Aerobic nitroreduction by flavoproteins: enzyme structure, mechanisms and role in cancer chemotherapy.

NQO1 (DT-diaphorase) and its truncated isoenzyme, the metalloenzyme NQO2, can reduce quinone substrates by two-electron transfer. While NQO1 is a known detoxification enzyme, the function of NQO2 is less well understood. Both rat NQO1 and human NQO2 reductively bioactivate the dinitroarene CB 1954 to a cytotoxic product that behaves as a difunctional DNA-crosslinking species with potent anti-tumour activity, although human NQO1 is much less effective. A FMN-dependent nitroreductase from E. coli B also reduces quinones and reductively bioactivates CB 1954. However, this enzyme reduces CB 1954 to the 2- and 4-hydroxylamines in equivalent yield, whereas NQO1 and NQO2 generate only the 4-isomer. The reduction profile is a key factor in the development of anti-tumour prodrugs, where distinct delivery strategies are being evaluated: prodrug therapy, antibody-, macromolecule and gene-directed enzyme prodrug therapy (ADEPT, MDEPT or GDEPT). The flavoprotein enzymes are explored in terms of structure and bioreduction mechanism, particularly for use in the design of novel prodrugs with potential application as chemotherapeutic agents.

Crystal structure of FMN-dependent nitroreductase from Escherichia coli B: a prodrug-activating enzyme.

The FMN-dependent flavoprotein nitroreductase from Escherichia coli B (NTR) is used in cancer chemotherapy to activate a range of prodrugs. The crystal structure of this enzyme has been determined, using molecular replacement methods and refined at 2.06 A resolution. The recombinant 24-kDa enzyme was crystallized in the tetragonal space group P4(1)2(1)2, with unit cell dimensions of a = b = 57.74 A and c = 275.51 A and two molecules in the asymmetric unit. The structure has a final R factor of 20.3% (R(free) = 26.7%), for all data between the resolution ranges of 10-2.06 A, and includes 4453 protein atoms, 230 water molecules, and 2 flavin mononucleotide (FMN) molecules. The functional unit is a homodimer, which forms the asymmetric unit in the crystal structure. The tertiary structures of these two monomers and their subunit interactions are nearly identical. The molecular replacement search model, the crystal structure of the major NAD(P)H:FMN oxidoreductase of Vibrio fisheri (FRase 1), was selected on the basis of its high sequence identity to that of NTR. The final superposition of these two enzymes revealed a very similar overall fold, with variation in the structures focused around surface loops and helices near the FMN cofactor. Helix G is implicated in substrate specificity and is better resolved in the present NTR structure than in the previously reported FRase 1 structure. The FMN binding pocket is also well-resolved, showing the presence of two channels leading into the active site. The amino acid side chains and main chain atoms interacting with the FMN are well-ordered. The structure of the substrate binding pocket has been used to examine substrate specificity and enzyme kinetics for prodrugs used in antibody-directed enzyme prodrug therapy (ADEPT) and gene-directed enzyme prodrug therapy (GDEPT).

Crystal structure of human DT-diaphorase: a model for interaction with the cytotoxic prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954).

The crystal structure of human DT-diaphorase (NAD(P)H oxidoreductase (quinone); EC 1.6.99.2) has been determined to 2.3 A resolution. There are only minor differences in shape and volume between the active sites of the rat and human enzymes and in the hydrophobic environment in the vicinity of the substrate. The isoalloxazine ring of the bound FAD is more buried in the human structure. Molecular modeling was used to examine optimal positions for the antitumor prodrug CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) in both the human and rat enzyme active sites. This suggests that the position of CB1954 in the active site of the human enzyme is very similar to that in the rat, although there are detailed differences in the predicted patterns of hydrogen bonding between side chains and the drug. Some of the differences are a consequence of the shift in position for the FAD molecule and may contribute to the observed differences in rate of the two-electron reduction of CB1954.

Purification, crystallization and preliminary X-ray analysis of human recombinant cytosolic serine hydroxymethyltransferase.

As an enzyme of the thymidylate synthase cycle, serine hydroxymethyltransferase (SHMT) has a key role in nucleotide biosynthesis. Elevated activities of SHMT have been correlated with the increased demand for nucleotide biosynthesis in tumors of human and rodent origin, making this enzyme a novel target for cancer chemotherapy. Here the purification and crystallization of recombinant human cytosolic SHMT are reported. Crystals belong to space group P6222 or P6422 with cell parameters a = b = 155.0, c = 235.5 A and diffract to at least 3.0 A resolution.

Equilibrium and kinetic measurements reveal rapidly reversible binding of Ras to Raf.

Raf is a serine/threonine kinase that binds through its amino-terminal regulatory domain to the GTP form of Ras and thereby activates the mitogen-activated protein kinase pathway. In this study, we have characterized the interaction of the Ras-binding domain of Raf with Ras using equilibrium binding methods (scintillation proximity assay and fluorescence anisotropy), rather than with more widely used nonequilibrium procedures (such as enzyme-linked immunosorbent assay and affinity precipitation). Initial studies using glutathione S-transferase fusion proteins with either residues 1-257 or 1-190 of Raf showed that although it was possible to detect Ras binding using an enzyme-linked immunosorbent assay or affinity precipitation, it was substoichiometric; under equilibrium conditions with only a small excess of Raf almost no binding was detected. This difference was probably due to the presence of a high percentage of inactive Raf protein. Further studies used protein containing residues 51-131 of Raf, which expressed in Escherichia coli as a stable glutathione S-transferase fusion. With this protein, binding with Ras could readily be measured under equilibrium conditions. The catalytic domain of neurofibromin inhibited binding of Ras to Raf, and Raf inhibited the binding of Ras to neurofibromin showing that Raf and neurofibromin cannot be bound simultaneously to Ras. The affinities of interaction of neurofibromin and Raf with Harvey-RasLeu-61 were similar. The rate constant for dissociation of Raf from Ras was estimated to be >1 min-1, suggesting that Ras, Raf, and neurofibromin may be in rapid equilibrium in the cell. In contrast to previous reports, under equilibrium conditions there was no evidence for a difference in affinity between the minimal Ras binding domain of Raf (residues 51-131) and a region containing an additional 16 carboxyl-terminal amino acids, suggesting that residues 132-147 do not form a critical binding determinant.

Crystallization and preliminary crystallographic data for an FMN-dependent nitroreductase from Escherichia coli B.

An FMN-dependent nitroreductase enzyme isolated from Escherichia coli B has been crystallized in a form suitable for high-resolution structural analysis. The crystals belong to the tetragonal space group P4(1)2(1)2 or its enantiomorph P4(3)2(1)2 with cell parameters a = b = 57.74 A, c = 275.51 A and two molecules per asymmetric unit. Diffraction extends to beyond 1.9 A.

Sequence-dependent effects in drug-DNA interaction: the crystal structure of Hoechst 33258 bound to the d(CGCAAATTTGCG)2 duplex.

The bis-benzimidazole drug Hoechst 33258 has been co-crystallized with the dodecanucleotide sequence d(CGCAAATTTGCG)2. The structure has been solved by molecular replacement and refined to an R factor of 18.5% for 2125 reflections collected on a Xentronics area detector. The drug is bound in the minor groove, at the five base-pair site 5'-ATTTG and is in a unique orientation. This is displaced by one base pair in the 5' direction compared to previously-determined structures of this drug with the sequence d(CGCGAATTCGCG)2. Reasons for this difference in behaviour are discussed in terms of several sequence-dependent structural features of the DNA, with particular reference to differences in propeller twist and minor-groove width.

Crystal structure of an oligonucleotide duplex containing G.G base pairs: influence of mispairing on DNA backbone conformation.

The structure of the G.G mispaired dodecanucleotide d(CGCGAATTGGCG)2 has been solved by x-ray crystallography and refined to an R factor of 18.8% at 2.2 A resolution for 3513 reflections. The dodecamer crystallizes as a B-type DNA double helix. It contains two G(anti).G(syn) base pairs--i.e., G-4/G-16(anti).G-21/G-9(syn). The Hoogsteen base pairing involves atoms O-6 and N-7 of the guanine in the syn conformation with atoms N-1 and N-2 of the anti-paired purine. One G.G base pair has a bifurcated hydrogen bond between G-4(N-1)...G-21(N-7) and G-4(N-1)...G-21(O-6). There is little overall structural distortion of the double helix induced as a consequence of the mispairing. The helical width is significantly increased by comparison with the structure of the native duplex, and the minor groove width in the 5'-AATT region is decreased. The G.G base pairing induces high-BII phosphate conformations at residues G-9 and T-20 in addition to more normal BII conformations at G-10 and G-22. It is suggested that these backbone aberrations provide signals for the facile repairability of G.G mispairs in DNA.

Molecular structure of the B-DNA dodecamer d(CGCAAATTTGCG)2. An examination of propeller twist and minor-groove water structure at 2.2 A resolution.

The crystal structure of the dodecanucleotide duplex d(CGCAAATTTGCG)2 has been solved to 2.2 A resolution and refined to an R-factor of 18.1% with the inclusion of 71 water molecules. The structure shows propeller twists of up to -20 degrees for the A.T base-pairs, although there is probably only one (weak) three-centre hydrogen bond in the six base-pair AT narrow minor-groove region. An extensive ribbon of hydration has been located in this groove that has features distinctive from the classic "spine of hydration". Solvation around phosphate groups is described, with several instances of water molecules bridging between phosphates.

Conformational properties of the G.G mismatch in d(CGCGAATTGGCG)2 determined by NMR.

The conformational properties of the DNA duplex d(CGCGAATTGGCG)2, which contains two noncomplementary G.G base pairs, have been examined in aqueous solution by 1H and 31P NMR as a function of temperature. The G.G mismatch is highly destabilizing, with a Tm value 35 K below that observed for the native EcoRI dodecamer. The dodecamer appears symmetric in the NMR spectra and exists largely as an average B-type DNA conformation. However, the 1H and 31P NMR spectra give evidence of considerable conformational heterogeneity at the mismatched nucleotides and their nearest neighbors, which increases with increasing temperature. There is no evidence for a significant population of the syn purine conformation. The imino protons of the mispaired bases G4 and G9 are degenerate, resonate at high field, and exchange readily with solvent. These results indicate that the mispaired bases are only weakly hydrogen-bonded and are only partially stacked into the helix. On raising the temperature, the duplex shows increasing exchange between two or more conformations originating from the mismatch sites. However, these additional conformations maintain their Watson-Crick hydrogen bonding. The increase in chemical exchange is consistent with a quasimelting process for which the G.G sites provide local nuclei. Extensive modeling studies by dynamic annealing have confirmed that the G(anti).G(anti) conformation is favored and that the mispairs are poorly stacked within the helix. The results explain both the poor thermal stability and low hypochromicity of this duplex.

Crystal structure and sequence-dependent conformation of the A.G mispaired oligonucleotide d(CGCAAGCTGGCG).

The crystal structure of the dodecanucleotide d(CGCAAGCTGGCG) has been determined to a resolution of 2.5 A and refined to an R factor of 19.3% for 1710 reflections. The sequence crystallizes as a B-type double helix, with two G(anti).A(syn) base pairs. These are stabilized by three-center hydrogen bonds to pyrimidines that induce perturbations in base-pair geometry. The central AGCT region of the helix has a wide (greater than 6 A) minor groove.

Crystal structure of a berenil-dodecanucleotide complex: the role of water in sequence-specific ligand binding.

The three-dimensional structure of a complex between the dodecanucleotide d(CGCGAATTCGCG) and the anti-trypanocidal drug berenil, has been determined to a resolution of 2.5 A. The structure has been solved by molecular replacement and refined to an R factor of 0.177. A total of 49 water molecules have been located. The drug is bound at the 5'-AAT-3' region of the oligonucleotide. At one end of the drug the amidinium group is in hydrogen-bonded contact with N3 of the adenine base complementary to the thymine of the AAT. The other amidinium group does not make direct interactions with the DNA. Instead, a water molecule mediates between them. This is in hydrogen-bonded contact with an amidinium nitrogen atom, N3 of the 5' end adenine base and the ring oxygen atom of an adjacent deoxyribose. Molecular mechanics calculations have been performed on this complex, with the drug at various positions along the sequence. These show that the observed position is only 0.8 kcal/mol higher in energy than the best position. It is suggested that there is a broad energy well in the AATT region for this drug, and that water molecules as well as the neighbouring sequence, will determine precise positioning. More general aspects of minor groove binding are discussed.

Conformational effects of nucleotide exchange in ras p21 proteins as studied by fluorescence spectroscopy.

The intrinsic fluorescence properties of the oncogene protein p21N-ras,p21H-ras and one of its transforming mutants, p21N-ras (Val12), have been investigated. A mutant containing a single tryptophan at position 28 in p21H-ras (Trp28) has been specifically engineered to provide a probe of protein conformation on nucleotide binding. The proteins produced essentially similar circular dichroism spectra typical of alpha/beta proteins. A decrease in the intensity of the fluorescence emission spectrum due to tyrosine occurred on GDP/GTP nucleotide exchange in the native and mutant proteins. Selective excitation of the single tryptophan in p21 produced a decrease in fluorescence intensity which was accompanied by a blue shift in the wavelength of maximum emission on nucleotide exchange. A reduction in the residual Mg2+ ion concentration enhanced this effect.

Preliminary crystallographic data for NAD(P)H quinone reductase isolated from the Walker 256 rat carcinoma cell line.

An NAD(P)H quinone reductase isolated from Walker rat 256 carcinoma cells has been crystallized in a form suitable for high-resolution structural analysis. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with cell parameters a = 168.15 A, b = 105.09 A and c = 67.38 A and contain four monomeric or two dimeric enzyme molecules per asymmetric unit. Diffraction extends beyond 2.3 A resolution.

The crystal structure of the DNA-binding drug berenil: molecular modelling studies of berenil-DNA complexes.

The crystal structure of the DNA minor-groove DNA-binding drug berenil has been determined. Molecular-modelling techniques have been used to establish plausible binding modes of the structure to A-T sequences. These have shown that specific hydrogen bonds are possible between the amidine groups of the drug molecule and 02 atoms of thymine, although global energy minimisations tended to emphasise electrostatic interactions with phosphate groups rather than these hydrogen bonds with bases.

Automated platelet counting--a re-evaluation of the sedimentation method.

Platelet counting by means of the Coulter Thrombocounter, using the Thrombofuge method for specimen preparation has been evaluated in order to determine the cause of discrepancies in results obtained in interlaboratory quality assessment surveys. The method was assessed for accuracy, precision, drift and carry-over. When the method was used, as in routine practice, some discrepancies occurred; an important source of error was identified as being due to the level of sedimented plasma layer from which samples are removed. When the method was performed strictly in accordance with the manufacturer's instructions results were generally satisfactory and comparable to those obtained with the whole blood reference method, although, even with this standardized technique, the Thrombo-fuge tended to give higher platelet counts than the reference method with counts above 200 X 10(9)/l. This discrepancy to be independent of PCV, MCV or other identifiable factors.

Platelet counting-development of a reference method and a reference preparation.

A reference method has been defined for platelet counting by counting chamber haemocytometry, and a procedure has been developed, using platelet rich plasma (PRP) and a Coulter ZBI counter which provides a reliable, rapid and relatively simple method for platelet counting which is closely comparable to the reference method and can thus be used both as a secondary reference method and a routine method. The optimal method for obtaining PRP from EDTA blood was by augmented sedimentation with Boyum's methyl cellulose-metrizoate mixture at sp. gr. 1.08. This was shown to yield a platelet suspension which reflects closely the platelet content of the original blood. Calibration of electronic counters for platelet counting requires material which is stable and which parallels natural human platelets in size, distribution and other physical characteristics. A suspension of glutaraldehyde-fixed human platelets in glycerol appears to be suitable as a reference preparation. Its method of production is described.