Anthranilate synthase (EC 4.1.3.27) has been purified from cell cultures of Catharanthus roseus by poly(ethy1ene glycol) precipitatiodfractionation and subsequent separation by anion exchange on Q-Sepharose, Orange A dye chromatography, Mono Q anion-exchange chromatography and Superose 6 gel filtration. By analogy to anthranilate synthases from other sources it does look like the enzyme is a tetramer composed of two large and two small subunits, with molecular mass 67 and 25.5 f 0.5 kDa, respectively. The molecular mass determined by gel filtration was 143 f 5 kDa. The enzyme had a PI of 5.1 determined by chromatofocusing. The pH optimum was between pH 7.5 and pH 8.3, but the type of buffer used affected the results. The enzyme could utilize NH,' as ammonium donor instead of glutamine. The enzyme showed normal Michaelis-Menten kinetics with respect to the substrates L-glutamine and chorismate, and the cofactor Mg", K,,, values for L-glutamine was determined to be 0.37f0.05 mM, for chorismate 67?3 pM, and for MgCl, 0.26 -C-0.03 mM respectively. Anthranilate synthase was inhibited by L-tryptophan, tryptamine and D-tryptophan (with L-tryptophan being the best inhibitor). The enzyme was allosterically regulated showing positive cooperativity of chorismate binding at higher concentrations of tryptophan. For a tryptophan concentration of 20pM the Hill coefficient was determined to be 2. The tryptophan binding sites showed positive cooperativity for higher concentrations of chorismate. The purified enzyme did not contain anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase activity and is thus not of the same type as the well characterized Salmonella typhimurium anthranilate synthase/phosphoribosyl pyrophosphate transferase bifunctional type.Anthranilate synthase (AS; EC 4.1.3.27) catalyzes the first reaction branching from the shikimate pathway toward the biosynthesis of tryptophan occurring in bacteria, fungi and plants [I] (Fig. 1).Tryptophan is a key precursor for the indole alkaloids, among which there are several pharmacologically important compounds [2], from Catharanthus roseus, for example, the two anticancer drugs vinblastine and vincristine. The availability of tryptophan might be a limiting factor in the production of indole alkaloids. In all plants and cell cultures where AS has been examined, the enzyme was found to be strongly feedback-inhibited by tryptophan ; in accordance with this, several studies have indicated that loss of feedback regulation of AS can lead to largely unregulated accumulation of tryptophan [3 -61.AS enzymes have been purified to homogeneity and characterized from a number of microorganisms. In all mi- crobial species studied, AS is composed of two nonidentical subunits [7-91. Component I (also called a-subunit) binds the substrate chorismate and catalyzes its aromatization; component I1 @-subunit) binds the other substrate glutamine and transfers an ammonia group from glutamine to component I (chorismate). This glutamine-dependent AS reaction requires both subunit...
A genomic expression library of Streptococcus pneumoniae was screened with a convalescent-phase serum for immunoreactive proteins. Six known and 17 unknown pneumococcal proteins were detected. Five of the known proteins were surface-located virulence factors, and eight of the unknown proteins were putative membrane proteins.Streptococcus pneumoniae is the major cause of otitis media, pneumonia, and meningitis. Recent studies have presented new insights into the pathogenesis of pneumococcal infection (17). In addition to the antiphagocytic polysaccharide capsule, several proteins have been described as virulence factors for S. pneumoniae, most of which are located on the pneumococcal cell surface and contribute to colonization, adherence, and invasion during infection of various animal models (12, 13). We constructed a genomic expression library of an S. pneumoniae strain (strain 3.B, serotype 1; collection of the Department of Medical Microbiology and Virology, University of Duesseldorf) which was isolated from a patient suffering from disseminated pneumococcal infection. Here we describe the screening of this library with a serum which was taken from the patient during the convalescent phase, 26 days after diagnosis.A number of pneumococcal proteins were highly reactive with immunoglobulin G (IgG) antibodies of the patient serum. Immunoblot analysis of pneumococcal cell lysates showed immunodominant bands from 60 up to 130 kDa (data not shown). To identify these immunoreactive proteins, an expression library of the pneumococcal genome was created in Escherichia coli. Pneumococcal DNA was digested partially by Sau3A, and fragments of 500 to 1,600 bp were ligated with the expression vector pET15b (Novagen) and used for transformation of E. coli. About 61,000 recombinants were screened for expression of proteins which were reactive with IgG antibodies of the convalescent-phase serum by colony immunoblot analysis. Seventy-eight recombinant proteins were detected. Western blot analysis of whole-cell lysates confirmed expression of immunoreactive proteins in 56 clones, from which plasmid DNA was isolated and the pneumococcal DNA was sequenced. The DNA sequences were used for a similarity search. The nucleotide sequence of the identified gene was completed by the sequence data obtained from the unfinished pneumococcal genome (available on the website of The Institute for Genomic Research [TIGR] [http://www.tigr.org]).
The gene coding for isochorismate synthase (EC 5.4.99.6) was amplified by the polymerase chain reaction fromEscherichia coli, cloned into a binary vector. and mobilised intoAgrobacterium rhizogenes LBA9402 which was used to transformRubia peregrina. Transgenic roots containing bacterial isochorismate synthase cDNA expressed twice as much isochorismate synthase activity (4.88 pkat/mg protein) as the control roots (2.45 pkat/mg protein) after 10 days in culture, and accumulatedca. 20% higher levels of total anthraquinones after 30 days in culture. Whilst the amount of total alizarin (free and bound) in the transgenic roots was 30% higher than in control roots, the level of free alizarin wasca. 85% higher.
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