrate Specificities by Substitutions at Tyr 218 in Bacterial Aminoglycoside 3'- Phosphotransferase – II

Mutant aminoglycoside 3'-phosphotransferase II enzymes were produced in which Tyr218 was changed to serine, aspartic acid, or phenylalanine. In each case the mutation resulted in increased bacterial susceptibility to neomycin and kanamycin, while simultaneously increasing the Km values for these substrates. For the Ser and Asp mutants, bacterial resistance to amikacin increased, with a concomitant increase in affinity for this drug. Initial velocity studies indicated that the wild-type and mutant enzymes all followed Michaelis-Menten kinetics. Although these mutagenic substitutions changed the substrate specificity of these enzymes they did not alter the enzyme affinity for Mg(2+)-ATP

Structure - Function Analyses for Aminoglycoside 3'-Phosphotransferase-II [APH(3')-II]

Mutant strains containing APH(3')-II were constructed via site-directed mutagenesis of the cloned gene and by random mutagenesis of a strain containing the APH(3')-II gene on a conjugative plasmid. Substitutions at highly conserved amino acid residues produced APH(3') enzymes which in general showed reduced activity and conferred reduced levels of resistance to their substrates. Substitutions at Tyr 218 altered substrate specificity for the enzymes. Random mutagenesis produced plasmid-borne mutations conferring amikacin resistance. Two of these mutations appeared to be localized to the APH(3')-II structural gene

High-level amikacin resistance in Pseudomonas aeruginosa associated with a 3'-phosphotransferase with high affinity for amikacin.

This work describes the characterization of the phosphotransferase enzymatic activity responsible for amikacin resistance in two clinical Pseudomona aeruginosa strains, isolated from a hospital that used amikacin as first-line aminoglycoside. Amikacin-resistant P. aeruginosa PA40 and PA43 (MIC: 128 mg/l) were shown to have APH activity with a substrate profile similar to that of APH(3')-VI. The enzyme from P. aeruginosa PA40 was purified to >70% homogeneity. The K(m) of amikacin for this enzyme was 1.4 M, the V(max)/K(m) ratio for amikacin was higher than for the other aminoglycosides tested and PCR and DNA sequencing ruled out the presence of aph(3')-IIps. Amikacin resistance in this strain was, therefore, associated with APH(3')-VI and the high affinity of this enzyme for amikacin could explain the high-level resistance that we observed

On the space of oriented affine lines in R^3.

We introduce a local coordinate description for the correspondence between the space of oriented affine lines in Euclidean and the tangent bundle to the 2-sphere. These can be utilised to give canonical coordinates on surfaces in, as we illustrate with a number of explicit examples

An interactive fluid model of jellyfish for animation

We present an automatic animation system for jellyfish that is based on a physical simulation. We model the thrust of an adult jellyfish, and the organism's morphology in its most active mode of locomotion. We reduce our model by considering only species that are axially symmetric so that we can approximate the full 3D geometry of a jellyfish with a 2D simulation. We simulate the organism's elastic volume with a spring-mass system, and the surrounding sea water using the semi-Lagrangian method. We couple the two representations with the immersed boundary method. We propose a simple open-loop controller to contract the swimming muscles of the jellyfish. A 3D rendering model is extrapolated from our 2D simulation. We add variation to the extrapolated 3D geometry, which is inspired by empirical observations of real jellyfish. The resulting animation system is efficient with an acceptable compromise in physical accuracy