The gene encoding the <I>E. coli</I> flavodoxin NADP<SUP>+</SUP> oxidoreductase (FLDR) has been overexpressed in <I>E. coli</I> and purified to homogeneity. The molecular mass of FLDR apoprotein was determined as 27648 Da. The midpoint reduction potentials of the oxidised/semiquinone and semiquinone/hydroquinone couples of FLDR (-308mV and -268mV, respectively) were measured using redox potentiometry. FLDR was fully characterised kinetically both by steady state and pre-steady state techniques. Arginines (R144, R174 and R184) in the proposed NADPH binding site of <I>E. coli</I> flavodoxin NADP<SUP>+ </SUP>oxidoreductase (FLDR) were replaced by alanines and the mutant enzymes fully characterised and studied by pre-steady-state and the steady-state kinetics. From our studies R174 and R184 appear to interact with the adenosine ribose 2' phosphate, while R144 is more likely to stabilise NADPH binding by interaction with the nicotinamide ribose 5' phosphate. R174A and R184A are more efficient enzymes than wild-type or R144A with NADPH as substrate, consistent with the proposed phosphate-binding roles for these residues. Arginine residues R237 and R238 in the proposed binding site for FLDR redox partner flavodoxin, have been mutated to alanine. These mutant enzymes have been characterised by pre-steady-state and steady-state kinetics, UV-Vis spectrophotometry, CD and florescence. These mutants are less efficient electron transfer proteins. In a separate project it was attempted to identify genes associated with the antibiotic biosynthetic pathway of aristeromycin from <I>Streptomyces citricolor</I>. An aristeromycin-induced protein was isolated from <I>S. citricolor</I> purified to homogeneity and an N-terminal sequence was determined. From this an oligonucleotide was designed and used to probe <I>S. citricolor</I> chromosomal DNA. A 1000bp fragment of DNA was isolated and sequenced, and the presence of part of an ORF identified.
Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:653741 |
Date | January 1999 |
Creators | Leadbeater, Claire |
Publisher | University of Edinburgh |
Source Sets | Ethos UK |
Detected Language | English |
Type | Electronic Thesis or Dissertation |
Source | http://hdl.handle.net/1842/15194 |
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