pH-dependent spectroscopic changes associated with the hydroquinone of FMN in flavodoxins.

Photoreduction with a 5-deazaflavin as the catalyst was used to convert flavodoxins from Desulfovibrio vulgaris, Megasphaera elsdenii, Anabaena PCC 7119, and Azotobacter vinelandii to their hydroquinone forms. The optical spectra of the fully reduced flavodoxins were found to vary with pH in the pH range of 5.0-8.5. The changes correspond to apparent pKa values of 6.5 and 5.8 for flavodoxins from D. vulgaris and M. elsdenii, respectively, values that are similar to the apparent pKa values reported earlier from the effects of pH on the redox potential for the semiquinone-hydroquinone couples of these two proteins (7 and 5.8, respectively). The changes in the spectra resemble those occurring with the free two-electron-reduced flavin for which the pKa is 6.7, but they are red-shifted compared with those of the free flavin. The optical changes occurring with flavodoxins from D. vulgaris and A. vinelandii flavodoxins are larger than those of free reduced FMN. The absorbance of the free and bound flavin increases in the region of 370-390 nm (Delta epsilon = 1-1.8 mM-1 cm-1) with increases of pH. Qualitatively similar pH-dependent changes occur when FMN in D. vulgaris flavodoxin is replaced by iso-FMN, and in the following mutants of D. vulgaris flavodoxin in which the residues mutated are close to the isoalloxazine of the bound flavin: D95A, D95E, D95A/D127A, W60A, Y98S, W60M/Y98W, S96R, and G61A. The 13C NMR spectrum of reduced D. vulgaris [2,4a-13C2]FMN flavodoxin shows two peaks. The peak due to C(4a) is unaffected by pH, but the peak due to C(2) broadens with decreasing pH; the apparent pKa for the change is 6.2. It is concluded that a decrease in pH induces a change in the electronic structure of the reduced flavin due to a change in the ionization state of the flavin, a change in the polarization of the flavin environment, a change in the hydrogen-bonding network around the flavin, and/or possibly a change in the bend along the N(5)-N(10) axis of the flavin. A change in the ionization state of the flavin is the simplest explanation, with the site of protonation differing from that of free FMNH-. The pH effect is unlikely to result from protonation of D95 or D127, the negatively charged amino acids closest to the flavin of D. vulgaris flavodoxin, because the optical changes observed with alanine mutants at these positions are similar to those occurring with the wild-type protein.

Integrin antagonists.

Integrins are a family of cell surface glycoproteins that mediate numerous cell-cell and cell-matrix interactions and are involved in biological processes such as tissue morphogenesis, leukocyte recirculation and migration, wound healing, blood clotting and immune response. Aberrant cell adhesion has been implicated in the pathogenesis of several diseases, including a number of inflammatory disorders such as rheumatoid arthritis, inflammatory bowel disease and asthma, as well as cancer and coronary heart disease. As such integrins are seen as excellent targets for the development of therapeutic agents. This report begins with an examination of the structure of integrin molecules and their ligands and then goes on to review the current state of development of antiintegrin antagonists.

Plasmodium chabaudi chabaudi and P. falciparum: inhibition of aminopeptidase and parasite growth by bestatin and nitrobestatin.

The major leucine aminopeptidase of the rodent malarial parasite Plasmodium chabaudi chabaudi was partially purified using a combination of high-pressure liquid chromatography on a size-exclusion column and affinity chromatography using the aminopeptidase-specific inhibitor bestatin as the ligand. The purified enzyme showed simple Michaelis-Menten kinetics when the fluorogenic peptide analogue leucyl-7-amino-4-methyl-courmarin served as the substrate, and it was strongly inhibited by both bestatin (Ki = 50.7 +/- 21.0 nM) and nitrobestatin (Ki = 2.51 +/- 0.2 nM) in a competitive manner. These inhibitors were also potent blockers of the growth of P. c. chabaudi and the human parasite P. falciparum in culture, and nitrobestatin was again the more potent. Therefore, the leucine aminopeptidase represents an important target to which novel anti-malarial agents could be directed.

Erythrocyte binding protein homologues of rodent malaria parasites.

Erythrocyte invasion by malaria parasites requires specific molecular interactions between the merozoite and erythrocyte surface receptors. A well-conserved, functionally important family of erythrocyte binding proteins is the EBP family. The EBP family includes the Plasmodium vivax, P. knowlesi Duffy binding protein (DBP) family and the P. falciparum erythrocyte binding antigen-175 (EBA-175). The EBP are transmembrane proteins, characterized by two conserved cysteine-rich domains, expressed in the micronemes of invasive merozoites. Oligonucleotide primers matching the region encoding the carboxyl cysteine-rich domain of the EBA-175 were used in a polymerase chain reaction to identify homologous genes in P. berghei and P. yoelii yoelii, leading to the isolation of a P. berghei partial genomic clone. This clone contained a 323 bp region that had high deduced amino acid sequence similarity to the amino acid sequences of the carboxyl cysteine-rich domains of the DBP family and EBA-175. The P. berghei carboxyl cysteine-rich domain was followed by a putative transmembrane domain and a cytoplasmic domain, demonstrating an exon-intron structure at the 3' end homologous to P. vivax dbp and P. falciparum eba-175. The carboxyl cysteine-rich domain is also highly conserved among P. berghei, P. y. yoelii, P. chabaudi and P. vinckei and is encoded by a single copy gene. Antisera prepared against the carboxyl cysteine-rich domain of the rodent malaria EBP homologues reacted with a 120 and 128 kDa protein doublet on Western blots of P. berghei parasite antigen and showed an apical localization pattern within merozoites by indirect immunofluorescence assays.

Cloning of peroxiredoxin, a novel antioxidant enzyme, from the helminth parasite Fasciola hepatica.

A cDNA was isolated from a cDNA expression library using serum prepared against a high molecular mass fraction of Fasciola hepatica excretory-secretory products. The full-length cDNA encodes a member of the recently described peroxiredoxin antioxidant family. Peroxiredoxin could be the major hydrogen peroxide removing antioxidant in F. hepatica since this parasite does not express a catalase and expresses little glutathione peroxidase activity. This novel antioxidant may be involved in functions such as protection against reactive oxygen species (ROS) generated by metabolic processes and/or protection of the parasite against ROS released by immune effector cells.

Functional expression of Fasciola hepatica cathepsin L1 in Saccharomyces cerevisiae.

A cDNA encoding the complete precursor of a Fasciola hepatica cathepsin L protease was isolated and sequenced. Functionally active enzyme was expressed and secreted by Saccharomyces cerevisiae transformed with a plasmid carrying the complete gene. Experiments with temperature-sensitive yeast mutants showed that the enzyme is trafficked through the yeast secretory pathway. Yeast transformed with a truncated gene, which lacked the pre-peptide-encoding and most of the pro-peptide-encoding sequences, did not express funtionally active enzyme. The yeast-expressed enzyme exhibited physicochemical properties in common with the native enzyme including, pH optimum for activity, stability at 37 degrees C and ability to cleave gelatin and immunoglobulin. Enzyme kinetic data showed that the native and yeast-expressed cathepsin L1 have similar specificities for substrates with hydrophobic residues in the P2 position. This is the first report of the functional expression of a cathepsin L proteinase in S. cerevisiae that did not require the use of yeast secretory signal sequences.

Kinetics and thermodynamics of the binding of riboflavin, riboflavin 5'-phosphate and riboflavin 3',5'-bisphosphate by apoflavodoxins.

The reactions of excess apoflavodoxin from Desulfovibrio vulgaris, Anabaena variabilis and Azotobacter vinelandii with riboflavin 5'-phosphate (FMN), riboflavin 3',5'-bisphosphate and riboflavin are pseudo-first-order. The rates increase with decreasing pH in the range pH 5-8, and, in general, they increase with increasing ionic strength to approach a maximum at an ionic strength greater than 0.4 M. The rate of FMN binding in phosphate at high pH increases to a maximum at an ionic strength of about 0.1 M, and then decreases as the phosphate concentration is increased further. The dissociation constants for the complexes with FMN and riboflavin decrease with an increase of ionic strength. Inorganic phosphate stabilizes the complex with riboflavin. The effects of phosphate on riboflavin binding suggest that phosphate interacts with the apoprotein at the site normally occupied by the phosphate of FMN. Redox potentials determined for the oxidized/semiquinone and semiquinone/hydroquinone couples of the riboflavin and FMN complexes were used with K delta values for the complexes with the oxidized flavins to calculate values for K delta for the semiquinone and hydroquinone complexes. The hydroquinone complexes are all less stable than the complexes with the two other redox forms of the flavin. Destabilization of the hydroquinone is less marked in the complexes with riboflavin, supporting a proposal that the terminal phosphate group of FMN plays a role in decreasing the redox potential of the semiquinone/hydroquinone couple.

Aminopeptidases from Plasmodium falciparum, Plasmodium chabaudi chabaudi and Plasmodium berghei.

Using fluorogenic substrates and polyacrylamide gels we detected in cell-free extracts of Plasmodium falciparum, Plasmodium chabaudi chabaudi and Plasmodium berghei only a single aminopeptidase. A comparative study of the aminopeptidase activity in each extract revealed that the enzymes have similar specificities and kinetics, a near-neutral pH optima of 7.2 and are moderately thermophilic. Each has an apparent molecular weight of 80,000 +/- 10,000, determined by high performance liquid chromatography on a calibrated SW500 column. Whilst the P. c. chabaudi and P. berghei activity co-migrate in native polyacrylamide gels, that of P. falciparum migrates more slowly. The three enzymes can be selectively inhibited by ortho-phenanthroline and are thus metallo-aminopeptidases; however, in contrast to other aminopeptidases the metal co-factor does not appear to be Zn2+.

Homonuclear and heteronuclear NMR studies of oxidized Desulfovibrio vulgaris flavodoxin. Sequential assignments and identification of secondary structure elements.

Recombinant Desulfovibrio vulgaris flavodoxin (molecular mass 16.3 kDa) was produced in Escherichia coli. The oxidized protein has been investigated with a combination of homonuclear and heteronuclear two-dimensional and heteronuclear three-dimensional NMR spectroscopy. Sequence-specific assignment of all backbone and most of the side chain 1H and 15N resonances has been obtained. The secondary structure has been inferred from the pattern of sequential, medium-, and long-range NOEs, together with information about slowly exchanging amide hydrogens and HN-H alpha spin-spin coupling constants. In solution, flavodoxin consists of a five-stranded parallel beta-sheet and four alpha-helices. Residues 3-9, 32-36, 52-58, 87-96, and 123-128 are involved in the beta-sheet whereas the a-helical regions comprise residues 13-28, 69-76, 104-114, and 134-148. Several proton resonances of the bound flavin mononucleotide cofactor have been assigned. NOE contacts between the prosthetic group and the apoprotein have been detected.

Redox and flavin-binding properties of recombinant flavodoxin from Desulfovibrio vulgaris (Hildenborough).

Flavodoxin from Desulfovibrio vulgaris (Hildenborough) has been expressed at a high level (3-4% soluble protein) in Escherichia coli by subcloning a minimal insert carrying the gene behind the tac promoter of plasmid pDK6. The recombinant protein was readily isolated and its properties were shown to be identical to those of the wild-type protein obtained directly from D. vulgaris, with the exception that the recombinant protein lacks the N-terminal methionine residue. Detailed measurements of the redox potentials of this flavodoxin are reported for the first time. The redox potential, E2, for the couple oxidized flavodoxin/flavodoxin semiquinone at pH 7.0 is -143 mV (25 degrees C), while the value for the flavodoxin semiquinone/flavodoxin hydroquinone couple (E1) at the same pH is -440 mV. The effects of pH on the observed potentials were examined; E2 varies linearly with pH (slope = -59 mV), while E1 is independent of pH at high pH values, but below pH 7.5 the potential becomes less negative with decreasing pH, indicating a redox-linked protonation of the flavodoxin hydroquinone. D. vulgaris apoflavodoxin binds FMN very tightly, with a value of 0.24 nM for the dissociation constant (Kd) at pH 7.0 and 25 degrees C, similar to that observed with other flavodoxins. In addition, the apoflavodoxin readily binds riboflavin (Kd = 0.72 microM; 50 mM sodium phosphate, pH 7.0, 5 mM EDTA at 25 degrees C) and the complex is spectroscopically very similar to that formed with FMN. The redox potentials for the riboflavin complex were determined at pH 6.5 (E1 = -262 mV, E2 = -193 mV; 25 degrees C) and are discussed in the light of earlier proposals that charge/charge interactions between different parts of the flavin hydroquinone play a crucial role in determining E1 in flavodoxin.

Effects of substituting asparagine for glycine-61 in flavodoxin from Desulfovibrio vulgaris (Hildenborough).

Gly-61 in flavodoxin from Desulfovibrio vulgaris (Hildenborough) has been changed to Asn by site-directed mutagenesis of the cloned gene. Values determined for the dissociation constant for the dissociation of the mutant protein into apoprotein and FMN, and for the redox potentials of the two 1-electron steps in the reduction of the bound flavin showed that FMN in all three redox states is bound more weakly in the mutant protein than in the wild-type flavodoxin. However, the greatest effect was on the semiquinone, for which Kd is 920 times larger in the mutant. The side-chain of Asn-61 in the mutant may hinder a redox-linked conformational change that occurs in this region of the protein, and which is thought to lead to formation of a hydrogen bond between N(5)H of FMNH and the backbone carbonyl group of amino acid-61.