Vitamin B-1 ( thiamine) is an essential cofactor for several key enzymes of carbohydrate metabolism. Mammals have to salvage this crucial nutrient from their diet to complement their deficiency of de novo synthesis. In contrast, bacteria, fungi, plants and, as reported here, Plasmodium falciparum, possess a vitamin B-1 biosynthesis pathway. The plasmodial pathway identified consists of the three vitamin B-1 biosynthetic enzymes 5-(2-hydroxy-ethyl)-4-methylthiazole (THZ) kinase (ThiM), 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP)/HMP-P kinase ( ThiD) and thiamine phosphate synthase (ThiE). Recombinant PfThiM and PfThiD proteins were biochemically characterised, revealing K-m(app) values of 68 mu M for THZ and 12 mu M for HMP. Furthermore, the ability of PfThiE for generating vitamin B1 was analysed by a complementation assay with thiE-negative E. coli mutants. All three enzymes are expressed throughout the developmental blood stages, as shown by Northern blotting, which indicates the presence of the vitamin B-1 biosynthesis enzymes. However, cultivation of the parasite in minimal medium showed a dependency on the provision of HMP or thiamine. These results demonstrate that the human malaria parasite P. falciparum possesses active vitamin B-1 biosynthesis, which depends on external provision of thiamine precursors.

Vitamin B6 is an essential cofactor for more than 100 enzymatic reactions. Mammalian cells are unable to synthesize vitamin B6 de novo, whereas bacteria, plants, fungi, and as shown here Plasmodium falciparum possess a functional vitamin B6 synthesis pathway. P. falciparum expresses the proteins Pdx1 and Pdx2, corresponding to the yeast enzymes Snz1-p and Sno1-p, which are essential for the vitamin B6 biosynthesis. An involvement of PfPdx1 and PfPdx2 in the de novo synthesis of vitamin B6 was shown by complementation of pyridoxine auxotroph yeast cells. Both plasmodial proteins act together in the glutaminase activity with a specific activity of 209 nmol min–1 mg–1 and a Km value for glutamine of 1.3 mm. Incubation of the parasites with methylene blue revealed by Northern blot analysis an elevated transcriptional level of pdx1 and pdx2, suggesting a participation of these proteins in the defenses against singlet oxygen. To be an active cofactor, vitamin B6 has to be phosphorylated by the pyridoxine kinase (PdxK). The recombinant plasmodial PdxK revealed Km values for the B6 vitamers pyridoxine and pyridoxal and for ATP of 212, 70, and 82 μm, respectively. All three enzymes expose a stage-specific transcription pattern within the trophozoite stage that guarantees the concurrent expression of Pdx1, Pdx2, and PdxK for the indispensable provision of vitamin B6. The occurrence of the vitamin B6 de novo synthesis pathway displays a potential new drug target, which can be exploited for the development of new chemotherapeutics against the human malaria parasite P. falciparum.

Aldose reductase (ALR2; EC 1.1.1.21)is a member of the aldoketo reductase superfamily and it catalyzes the NADPH-dependent reduction of aldeydes to their corresponding alcohols.It is implicated in the polyol pathway and in diabetic complication.The crystal structure of native aldose reductase has been determined to a resolution of 0.82 Å with a final R = 9.50 and R free = 10.90 and a mean coordinate error for the fully occupied sites of the protein of 0.011 Å (from fully matrix inversion).The structure contains a large number of multiple conformations: 78 out of 316 residues were modeled in two conformations.The overall structure folds into an eight-stranded / barrel with the active site located at the C-terminal end of the barrel and the NADP + -binding site near the hydrophobic binding pocket [1].The cofactor is held in place by the so-called 'safety-belt' (a loop between residue 216 and 227 of the canonical / barrel) [2].The active site of the structure contains a citrate molecule in two conformations.One of the conformations stabilizes the closed position of the safety-belt, whereby the other permits the safety-belt to open.Due to the high resolution, the partially opened conformation of the safety-belt can be observed in the electron density.

The polyamine synthesis enzyme spermidine synthase (SPDS) has been cloned from the model nematode Caenorhabditis elegans. Biochemical characterisation of the recombinantly expressed protein revealed a high degree of similarity to other eukaryotic SPDS with the exception of a low affinity towards the substrate decarboxylated S‐adenosylmethionine (K m = 110 μM) and a less pronounced feedback inhibition by the second reaction product 5’‐methylthioadenosine (IC50 = 430 μM). The C. elegans protein that carries a nematode‐specific insertion of 27 amino acids close to its N‐terminus was crystallized, leading to the first X‐ray structure of a dimeric eukaryotic SPDS.

The effects of oxidative insult on gene transcript levels in the filarial nematode Onchocerca volvulus were investigated using differential display RT-PCR. Oxidative stress was applied with the reagents paraquat, plumbagin and xanthine-xanthine oxidase. In all three cases, a cDNA fragment encoding a novel glutathione S-transferase (GST) resembling members of the theta-class was identified as upregulated (PQ29, PG112, XOD26). The subsequently isolated full-length cDNA harbors a 753-bp open reading frame encoding a GST with 268 amino acid residues and a predicted molecular mass of 31 kDa. This stress-responsive GST (Ov-GST-3) possesses only 14 and 21% sequence identity with the other O. volvulus GSTs (Ov-GST-1 and Ov-GST-2, respectively). Interestingly, Ov-GST-3 shares higher sequence identity with GSTs that are upregulated due to environmental stress. In order to confirm the specific upregulation of the Ov-GST-3 transcripts identified by differential display and to analyze the mRNA levels of the other Ov-GSTs (Ov-GST-1 and Ov-GST-2) under elevated stress conditions, a semi-quantitative polymerase chain reaction-enzyme-linked immunosorbent assay was performed. The Ov-GST-3 gene transcript level increased dramatically in response to xanthine-xanthine oxidase and to a lesser extent with paraquat and plumbagin. In contrast, Ov-GST-1 and Ov-GST-2 did not show any significant alterations in their steady-state mRNA levels in response to oxidative stress when examining the same mRNA samples. The present study clearly demonstrates that Ov-GST-3 is a critical enzyme in the defense against oxidative stress.

In order to identify genes that are differentially expressed as a consequence of oxidative stress due to paraquat we used the differential display technique to compare mRNA expression patterns in Caenorhabditis elegans . A C.elegans mixed stage worm population and a homogeneous larval population were treated with 100 mM paraquat, in parallel with controls. Induction of four cDNA fragments, designated L-1, M-47, M-96 and M-132, was confirmed by Northern blot analysis with RNA from stressed and unstressed worm populations. A 40-fold increase in the steady-state mRNA level in the larval population was observed for the L-1/M-47 gene, which encodes the detoxification enzyme glutathione S-transferase. A potential stress-responsive transcription factor (M-132) with C2H2-type zinc finger motifs and an N-terminal leucine zipper domain was identified. The M-96 gene encodes a novel stress-responsive protein. Since paraquat is known to generate superoxide radicals in vivo , the response of the C.elegans superoxide dismutase (SOD) genes to paraquat was also investigated in this study. The steady-state mRNA levels of the manganese-type and the copper/zinc-type SODs increased 2-fold in the larval population in response to paraquat, whereas mixed stage populations did not show any apparent increase in the levels of these SOD mRNAs.

This study describes the histological localization of two CuZn superoxide dismutases (SOD1 and SOD2) in the parasitic nematode Onchocerca volvulus, and a functional characterization of the `extracellular' form of this enzyme (SOD2) which provides evidence that it is involved in the defense against environmental superoxide anion radicals. These essential enzymes are detected in larval and adult stages of the parasite, determined at the mRNA and protein levels by in situ hybridization and immunolocalization studies. These proteins are distributed throughout the worm, at various concentrations with particularly high levels produced in the hypodermis. In vitro maintenance of parasites indicated that SOD2 was secreted outside the parasite into the medium. Baculovirus constructs designed to test the ability of the SOD2 hydrophobic N-terminal region to function in processing and secretion confirmed the ability of this polypeptide sequence to direct the secretion of a marker protein, as well as of the mature SOD2 enzyme. Analyses of the native, mature SOD2 enzyme molecular mass, and the primary and quaternary structure, indicate that unlike other extracellular SODs, the SOD2 is active as a non-glycosylated dimer, rather than as a tetrameric glycoprotein. The detection of SOD2 outside of the parasite maintained in vitro, and the confirmation that the SOD2 is a secreted enzyme, indicate that this enzyme plays a role in the interactive biology of parasitic nematodes with their hosts.

LacY-ompA fusions, encoding the N-terminal 50, 71 or 143 residues of lactose permease, were constructed. The observed orientation of the OmpA part of each hybrid protein with respect to the plasma membrane supports current models of the N-terminus of Lac permease. Hybrids possessing the entire mature OmpA were very stable; those with only a part thereof were much less stable. Due to their in vivo stability and accessibility to antibody it is proposed that such hybrids may represent potential models to investigate the assembly pathway of lactose permease.

The T-even type Escherichia coli phage Ox2 uses the outer membrane protein OmpA as a receptor. The protein is recognized with the ends of the virion's long tail fibers. The 266 residue protein 38 is located at this site and acts as an adhesin. Host-range mutants had previously been isolated from Ox2. Mutant Ox2h5 is able to infect cells possessing an altered OmpA protein, which renders the cell resistant to Ox2. Ox2h10 was selected from Ox2h5. This phage recognizes the OmpC protein in addition to the OmpA protein. Ox2h12, which stems from Ox2h10, binds to OmpC with high affinity, but has lost efficient binding to OmpA. The mutational alterations caused in genes 38 are: Asp231 → Asn(h5) and His170 → Arg(h10). The triple mutant Ox2h12 possesses an insertion of a Gly residue next to Gly121. The three mutants have additionally acquired mutations affecting their base plate, making them "trigger-happy". When protein 38 was compared with the same protein derived from other E. coli phages, it was found to contain two constant and one variable domains, the latter harboring four hypervariable regions flanked by a largely conserved glycine-rich sequence. The h5 and h10 mutations occurred within two hypervariable areas, while the additional Gly residue was present in one of the flanking conserved sequences. On the basis of these results, as well as those obtained from host-range mutants analyzed previously, a model for such adhesins is proposed. Receptor recognition is most likely performed via the hypervariable regions, which may form loops held together in close proximity by the oligoglycine sequences. The latter may achieve this by being part of highly compact omega loops.

A series of overlapping deletions has been constructed in the ompA gene which encodes the 325-residue Escherichia coli outer membrane protein OmpA. Immunoelectron microscopy showed that the OmpA fragments were either located in the periplasmic space or were associated with the outer membrane. Apparently an area between residues 154 and 180 is required for this association; all proteins missing this area were found to be periplasmic. The nature of this association remained unknown; no membrane-protected tryptic fragments could be identified for any of these polypeptides. Hybrid genes were constructed encoding parts of the periplasmic maltose binding protein and an area of the ompA gene coding for residues 154-274. The corresponding proteins were not localized to the outer membrane but remained attached to the outer face of the plasma membrane, possibly because the normal mechanism of release from this membrane was impaired. In the OmpA protein the conspicuous sequence Ala180-Pro-Ala-Pro-Ala-Pro-Ala-Pro187 exists. Frameshift mutants were constructed to eliminate this sequence. There was no effect on the incorporation of the mutant proteins into the outer membrane. Thus, this hinge region is not involved in sorting. A proposal suggesting the existence of a sorting signal common to several outer membrane proteins (Benson, S. A., Bremer, E., and Silhavy, T. J. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 3830-3834) was subsequently rejected (Bosch, D., Leunissen, J., Verbakel, J., de Jong, M., van Erp, H., and Tommassen, J. (1986) J. Mol. Biol. 189, 449-455; Freudl, R., Schwarz, H., Klose, M., Movva, N. R., and Henning, U. (1985) EMBO J. 4, 3593-3598). Although it is not known whether or not the outer membrane association observed represents a step in the normal sorting mechanism, it is concluded that it remains an open question whether or not a sorting signal, as proposed originally, exists in outer membrane proteins.

The 325-residue outer membrane protein, OmpA, of Escherichia coli, like most other outer membrane proteins with known sequence, contains no long stretch of hydrophobic amino acids. A synthetic oligonucleotide, encoding the sequence Leu-Ala-Leu-Val, was inserted four times between the codons for amino acid residues 153 and 154 and two, three, or four times between the codons for residues 228 and 229, resulting in the OmpA153-4, OmpA-228-2, -3, and -4 proteins, respectively. In the first case, the lipophilic sequence anchored the protein in the plasma membrane. In the OmpA228 proteins, 16 but not 12 or 8 lipophilic residues most likely also acted as an anchor. By removal of the NH2-terminal signal peptide, the function of the insert in OmpA153-4 was converted to that of a signal-anchor sequence. Possibly due to differences in amino acid sequences surrounding the insert, no signal function was observed with the insert in OmpA228-4. Production of the OmpA153-4 protein, with or without the NH2-terminal signal sequence, resulted in a block of export of chromosomally encoded OmpA. Clearly, long hydrophobic regions are not permitted within proteins destined for the bacterial outer membrane, and these proteins, therefore, have had to evolve another mechanism of membrane assembly.

Proteins 38 of bacteriophages T2, K3, Ox2 and M1 are located at the free ends of their long tail fibers and function as adhesins, i.e. they mediate binding to the bacterial receptors. The latter three phages use the Escherichia coli outer membrane protein OmpA as a receptor, while T2 uses the outer membrane proteins OmpF or Ttr. The DNA sequences of genes 38 of phages Ox2 and M1 have been determined and are compared with those known for T2 and K3. The genes encode 262(T2), 260(K3), 266(Ox2) and 262(M1) amino acid residues. Three domains are distinguishable in these proteins. There are two conserved regions encompassing about 120 NH2-terminal and about 25 CO2H-terminal residues, respectively. The area between these was found to be hypervariable, and it is shown that a very large number of amino acid substitutions, deletions and/or insertions have occurred. Glycine-rich stretches are present within and flanking these areas. Their positions are essentially conserved, indicating an important structural role in receptor recognition. The hypervariability, most likely caused by a constant struggle with bacterial phage-resistant mutants, is so drastic that one cannot discern that T2 uses different receptors from those of the other phages. The partially known sequence of gene 38 of phage T4 has been completed. The gene encodes a protein consisting of 183 amino acid residues. The amino acid composition and sequence of this protein is completely different from those of phages T2, K3, Ox2 and M1. Also, the protein is functionally unrelated to the other proteins 38: it is not present in phage T4 and, unlike the other proteins 38, is required for the efficient dimerization of protein 37. All phages under study are of the same morphology and the genomic organization of the tail fiber genes is identical, with genes 36, 37 and 38 most likely representing, in this order, a transcriptional unit. Sequence similarities between the CO2H-termini of genes 37 of the non-T4 phages and gene 38 of phage T4 were found; this part of gene 37 does not exist in T4. It is suggested that gene 38 of phage T4 originated from a segment of gene 37 of a T2-type phage. Gene 38 of phage T4 is not unique, DNA-DNA hybridization experiments indicated that two other T-even type phages, TuIa and TuIb, possess a T4-type gene 38.

The OmpA protein is one of the major outer membrane proteins of Escherichia coli. Among other functions the protein serves as a receptor for several phages and increases the efficiency of F-mediated conjugation when present in recipient cells. TraT is an F-factor-coded outer membrane lipoprotein involved in surface exclusion, the mechanism by which E. coli strains carrying F-factors become poor recipients in conjugation. To determine a possible interaction of TraT with OmpA, the influence of TraT on phage binding to cells was measured. Because TraT inhibits inactivation of OmpA-specific phages it is suggested that TraT interacts directly with OmpA. Sequence homology of TraT with proteins 38, the phage proteins recognizing outer membrane proteins, supports this finding. A model of protein interactions is discussed.

The classical T-even bacteriophages recognize host cells with their long tail fibers. Gene products 35, 36, and 37 constitute the distal moiety of these fibers. The free ends of the tail fibers, which are formed by the CO2H terminus of gene product 37, possess the host range determinants. It was found that 4 out of 10 different strains of Escherichia coli K-12 contained regions of chromosomal DNA which hybridized with a probe consisting of genes 35, 36, and 37 of the T-even phage K3. From one strain this homologous DNA, which was associated with an EcoRI fragment of about 5 kilobases, was cloned into plasmid pUC8. Two independently recovered hybrid plasmids had undergone a peculiar rearrangement which resulted in the loss of about 3 kilobases of cloned DNA and a duplication of both the vector and the remaining chromosomal DNA. The mechanisms causing this duplication-deletion may be related to that of transposases. The cloned DNA was capable of recombination with phage T4 gene 36 and a phage T2 gene 37 amber mutant. DNA sequencing revealed the existence of regions of identity between the cloned DNA and genes 36 and 37 of phage T2. In addition, after growth of a derivative of phage K3 on a strain harboring T2 DNA, it was found that this phage contained the same parts of the T2 tail fiber genes which had been recovered from the bacterial chromosome. There appears to be little doubt that the phage had picked up this DNA from the host. The possibility is considered that a repertoire of parts of genes 36 and 37 of various T-even-type phages is present in their hosts, allowing the former to change their host ranges.

Genes 36 have been cloned from phage T2 and the T-even type phages K3 and Ox2. The products of these genes are part of the long tail fibers of the phages, they form the proximal moiety of the distal half fiber. The genes have been sequenced, the nucleotide sequence of gene 36 of phage T4 is known (Oliver, D.B. & Crowther, R.A. (1981) J.Mol.Biol. 153, 545-568). Comparison of the deduced amino acid sequences of the four proteins revealed a surprising pattern. These sequences can be divided into two highly conserved and one very variable region. The former consist of about 60 NH2-terminal and 70 CO2H-terminal residues flanking the variable middle region of about 100 residues. Thus, an identical and unique morphology can be formed by a number of different primary structures. It is proposed that the conserved areas are involved in binding of the proteins to the neighboring products of genes 35 and 37 and that this function has put constraints on the variability of the primary protein structure. The overall amino acid composition of the proteins is rather similar; the codon usage is that known for phage T4. The intercistronic region between genes 35 and 36 consisting of 62 base pairs and containing a presumed terminator for g35 transcription and the 'late type' promoter for transcription of genes 36, 37, and 38, is almost completely identical in the four phages.