The three genes believed to encode the redox system for cytochrome P450 BioI have been cloned (<i>bioI</i>, <i>orf2 </i>and <i>fer</i>). Protein overexpression of both BioI and the ferredoxin has been achieved to high levels and purification protocols determined for both. Characterisation of BioI using a variety of spectroscopic methods has proved that the enzyme is indeed a P450, showing typical spectral shifts on binding of exogenous ligands such as CO and NO. Ligand binding studies have indicated a high affinity of the protein for long-chain saturated fatty acids, with the optimal ligand being myristate, C14 Crystals of BioI have also been obtained, but solution of the structure has so far proved elusive. The <i>Bacillus subtilis</i> ferredoxin appeared to be the only candidate electron donor to BioI and sequence similarities suggested it contained a 4Fe-4S cluster. Characterisation of the ferredoxin by UV-visible spectroscopy, CD and EPR (in the reduced form) confirmed this. ICP-AES and the oxidised EPR data seemed to suggest the presence of at least some 3De-4S cluster, which appears to suggest oxidative degradation of the cluster. Using <i>E. coli</i> flavodoxin reductase (FLDR) as a redox partner alongside the ferredoxin, electron transfer to BioI has been demonstrated, albeit very slowly. Electron transfer from the FLDR to the ferredoxin was shown to be very rapid, indicating the slow step to be electron transfer from ferredoxin to BioI or steps after this. The ability of this system to hydroxylate fatty acids has also been demonstrated using the fatty acid chromophore, <i>p</i>-nitrophenoxydodecanoic acid (Schwaneberg <i>et al</i>., 1999).

The carbocyclic nucleosides aristeromycin I and neplanocin A II are produced from D-glucose by <i>Streptomyces citricolor</i>. (Fig. 8570A) To investigate the mechanism of the biosynthesis, an effective route to a putative intermediate III has been developed. By modifications to a known procedure, a single low yielding and unreliable reaction has been improved considerably. (Fig. 8570B) By using a <i>tert-</i>butyl-diphenyl silyl group in place of the acetyl group present in the original procedure, a more stable compound was obtained, which was tolerant to the insertion of a protected hydroxy methyl moiety. Yields for this reaction were increased from £ 20% to 76%. An efficient route to enantiomerically pure 3-benzyloxymethylcyclopentene VI has been demonstrated. Using <i>Rhizopus arrhizus</i> ATCC 11145, ethylcyclopentanone-2-carboxylate was reduced to give a single enantiomer of 2-hydroxy-cyclopentanecarboxylic acid ethyl ester VII which was converted in 5 steps to the desired substrate. (Fig. 8570C) It was observed that VI could be hydroxylated at C-5 by incubation with <i>Rhodococcus rhodochrous </i>NCIMB 9703, to give the corresponding alcohol VIII suitable for further elaboration to carbocyclic nucleosides.

The two distinct domains of flavocytochrome <I>b<SUB>2</SUB> </I>(L-lactate : cytochrome <I>c</I> oxidoreductase) are connected by a typical hinge peptide. Kinetic experiments have illustrated the importance of maintaining the structural integrity of the hinge for efficient interdomain electron transfer. To probe the role of the hinge in a more subtle manner, three mutant enzymes have been constructed; HΔ3, HΔ6 and HΔ9 which have three, six and nine amino acids deleted from the hinge region, respectively. Intra- (interdomain) and inter-protein (between flavocytochrome <I>b<SUB>2</SUB> </I>and cytochrome <I>c</I>) electron-transfer was investigated by steady-state and stopped-flow kinetic analysis. All three hinge-deleted enzymes remained good L-lactate dehydrogenases as was evident from steady-state experiments with ferricyanide as electron acceptor (k<SUB>cat</SUB> = 256 s<SUP>-1</SUP>, 276 s<SUP>-1</SUP> and 400 s<SUP>-1</SUP> for HΔ3, HΔ6, and HΔ9, respectively, compared to 400s<SUP>-1</SUP> for the wild-type enzyme) and from stopped-flow experiments monitoring flavin reduction (k<SUB>cat</SUB> = 516 s<SUP>-1</SUP>, 520 s<SUP>-1</SUP> and 715 s<SUP>-1</SUP> for HΔ3, HΔ6 and HΔ9, respectively, compared to 600 s<SUP>-1</SUP> for the wild-type enzyme). The global effect of these deletions is to lower the enzymes' effectiveness as cytochrome <I>c</I> reductases. This property of HΔ6 and HΔ9 flavocytochromes <I>b<SUB>2</SUB><SUP> </SUP> </I>is manifested at the interdomain electron-transfer step, where the rate of haem reduction is the same within experimental error as the steady-state rate of cytochrome <I>c</I> reduction: interdomain electron-transfer has become rate-limiting in the case of these two hinge-deleted enzymes, compared to the wild-type enzyme, where H-abstraction from C2 of L-lactate is rate-limiting. The situation for HΔ3 is more complicated; the rate of haem reduction has fallen 35-fold compared with the wild-type enzyme (from 1500 s<SUP>-1</SUP> to 91 s<SUP>-1</SUP>) and, secondly, the steady-state rate of cytochrome <I>c</I> reduction has fallen 5-fold (from 207 s<SUP>-1</SUP> to 39 s<SUP>-1</SUP>). This implies that, for HΔ3, interdomain electron-transfer from fully reduced flavin to haem cannot be rate-limiting, as is the case for HΔ6 and HΔ9, but some other step, such as flavin semiquinone to haem electron-transfer must be involved. These data, along with the measured kinetic isotope effects imply that complete structural integrity within the hinge region is essential for efficient interdomain communication.

The S polypeptide of the <I>Eco</I>KI Type I methyltransferase (mtase) has the function of recognising DNA. Specific contacts between <I>Eco</I>Ki (mtase) and the DNA containing its recognition sequence (-AAC-(N)<SUB>6</SUB>-GTGC-) have been investigated using various substituted oligonucleotides, namely 4-thiothymidine, 5-bromodeoxyuridine, 5-iododeoxyuridine and deoxyuridine. After ultraviolet irradiation, the DNA-S polypeptide crosslinked bands were observed on the specific target recognition DNA, but not on the non-specific DNA by SDS-PAGE gel electrophoresis. The crosslinking of DNA and mtase occurs only between the bottom-strand of the DNA and the S polypeptide as illustrated by labelling the different strands of DNA with γ<SUP>32</SUP>P. The crosslinking efficiency between DNA-mtase increased only in one of the nine single sites of BrdU substitution in which the BrdU was substituted at the residue complementary to the first adenine in the -AAC- sequence. This demonstrates a close contact between this sequence and the S subunit at the major groove. Peptide sequencing of various trypsin digested samples of the crosslinked protein-DNA complex further illustrates that the Tyr-27 of the NH<SUB>2</SUB>-terminal domain of the S polypeptide is crosslinked with DNA. This is consistent with the role of the N terminal domain of the S subunit in recognizing the -AAC- sequence. By amino acid sequence comparison, Tyr-27 and other small sequence segments can be found at corresponding positions in other two type I enzymes, which have a similar trinucleotide recognition site. In addition the comparison reveals that motif LP-GWEW is partly retained not only in the amino recognition domain but also in the carboxyl recognition domain of the S subunit of type I enzymes.

Hypoxanthine phosphoribosyltransferase (EC 2.7.2.8) of P.falciparum has been studied for its biological properties and cellular location. The enzyme plays an important role in the parasite's life, and therefore is a putative target for chemotherapy against malaria. Due to the difficulty in obtaining large amounts of the enzyme from the parasite, it was over-expressed in E.coli, first as a fusion protein with E.co/i-j8-galactosidase. This facilitated the one step purification of the protein, using /3-galactosidase substrate affinity chromatography, for making antibodies against the enzyme. The antibodies thus made were used to investigate the cellular location, a prerequisite for a successful drug design against the parasite enzyme. Im-munofluorescent microscopy (IFA) and immunogold electron microscopy revealed that; 1. the enzyme is expressed at all the stages of the parasite's life, 2. the enzyme is concentrated in some vesicular bodies of unknown origin, 3. in the sporozoite, it may be released into the intrapellicular spaces. Subsequently, the enzyme was over-expressed directly in E.coli, as a non-fusion protein and retained its enzymatic activity. This opened the way to study the enzyme for its biochemical properties and structure-activity relationship. The active E.co/i-expressed P.falciparum HPRT compensated a S.typhimurium hpt mutation. The strain, named S.lyphimurium SH4 can now be used to screen large numbers of putative antimalarial drugs which might act against the parasite HPRT. This will be both simple and inexpensive. The cell free extract of induced SH4 was used to study some biochemical properties of the enzyme. Such a study confirmed the finding that unlike any other Plasmodia studied so far, P.falciparum HPRT can use xanthine as its substrate in addition to hypoxanlhine and guanine. However, competitive inhibition studies revealed that hypoxanthine is the most favourable substrate. The possible biological significance of such properties is discussed.

No description available.

The gene encoding the <i>E. coli</i> flavodoxin NADP⁺ oxidoreductase (FLDR) and flavodoxin (FLD) have been overexpressed in <i>E. coli</i> and the gene products purified to homogeneity. Physical characterisation of both proteins was carried out using steady-step and stopped flow kinetics, circular dichroism and fluorimetric studies. The molecular masses of the apoproteins were determined as 27648Da and 19606Da and the isoelectric points as 4.8 and 3.5, respectively. The midpoint reduction potentials of the oxidised/semiquinone and semiquinone/hydroquinone couples of both FLDR (-308mV and -268mV) and FLD (-254mV and -433mV) were measured using redox potentiometry. This confirms the electron-transfer route as NADPH (r) FLDR (r) FLD. Binding of 2' adenosine monophosphate increases the midpoint reduction potentials for both FLDR couples. These data highlight the strong stabilisation of the flavodoxin semiquinone with respect to the hydroquinone state and indicate that FLD must act as a single electron shuttle from the semiquinone form in its support of cellular functions. FLDR and FLD were covalently crosslinked using 1-ethyl-3(dimethylamino-propyl) carodiimide. A single species was formed with an apparent molecular weight of 47kD. This complex was catalytically active in that it could reduce cytochrome <i>c</i> and potassium ferricyanide almost as efficiently as the individual proteins. The gene encoding <i>E. coli</i> biotin synthase (<i>bio</i>B) has been expressed as a histidine-fusion protein and the protein purified in a single step using IMAC. The His₆-tagged protein was fully functional in <i>in vitro</i> and <i>in vivo</i> biotin production assays. Analysis of all the published <i>bio</i>B sequences identified a number of conserved residues. Single point mutations, to either serine or threonine, were carried out on the four conserved (Cys-53, Cys-57), Cys-60 and Cys-188) and on non-conserved (Cys-288) cysteine residues and the purified mutant proteins tested both for ability to reconstitute the [2Fe-2S] clusters of the native (oxidised) dimer and enzymatic activity.

No description available.

A purification procedure has been developed for a novel extradiol dioxygenase, designated as 3-methylcatechol 2,3-dioxygenase. The enzyme which is expressed in <i>Escherichia coli</i>, was originally derived from a Pseudomonas putida strain able to grow on toluidine. 3-Methylcatechol 2,3-dioxygenase was purified to homogeneity as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Physical and kinetic properties of the purified enzyme were investigated. The enzyme consists of a single subunit type of M<SUB>r =</SUB> 33,500 ± 2,000 by SDS-PAGE. Gel filtration indicated a molecular weight, under non-denaturing conditions, of 120,000 ± 20,000 consistent with the native enzyme existing as a tetramer of identical subunits. The NH<SUB>2</SUB>-terminal sequence (35 residues) has been determined and shows 50% identity with other extradiol dioxygenases. The structural characterisation of 3-methylcatechol 2,3-dioxygenase at the primary, secondary and quaternary levels indicates that the enzyme is typical of the extradiol dioxygenases. The kinetics of 3-methylcatechol 2,3-dioxygenase were investigated using UV/visible spectrophotometry and oxygen electrode polarography. Measurements were made under both standard and modified conditions. Typical saturation kinetics were observed for catechol, 3-methylcatechol and 4-methylcatechol as substrates. Data were analysed to give values of V<SUB>max</SUB> and K<SUB>m</SUB>. The substrate specificity for this enzyme was somewhat different from that seen for other catechol 2,3-dioxygenases, with 3-methylcatechol being cleaved at the highest rate. The K<SUB>m</SUB> values for the organic substrates were all around 0.3 μM, the lowest found for any dioxygenase to date. The K_m for dioxygen was determined to be ≤ 10<SUP>-6</SUP>M. The enzyme consumed one mole of oxygen per mole of substrate in all three cases.

Flavocytochrome <i>b</i><SUB>2</SUB> is a respiratory enzyme found in the intermembrane space of yeast mitochondria, where it catalyses the oxidation of L-lactate to pyruvate, transferring electrons to cytochrome <i>c</i>. The enzyme is a tetramer. Each subunit comprises two functionally-distinct domains, one containing flavin mononucleotide and the other containing protohaem IX. The lactate-binding site of the enzyme is located within the flavin-containing domain. This domain is essential for L-lactate dehydrogenase activity. The haem domain is essential for electron transfer to cytochrome <i>c</i>. The haem- and FMN-containing domains of flavocytochrome <i>b</i><SUB>2</SUB> have now been expressed independently in <i>E.coli</i>. This thesis describes the development of effective purification procedures for the two independently-expressed domains and their characterization by biophysical and biochemical techniques. The characterization of a point mutant of the isolated <i>b</i><SUB>2</SUB>-flavin domain is also discussed. The independently-expressed haem domain was purified to a high level by column chromatography. The isolated protein was found to be monomeric with a molecular weight of 10,500. It had no detectable enzyme activity and failed to accept electrons from either the isolated <i>b</i><SUB>2</SUB>-flavin domain or the holoenzyme. Isolated <i>b</i><SUB>2</SUB>-haem domain was found to be stable over a wide temperature and pH range. Its redox potential was found to be -31+ /-2mV, in agreement with a previously-determined value for a similar fragment isolated from the holoenzyme by proteolytic methods. High-field <SUP>1</SUP>H-NMR studies have been carried out on the isolated <i>b</i><SUB>2</SUB>-haem domain. The pK<SUB>A</SUB> values of the haem propionates were found by NMR to be 4.8 and 4.6, consistent with these groups being exposed to solvent. NMR studies were also used to determine an electron self-exchange rate constant of 2.3x10<SUP>6</SUP>M<SUP>-1</SUP>s<SUP>-1</SUP> for the <i>b</i><SUB>2</SUB>-haem domain. The interactions of cytochrome <i>c</i> with both the holoenzyme and the isolated haem domain were studied by NMR. It was found that, whereas cytochrome <i>c</i> bound to the holoenzyme, it failed to bind to the isolated haem domain.