1131 Crystal structure analysis of 2 : NaC,,H,,N,, Pi, u = 876.9(2), b = 988.8(4), c = 1274.1(3) pm, a = 100.52(2), fi = 94.66(2), y = 94.13(2)", V = 1078.3(5) x lo6 pm3, psalsd = 1.089 g~m -~, Z = 2, crystal size 0.3 x 0.3 x 0.9 mm3, 173 K, Cu,,, radiation, Enraf-Nonius CAD4, scan width 2.25 <8 < 76.5",4202 symmetry-independent and significant reflections [IF1 >3u(F)] structure solution by direct methods (SHELXS-86 [6]) supplemented by difference Fourier and LSQ calculations (SHELX-76) [6], 288 refined parameters, R = 0.0610, R , = 0.0608 ( w = l/u2(fl), all non-hydrogen atoms were refined with anisotropic temperature factors; the positions of H122 and H123 were determined directly, those of the remaining H atoms were place at calculated positions in the refinement
Dihydrodipicolinate synthase (EC 4.2.1.52), the first enzyme unique to lysine biosynthesis in bacteria and higher plants, has been purified to homogeneity from etiolated pea (Pisum sativum) seedlings using a combination of conventional and affinity chromatographic steps. This is the first report on a homogeneous preparation of native dihydrodipicolinate synthase from a plant source. The pea dihydrodipicolinate synthase has an apparent molecular weight of 127,000 and is composed of three identical subunits of 43,000 as determined by gel filtration and crosslinking experiments. The tnmenc quaternary structure resembles the trimenc structure of other aldolases, such as 2-keto-3-deoxy-6-phosphogluconic acid aldolase, which catalyze similar aldol condensations. The amino acid compositions of dihydrodipicolinate synthase from pea and Escherichia coli are similar, the most significant difference concems the methionine content: dihydrodipicolinate synthase from pea contains 22 moles of methionine residue per mole of native protein, contrary to the E. coli enzyme, which does not contain this amino acid at all. Dihydrodipicolinate synthase from pea is highly specific for the substrates pyruvate and L-aspartate-fl-semialdehyde; it follows Michaelis-Menten kinetics for both substrates. The pyruvate and L-aspartate-ft-semialdehyde have Michaelis constant values of 1.70 and 0.40 millimolar, respectively. L-Lysine, S-(2-aminoethyl)-L-cysteine, and La-(2-aminoethoxyvinyl)glycine are strong allostenc inhibitors of the enzyme with 50% inhibitory values of 20, 160, and 155 millimolar, respectively. The inhibition by L-lysine and L-a-(2-aminoethoxyvinyl)glycine is noncompetitive towards L-aspartateBl-semialdehyde, whereas S-(2-aminoethyl)-L-cysteine inhibits dihydrodipicolinate synthase competitively with respect to L-aspartate-jt-semialdehyde. Furthermore, the addition of (2R,3S,6S)-2,6-diamino-3-hydroxy-heptandioic acid (1.2 millimolar) and (2S,6R/S)-2,6-diamino-6-phosphono-hexanic acid (1.2 millimolar) activates dihydrodipicolinate synthase from pea by a factor of 1.4 and 1.2, respectively. This is the first reported activation process found for dihydrodipicolinate synthase.
3'-Demethoxy-3'-hydroxystaurosporine, 1 (CGP58 546), a novel staurosporine analogue, was isolated from a mutant of Streptomyces longisporoflavus R19 blocked in the last step of the biosynthetic pathway. CGP58 546 was less potent than staurosporine, but it showed a more selective inhibition pattern against various subtypes of protein kinase C. Staurosporine, 2, an indolo (2,3-a) carbazole alkaloid, was first isolated in 1977 by S. Omura and co-workers from Streptomyces sp. AM-22821}, later reclassified as Saccharothrix sp. Its structure and relative stereochemistry were determined by X-ray crystallographic analysis2'3) and by XH and 13C NMRstudies4). The
3-Amino-5-hydroxybenzoic acid was investigated for its ability to induce rifamycin biosynthesis in an appropriate mutant of Nocardia mediterranei and identified as a direct precursor of the seven-carbon amino starter-unit for the biosynthesis of ansamycins.A model for the biosynthesis of different types of ansamycins is presented and discussed.As described in part III of this series of papers1)we could isolate a very early aromatic ansamycinprecursor containing the seven-carbon amino starter-unit and three initial acetate/propionate-units of the ansa chain. This precursor was designated product P8/1-OG and identified as 2,6-dimethyl-3,5,7-trihydroxy-7-(3'-amino-5'-hydroxyphenyl)-2,4-heptadienoic acid. The structural comparison between product P8/1-OG and rifamycin S or rifamycin W clearly demonstrated a common biogenetic origin. 3-Amino-5-hydroxybenzoyl-coenzyme A was proposed as a starter-molecule for both the biosynthesis of product P8/1-OG and the biosynthesis of rifamycins (ansamycins).In order to prove this hypothesis, supplementation studies and cosynthesis experiments were carried out using P--strains1)of Nocardia mediterranei and the transketolase--mutant N. mediterranei A82). Experiments and ResultsSynthesis of 3-Amino-5-hydroxybenzoic Acid 3-Amino-5-hydroxybenzoic acid is not commercially available. The compound was synthesized starting from 3,5-dinitrobenzoic acid according to the following method (modified method based on BRAY et al.3) and BICKEL et al.4)). A solution of 100 g 3,5-dinitrobenzoic acid (0.472 moles) in 750 ml of methanol and a solution of 100g NaHS • H2O (1.35 moles) in 1,500 nil of 65%methanol were combined and the reaction mixture was stirred for 20 minutes; 300 ml of 4 N HCl were then added, the mixture was filtered and the filtrate evaporated to dryness under reduced pressure. The residue was dissolved in 1,000 ml of methanol and the insoluble NaCl was separated by filtration and the filtrate again evaporated to dryness. This operation was repeated with 500 ml of acetone and 90 g of 3-amino-5-nitrobenzoic acid-hydrochloride was obtained. The product was tested for purity by TLC on silica gel plates using solvent system 12). On the chromatogram (after air-drying) no 3,5-dinitrobenzoic acid (Rf 0.80, UV-254 nm), but only 3-amino-5-nitrobenzoic acid (Rf 0.85, orange) and traces of 3,5-diaminobenzoic acid (Rf 0.56, brown) were detected.3-Amino-5-nitrobenzoic acid -hydrochloride (90 g, 0.413 moles) were suspended in 800 ml of pyridine and 170 ml of acetic anhydride (~1.5 moles) were added in small portions under shaking.
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