Characterization of genes with a putative key role in the parasitic lifestyle of the nematode Strongyloides ratti.

Parasitic nematodes are significant pathogens of humans and other animals. The molecular and genetic basis of animal parasitism is not yet fully understood. Strongyloides spp. are a genus of gastrointestinal nematodes of which species infect approximately 100­200 million people worldwide. S. ratti is a natural parasite of the rat, and a useful and amenable laboratory model. Previous EST and microarray analyses of the S. ratti life cycle have identified genes whose expression was specific, or biased, to the parasitic adult stage, suggesting that they may play a key role in parasitism in this species. Here we have further investigated the expression of these genes (by RT-PCR) throughout the S. ratti life-cycle. We produced recombinant proteins in vitro for a subset of these genes, which were used in Western blot analyses to investigate the distribution of the gene products among different stages of the S. ratti life cycle. We tested the efficacy of these recombinant proteins as anti-S. ratti vaccines. One of the proteins was detected in the excretory/secretory products of the parasitic stages.

The crystal structure of the L1 metallo-beta-lactamase from Stenotrophomonas maltophilia at 1.7 A resolution.

The structure of the L1 metallo-beta-lactamase from the opportunistic pathogen Stenotrophomonas maltophilia has been determined at 1.7 A resolution by the multiwavelength anomalous dispersion (MAD) approach exploiting both the intrinsic binuclear zinc centre and incorporated selenomethionine residues. L1 is unique amongst all known beta-lactamases in that it exists as a tetramer. The protein exhibits the alphabeta/betaalpha fold found only in the metallo-beta-lactamases and displays several unique features not previously observed in these enzymes. These include a disulphide bridge and two substantially elongated loops connected to the active site of the enzyme. Two closely spaced zinc ions are bound at the active site with tetrahedral (Zn1) and trigonal bipyramidal (Zn2) co-ordination, respectively; these are bridged by a water molecule which we propose acts as the nucleophile in the hydrolytic reaction. Ligation of the second zinc ion involves both residues and geometry which have not been previously observed in the metallo-beta-lactamases. Simulated binding of the substrates ampicillin, ceftazidime and imipenem suggests that the substrate is able to bind to the enzyme in a variety of different conformations whose common features are direct interactions of the beta-lactam carbonyl oxygen and nitrogen with the zinc ions and of the beta-lactam carboxylate with Ser187. We describe a catalytic mechanism whose principal features are a nucleophilic attack of the bridging water on the beta-lactam carbonyl carbon, electrostatic stabilisation of a negatively charged tetrahedral transition state and protonation of the beta-lactam nitrogen by a second water molecule co-ordinated by Zn2. Further, we propose that direct metal:substrate interactions provide a substantial contribution to substrate binding and that this may explain the lack of specificity which is a feature of this class of enzyme.

Enzyme kinetics and biochemical analysis of ImiS, the metallo-beta-lactamase from Aeromonas sobria 163a.

The metallo-beta-lactamase from Aeromonas sobria 163a, ImiS, was isolated in a two stage purification procedure using protein affinity columns. Enzyme kinetics show that ImiS hydrolyses the carbapenems but displays poor activity against other beta-lactams. ImiS possesses the narrowest spectrum of activity of the Group 3 enzymes that have been analysed. Sequencing of the 40 N-terminal amino acids show this region to be identical to that of the CphA metallo-beta-lactamase from Aeromonas hydrophila (Massidda, Rossolini & Satta, 1991). Light scattering analysis indicates that ImiS is functionally active as a monomer.

Construction of a stable dimer of Bacillus stearothermophilus lactate dehydrogenase.

A molecular graphics analysis of the features which prevent cytosolic malate dehydrogenase dimers from forming tetramers was evaluated by its success in predicting the synthesis of a version of the LDH framework which is a stable dimer. Surface residues responsible for malate dehydrogenases being dimers were revealed by superimposing the structures of two dimers of pig cytosolic malate dehydrogenase on one homologous tetramer of L-lactate dehydrogenase from Bacillus stearothermophilus. Four regions were identified as composing the P-axis dimer-dimer interface. Two regions of the dimer were surface loops that collided when built as a tetramer: a large loop (residues 203-207, KNOBI) and a small loop (residues 264-269, KNOBII), and these were candidates to explain the dimeric character of malate dehydrogenase. The analysis was tested by constructing a synthetic B. stearothermophilus lactate dehydrogenase (KNOBI) containing the large malate dehydrogenase loop (residues 203-207 being AYIKLQAKE, and extra four amino acids). The new construct was thermotolerant (90 degrees C) and enzymically active with kcat and KM (pyruvate) values similar to those of the wild-type enzyme. However, whereas the allosteric activator fructose 1,6-bisphosphate decreased KM 100 times for wild type, it had no influence on KNOBI. The molecular volumes of 1-120 microM concentrations of the construct were measured by time-resolved decay of tryptophan fluorescence anisotropy and by gel filtration. Both methods showed the molecular weight of wild type increased from dimer to tetramer with Kd about 20 microM dimer. KNOBI remained a dimer under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Charge balance in the alpha-hydroxyacid dehydrogenase vacuole: an acid test.

The proposal that the active site vacuole of NAD(+)-S-lactate dehydrogenase is unable to accommodate any imbalance in electrostatic charge was tested by genetically manipulating the cDNA coding for human muscle lactate dehydrogenase to make a protein with an aspartic acid introduced at position 140 instead of the wild-type asparagine. The Asn 140-Asp mutant enzyme has the same kcat as the wild type (Asn 140) at low pH (4.5), and at higher pH the Km for pyruvate increases 10-fold for each unit increase in pH up to pH 9. We conclude that the anion of Asp 140 is completely inactive and that it binds pyruvate with a Km that is over 1,000 times that of the Km of the neutral, protonated aspartic-140. Experimental results and molecular modeling studies indicate the pKa of the active site histidine-195 in the enzyme-NADH complex is raised to greater than 10 by the presence of the anion at position 140. Energy minimization and molecular dynamics studies over 36 ps suggest that the anion at position 140 promotes the opening of and the entry of mobile solvent beneath the polypeptide loop (98-110), which normally seals off the internal active site vacuole from external bulk solvent.

Duck liver 'malic' enzyme. Expression in Escherichia coli and characterization of the wild-type enzyme and site-directed mutants.

A cDNA for duck liver 'malic' enzyme (EC 1.1.1.40) was subcloned into pUC-8, and the active enzyme was expressed in Escherichia coli TG-2 cells as a fusion protein including a 15-residue N-terminal leader from beta-galactosidase coded by the lacZ' gene. C99S and R70Q mutants of the enzyme were generated by the M13 mismatch technique. The recombinant enzymes were purified to near homogeneity by a simple two-step procedure and characterized relative to the enzyme isolated from duck liver. The natural duck enzyme has a subunit molecular mass of approx. 65 kDa, and the following kinetic parameters for oxidative decarboxylation of L-malate at pH 7.0: Km NADP+ (4.6 microM); Km L-malate (73 microM); kcat (160 s-1); Ka (2.4 microM) and Ka' (270 microM), dissociation constants of Mn2+ at 'tight' (activating) and 'weak' metal sites; and substrate inhibition (51% of kcat. at 8 mM-L-malate). Properties of the E. coli-derived recombinant wild-type enzyme are indistinguishable from those of the natural duck enzyme. Kinetic parameters of the R70Q mutant are relatively unaltered, indicating that Arg-70 is not required for the reaction. The C99S mutant has unchanged Km for NADP+ and parameters for the 'weak' sites (i.e. inhibition by L-malate, Ka'); however, kcat. decreased 3-fold and Km for L-malate and Ka each increased 4-fold, resulting in a catalytic efficiency [kcat./(Km NADP+ x Km L-malate x Ka)] equal to 3.7% of the natural duck enzyme. These results suggest that the positioning of Cys-99 in the sequence is important for proper binding of L-malate and bivalent metal ions.

Structure and expression of the rat epididymal secretory protein I gene. An androgen-regulated member of the lipocalin superfamily with a rare splice donor site.

The complete rat epididymal secretory protein I (ESP I) gene was isolated from a genomic library constructed in bacteriophage lambda Charon 4A. The complete nucleotide sequence of the gene and its immediate 5' and 3' flanking sequences were determined. Interesting features include the presence of a rare, but functional, splice donor site (...GC) and the presence of a putative androgen-receptor-binding element. A detailed analysis of ESP I regulation was carried out after castration and subsequent testosterone treatment, demonstrating the requirement for androgens. Efferent-duct ligation and cryptorchism, on the other hand, had no effect on the steady-state concentrations of ESP I transcripts. Comparison of the exon/intron organization of the ESP I gene with those of members of the lipocalin superfamily provides strong support for a common ancestral origin.

cDNA cloning of porcine interleukin 2 by polymerase chain reaction.

Porcine interleukin 2 (IL-2) cDNA was cloned by polymerase chain reaction (PCR), using primers derived from the corresponding bovine sequence. The resulting porcine DNA sequence encodes a 154 residue IL-2 primary translation product. Comparison of the mature, secreted form of porcine IL-2 with those of other species was carried out in an attempt to identify differences that might contribute to the observed differing species specificities.

Organization and sequence of the human alpha-lactalbumin gene.

A recombinant bacteriophage containing the entire alpha-lactalbumin gene was isolated from a human genomic library constructed in bacteriophage lambda L47. Within this recombinant the 2.5 kb alpha-lactalbumin gene is flanked by about 5 kb of sequence on either side. The complete nucleotide sequence of the gene and its immediate flanking sequences were determined and compared with those of the rat alpha-lactalbumin gene. These studies showed that the size, organization and sequence of the exons have been highly conserved, whereas the introns have diverged considerably. In particular, the first intron of the human gene was found to contain an Alu repetitive sequence not present in the rat. A high degree of homology (67%) was also observed in the 5' flanking regions, extending as far as 655 nucleotide residues upstream of the transcriptional initiation site. Comparison of the 5' flanking sequences of these two alpha-lactalbumin genes with those of five casein genes has revealed the presence of a highly conserved region [consensus sequence: RGAAGRAAA(N)TGGACAGAAATCAA(CG)TTTCTA], extending from position -140 to -110 in all seven sequences examined, suggesting a possible regulatory role in the hormonal control or tissue-specific expression of milk protein genes in the mammary gland.