Alfred Scheidegger

A Novel Class of Herbicides (Specific Inhibitors of Imidazoleglycerol Phosphate Dehydratase)

A new mode of herbicidal action was established by finding specific inhibitors of imidazoleglycerol phosphate dehydratase, an enzyme of histidine (His) biosynthesis. Three triazole phosphonates inhibited the reaction of the enzyme with Ki values of 40 [plus or minus] 6.5, 10 [plus or minus] 1.6, and 8.5 [plus or minus] 1.4 nM, respectively, and were highly cytotoxic to cultured plant cells. This effect was completely reversed by the addition of His, proving that the cytotoxicity was primarily caused by the inhibition of His biosynthesis. These inhibitors showed wide-spectrum, postemergent herbicidal activity at application rates ranging from 0.05 to 2 kg/ha.

Isolation and Characterization of cDNAs Encoding Imidazoleglycerolphosphate Dehydratase from Arabidopsis thaliana

cDNA clones encoding imidazoleglycerolphosphate dehydratase (IGPD; EC 4.2.1.19) from Arabidopsis thaliana were isolated by complementation of a bacterial auxotroph. The predicted primary translation product shared significant identity with the corresponding sequences from bacteria and fungi. As in yeast, the plant enzyme is monofunctional, lacking the histidinol phosphatase activity present in the Escherichia coli protein. IGPD mRNA was present in major organs at all developmental stages assayed. The Arabidopsis genome appears to contain two genes encoding this enzyme, based on DNA gel blot and polymerase chain reaction analysis.

Purification and characterization of histidinol dehydrogenase from cabbage

Histidinol dehydrogenase (EC 1.1.1.23) activity was determined in several plant species and in cultured plant cell lines. The enzyme was purified from cabbage (Brassica oleracea) to apparent homogeneity. To render complete purification, a new, specific histidinol-Sepharose 4B affinity chromatography was developed. The apparent molecular mass of the protein is 103 kDa. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein migrated as a single band with a molecular mass of 52 kDa, giving evidence for a dimeric quaternary structure. By isoelectric focusing, the enzyme was separated into six protein bands, five of which possessed the dehydrogenase activity when examined by an activity staining method. The Km values for L-histidinol and NAD+ were 15.5 and 42 microM, respectively. Enzyme activity was stimulated by addition of Mn2+, but was inhibited in the presence of Ba2+, Mg2+, Ni2+, Ca2+, Zn2+, or Cu2+. Histidinol dehydrogenase is the first histidine enzyme that has been purified to homogeneity and characterized from plants. This plant enzyme catalyzes the NAD-linked four-electron dehydrogenase reaction leading from histidinol to His. The results indicate a similar pathway of His in plants and show furthermore the last two reaction steps to be identical to those in microorganisms.

Purification and Properties of a Monofunctional Imidazoleglycerol-Phosphate Dehydratase from Wheat

Imidazoleglycerol-phosphate dehydratase (EC 4.2.1.19) activity was detected in extracts of several monocotyledonous and dicotyledonous plants using a newly developed assay method. The enzyme was purified 114,000-fold (to apparent homogeneity) from wheat germ by five chromatographic steps. Its native relative molecular weight (Mr) was determined to be 600,000 to 670,000, and it consists of identical subunits of Mr 25,500. In wheat germ, the dehydratase, unlike those of prokaryotic origin, is not associated with histidinol phosphatase activity. The reaction product was identified as imidazoleacetol phosphate (IAP) by comparing it with synthetic IAP as an authentic reference. The Km value for imidazoleglycerol phosphate was 0.36 mM at the optimal pH of 6.6. The enzyme required a reducing agent, such as 2-mercaptoethanol or dithiothreitol, and Mn2+ for maximal activity. 3-Amino-1,2,4-triazole competitively inhibited the activity with a Ki value of 46 [mu]M. The purification of imidazoleglycerol-phosphate dehydratase from wheat germ and histidinol dehydrogenase from cabbage (A. Nagai, A. Scheidegger [1991] Arch Biochem Biophys 284: 127-132) suggests that at least the second half of the histidine biosynthesis in plants is identical to that in microorganisms.

Overexpression of plant histidinol dehydrogenase using a baculovirus expression vector system

A cDNA encoding cabbage histidinol dehydrogenase, including the chloroplast transit peptide sequence, was overexpressed using a baculovirus expression vector system. The maximum level of the expression of histidinol dehydrogenase was reached 5 days after infection of the insect cells. Two forms of recombinant histidinol dehydrogenase with molecular masses of 53 and 52 kDa, respectively, were obtained by a one-step purification from the cell homogenate. Compared with the 52-kDa form, the 53-kDa form contained 10 additional amino acids at the N-terminus derived from the transit peptide. By incubating the cell homogenate for 2 h at 30 degrees C, the 53-kDa form could be completely converted to the 52-kDa form. This conversion was blocked by leupeptin. Eighty percent of the converted 52-kDa form had Cys at position 31 at the N-terminal amino acid and the rest had Met 33. Kinetic properties of the recombinant enzyme were virtually identical to those of histidinol dehydrogenase isolated from cabbage plants. The overexpression of recombinant cabbage histidinol dehydrogenase in insect cells, the proteolytic processing of the preprotein next to the N-terminus (compared to the mature cabbage enzyme), and its easy purification allow the preparation of large amounts of the active enzyme for structural and functional studies.

Investigation of acetyl-CoA: Deacetylcephalosporin C O-acetyltransferase of Cephalosporium acremonium

Abstract a new, rapid test system to measure the activity of acetyl-CoA : deacetylcephalosporin C O -acetyltransferase (DAC-acetyltransferase) was established. The reaction product cephalosporin C could be easily analyzed by HPLC. The DAC-acetyltransferase was partially purified by means of fractionated (NH 4 ) 2 SO 4 precipitation, Sephadex G-100 gel chromatography and isoelectric focusing. Molecular weight was determined to be 70 000 ± 5 000 and the p I 4.3 ± 0.2. Besides the two substrates acetyl-CoA and deacetylcephalosporin C, no other factor necessary for the reaction could be found. The enzyme was inhibited by coenzyme A (61%), deacetoxycephalosporin C (57%), penicillin N (14%), 2,6-dihydroxybenzoic acid (27%) and pyruvate (37%) at a concentration of 0.1 mM in each case.

Effect of glucose and oxygen on β-lactam biosynthesis by Cephalosporium acremonium

Abstract The effects of glucose consumption rate ( q s ) and oxygen limitation on the control of cephalosporin C (Ceph C) biosynthesis and the activities of deacetoxycephalosporin C synthetase/hydroxylase (DAOC-SH) and acetyl coenzyme A: deacetylcephalosporin C o -acetyltransferase (DAC-AT) were investigated in cultivations of the highly productive Cephalosporium acremonium strain TR87 under conditions similar to those used in industrial production. A carefully optimised time course of q s during the first part of fed batch cultivations was essential for maximal Ceph C production. The actual glucose concentration in the medium was of secondary importance. A decrease of q s between 20 and 35 h of cultivation was found to induce the early onset of antibiotic synthesis. By subsequently maintaining q s at a relatively low level using a controlled feed of glucose and a limiting amount of phosphate, maximal production rates were obtained. Oxygen starvation after the onset of Ceph C production led to a pronounced increase in penicillin N formation, a reduced Ceph C yield (−30%) and a strongly reduced activity of the two enzymes tested. In general, neither the time course nor the absolute levels of the two enzyme activities directly correlated with the actual production rates of Ceph C. This is the first time where an independent parameter ( q s ) has been demonstrated to be responsible for triggering the synthesis of an antibiotic.

Biotechnology in Japan--towards the year 2000 ... and beyond.

Abstract In the forthcoming period of paradigmatic change in technology, the ongoing development in information-related technologies will continue, while later, we will witness a full flowering of biotechnology, marking the beginning of serious approaches to the hitherto unexplored microcosm of life, an area in which a tremendous change affecting the future of Homo sapiens may well occur in the twenty-first century. If that happens, Japan will surely be one of the most powerful leaders of that change, and accordingly required to play a role worthy of its leadership. This role will be to aggressively tackle new challenges.

Biotechnology in Japan: A lesson in logistics? Part I: The political substrate

Japan, like Europe and the USA, has accepted the challenge of biotechnology, which opens up the way towards new fundamental knowledge and capabilities. Government and industry have joined forces to exploit biotechnology as a means of restructuring whole sectors of the countrys economy, to ensure their continued viability in the coming decades. The strategy the Japanese have adopted in their bid for world leadership in this area differs from those of the Western countries. The extraordinarily complex infrastructure created for this purpose and its functional efficiency are unparalleled and evince the innate force and ingenuity of Japanese competition.

AIDS in Japan: facts and figures

References 1 Weaver, W. (1938) Report of the Rockefeller Foundation 2 Weaver, W. (1970) Science 170, 581582 3 Astbury, W. T. (1961) Nature 190, 1124 40 lby , R. (1974) The Path to the Double Helix, Macmillan Press 5 Judson, H. F. (1979) TheEighth Dayof Creation, Jonathan Cape 6 Portugal, F. H. and Cohen, J. S. (1977) A Century ofDNA, MIT Press 7 Cairns, J., Stent, G. S. and Watson, J. D. (1966) Phage and the Origins of Molecular Biology, Cold Spring Harbor Laboratory Press 8 Lwoff, A. and Ullman, A. (1979) Origins of Molecular Biology: A Tribute to Jaques Monod, Academy Press 9 Srinivasan, P. R., Fruton, J. S. and Edsall, J. T., eds (1979) The Origins of Modern Biochemistry: A Retrospect on Proteins, Ann. NY Acad. Sci. 325, 1-375 10 Whitehouse, H. L. K. (1969) Towards an Understanding of the Mechanism of Heredity, Edward Arnold 11 Watson, J. D., Hopkins, N. H., Roberts, J. W., Steitz, J. A. and Weiner, A. M. (1987) Molecular Biology of the Gene, Benjamin/Cummings 12 Watson, J. D. (1968) The Double Helix, Athenaeum 13 McCarty, M. (1985) The Transforming Principle, W. W. Norton 14 Angler, N. (1988) Natural Obsessions: The Search for the Oncogene, Houghton Mifflin 15 Rogers, M. (1977) Biohazard, Alfred A. Knopf 16 Watson, J. D. and Tooze, J. (1981) The DNA Story: A Documentary History of Gene Cloning, W. H. Freeman