Biomedical subjects

Sarah J Lipscomb

Publications and source records attributed to Sarah J Lipscomb.

3 recordsLinked to original sources

PubMed2006

SERRS labelled beads for multiplex detection.

Beads labelled using surface enhanced resonance Raman scattering (SERRS) are highly sensitive and specific tags, with potential applications in biological assays, including molecular diagnostics. The beads consist of a nucleus containing dye labelled silver-nanoparticle aggregates surrounded by a polymer core. The nuclei generate strong SERRS signals. To illustrate the coding advantage created by the sharp, molecularly specific SERRS signals, four specially designed SERRS dyes have been used as labels and three of these have been combined in a multiplex analysis. These dyes use specific groups such as benzotriazole and 8-hydroxyquinoline to improve binding to the surface of the silver particles. The aggregation state of the particles is held constant by the polymer core, this nucleus also contains many dye labels, yielding a very high Raman scattering intensity for each bead. To functionalise these beads for use in biological assays an outer polymer shell can be added, which allows the attachment of oligonucleotide probes. Oligonucleotide modified beads can then be used for detection of specific oligonucleotide targets. The specificity of SERRS will allow for the detection of multiple targets within a single assay.

Metal Nanoparticles↗

PubMed2004

Controlling the substrate selectivity of deacetoxycephalosporin/deacetylcephalosporin C synthase.

Deacetoxycephalosporin/deacetylcephalosporin C synthase (DAOC/DACS) is an iron(II) and 2-oxoglutarate-dependent oxygenase involved in the biosynthesis of cephalosporin C in Cephalosporium acremonium. It catalyzes two oxidative reactions, oxidative ring-expansion of penicillin N to deacetoxycephalosporin C, and hydroxylation of the latter to give deacetylcephalosporin C. The enzyme is closely related to deacetoxycephalosporin C synthase (DAOCS) and DACS from Streptomyces clavuligerus, which selectively catalyze ring-expansion or hydroxylation reactions, respectively. In this study, structural models based on DAOCS coupled with site-directed mutagenesis were used to identify residues within DAOC/DACS that are responsible for controlling substrate and reaction selectivity. The M306I mutation abolished hydroxylation of deacetylcephalosporin C, whereas the W82A mutant reduced ring-expansion of penicillin G (an "unnatural" substrate). Truncation of the C terminus of DAOC/DACS to residue 310 (Delta310 mutant) enhanced ring-expansion of penicillin G by approximately 2-fold. A double mutant, Delta310/M306I, selectively catalyzed the ring-expansion reaction and had similar kinetic parameters to the wild-type DAOC/DACS. The Delta310/N305L/M306I triple mutant selectively catalyzed ring-expansion of penicillin G and had improved kinetic parameters (K(m) = 2.00 +/- 0.47 compared with 6.02 +/- 0.97 mm for the wild-type enzyme). This work demonstrates that a single amino acid residue side chain within the DAOC/DACS active site can control whether the enzyme catalyzes ring-expansion, hydroxylation, or both reactions. The catalytic efficiency of mutant enzymes can be improved by combining active site mutations with other modifications including C-terminal truncation and modification of Asn-305.

Acremonium↗

PubMed2002

The role of arginine residues in substrate binding and catalysis by deacetoxycephalosporin C synthase.

Deacetoxycephalosporin C synthase (DAOCS) catalyses the oxidative ring expansion of penicillin N, the committed step in the biosynthesis of cephamycin C by Streptomyces clavuligerus. Site-directed mutagenesis was used to investigate the seven Arg residues for activity (74, 75, 160, 162, 266, 306 and 307), selected on the basis of the DAOCS crystal structure. Greater than 95% of activity was lost upon mutation of Arg-160 and Arg266 to glutamine or other residues. These results are consistent with the proposed roles for these residues in binding the carboxylate linked to the nucleus of penicillin N (Arg160 and Arg162) and the carboxylate of the alpha-aminoadipoyl side-chain (Arg266). The results for mutation of Arg74 and Arg75 indicate that these residues play a less important role in catalysis/binding. Together with previous work, the mutation results for Arg306 and Arg307 indicate that modification of the C-terminus may be profitable with respect to altering the penicillin side-chain selectivity of DAOCS.

Amino Acid Substitution↗