In vivo expression technology for selection of bacterial genes specifically induced in host tissues.

We have developed a genetic system, termed IVET (in vivo expression technology), designed to identify bacterial genes that are induced when a pathogen infects its host. A subset of these induced genes should include those that encode virulence factors, products specifically required for the infection process. The system is based on complementation of an attenuating auxotrophic mutation by gene fusion, and it is designed to be of use in a wide variety of pathogenic organisms. In Salmonella typhimurium, we have successfully used the system to identify a number of genes that are induced in BALB/c mice, and that, when mutated, confer a virulence defect. The IVET system has several applications in the area of vaccine and antimicrobial drug development. The technique was designed for the identification of virulence factors and thus may lead to the discovery of new antigens useful as vaccine components. The IVET system facilitates the isolation of mutations in genes involved in virulence and, therefore, should aid in the construction of live attenuated vaccines. In addition, the identification of promoters that are optimally expressed in animal tissues provides a means of establishing in vivo regulated expression of heterologous antigens in live vaccines, an area that has been previously problematic. Finally, we expect that our methodology will be used to uncover many biosynthetic, catabolic, and regulatory genes that are required for growth of microbes in animal tissues. The elucidation of these gene products should provide new targets for antimicrobial drug development.

Recombinant mouse muscle adenylosuccinate synthetase: overexpression, kinetics, and crystal structure.

Vertebrates possess two isozymes of adenylosuccinate synthetase. The acidic isozyme is similar to the synthetase from bacteria and plants, being involved in the de novo biosynthesis of AMP, whereas the basic isozyme participates in the purine nucleotide cycle. Reported here is the first instance of overexpression and crystal structure determination of a basic isozyme of adenylosuccinate synthetase. The recombinant mouse muscle enzyme purified to homogeneity in milligram quantities exhibits a specific activity comparable with that of the rat muscle enzyme isolated from tissue and K(m) parameters for GTP, IMP, and l-aspartate (12, 45, and 140 microm, respectively) similar to those of the enzyme from Escherichia coli. The mouse muscle and E. coli enzymes have similar polypeptide folds, differing primarily in the conformation of loops, involved in substrate recognition and stabilization of the transition state. Residues 65-68 of the muscle isozyme adopt a conformation not observed in any previous synthetase structure. In its new conformation, segment 65-68 forms intramolecular hydrogen bonds with residues essential for the recognition of IMP and, in fact, sterically excludes IMP from the active site. Observed differences in ligand recognition among adenylosuccinate synthetases may be due in part to conformational variations in the IMP pocket of the ligand-free enzymes.

Overproduction, purification, and characterization of adenylosuccinate synthetase from Escherichia coli.

Adenylosuccinate synthetase, encoded by the purA gene of Escherichia coli, catalyzes the first committed step toward AMP in the de novo purine biosynthetic pathway and plays an important role in the interconversion of purines. A 3.2-kb DNA fragment, which carries the purA gene, was cloned into the temperature-inducible, high-copy-number plasmid vector, pMOB45. Upon temperature induction, cells containing this plasmid produce adenylosuccinate synthetase at approximately 40 times the wild-type level. A scheme is presented for the purification of the overproduced adenylosuccinate synthetase to homogeneity in amounts sufficient for studies of its structure and mechanism. The wild-type and the overproduced adenylosuccinate synthetase enzyme preparations were judged to be identical by the following criteria. The amino acid sequence at the N-terminus of the overproduced enzyme proved identical to the corresponding sequence of the wild-type enzyme. Michaelis constants for both the wild-type and overproduced enzyme preparations were the same. And (iii) both proteins shared similar chromatographic behavior and the same mobility during sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Results from size-exclusion chromatography and SDS-polyacrylamide gel electrophoresis suggest that adenylosuccinate synthetase exists as a dimer of identical, 48,000-Da, subunits.

Characterization of the putative GTP-binding site residues of Escherichia coli adenylosuccinate synthetase by site-directed mutagenesis.

Adenylosuccinate synthetase contains amino acid sequences in its GTP-binding domain that are homologous to other G-proteins. This homology includes a glycine-rich phosphate-binding loop, GXXXXGK, and a guanine-specific binding region, (N/T/Q)KXD; however, virtually no other sequence homology exists between other G-proteins and adenylosuccinate synthetase. On the basis of X-ray diffraction studies, the folding topologies of the synthetase and the p21 ras proteins are different. Yet, residues that interact with GTP in the p21 ras proteins are present in the synthetase in nearly identical positions. We chose therefore to study the G15V mutant, a phosphate-binding loop mutant, and K331L and K331R, two mutants of Lys331 that are involved in guanine ring binding. The Km values for GTP of adenylosuccinate synthetase mutants, K331L and K331R, when compared to those of the wild-type enzyme, were 27- and 20-fold increased, respectively, without any significant change in the Km values for IMP. Because both mutations affected the Km values for GTP similarly, whereas the kcat and secondary structure were essentially unchanged, it is suggested that Lys331 is located in the GTP-binding site of adenylosuccinate synthetase and the terminal N zeta of the Lys is not necessarily important in GTP-binding on the enzyme. Therefore, Lys331 may interact with GTP through hydrophobic interactions between its linear side chain and the aromatic ring of the guanine base of GTP. Also, structural characterization of the G15V mutant was carried out using circular dichroism (CD) spectrometry, NMR spectroscopy, and spectrofluorometry. The CD spectral data indicated that the secondary structure of the G15V mutant was significantly altered by GTP and IMP, whereas that of the wild-type enzyme is not changed; however, the two enzymes exhibited similar secondary structures in the absence of substrates. The NMR spectra of both enzymes were also similar in the absence of substrates. The dissociation constant (Kd) for IMP of the G15V mutant was 4.8-fold larger than its Km value which was 1.5-fold increased compared to the wild-type enzyme. From these findings, it was concluded that the phosphate-binding region of adenylosuccinate synthetase is involved in a conformational change induced by GTP and IMP binding, and that GTP and IMP binding depend on the presence of the other substrate at the active site of the enzyme. These results suggest that the Lys331 of adenylosuccinate synthetase may play similar roles in the function and structure to that of GTP-binding proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Purification and characterization of recombinant Plasmodium falciparum adenylosuccinate synthetase expressed in Escherichia coli.

Most parasitic protozoa lack the de novo purine biosynthetic pathway and rely exclusively on the salvage pathway for their purine nucleotide requirements. Enzymes of the salvage pathway are, therefore, candidate drug targets. We have cloned the Plasmodium falciparum adenylosuccinate synthetase gene. In the parasite, adenylosuccinate synthetase is involved in the synthesis of AMP from IMP formed during the salvage of the purine base, hypoxanthine. The gene was shown to code for a functionally active protein by functional complementation in a purA mutant strain of Escherichia coli, H1238. This paper reports the conditions for hyperexpression of the recombinant protein in E. coli BL21(DE3) and purification of the protein to homogeneity. The enzyme was found to require the presence of dithiothreitol during the entire course of the purification for activity. Glycerol and EDTA were found to stabilize enzyme activity during storage. The specific activity of the purified protein was 1143.6 +/- 36.8 mUnits/mg. The K(M)s for the three substrates, GTP, IMP, and aspartate, were found to be 4.8 microM, 22.8 microM, and 1.4 mM, respectively. The enzyme was a dimer on gel filtration in buffers of low ionic strength but equilibrated between a monomer and a dimer in buffers of increased ionic strength.