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R Welle

Publications and source records attributed to R Welle.

7 recordsLinked to original sources
PubMed1993

Isoprenoid biosynthesis in bacteria: two different pathways?

The biosynthesis of isopentenylpyrophosphate, a central intermediate of isoprenoid formation, was investigated in six different bacterial organisms. Cell-free extracts of Myxococcus fulvus, Staphylococcus carnosus, Lactobacillus plantarum and Halobacterium cutirubrum converted [14C]acetyl-CoA or [14C]hydroxymethylglutaryl-CoA to [14C]mevalonic acid. Furthermore, [14C]mevalonic acid, [14C]mevalonate-5-phosphate and [14C]mevalonate-5-pyrophosphate were metabolized to [14C]isopentenylpyrophosphate. These data demonstrated the in vitro operation of acetoacetate pathway for the formation of isopentenylpyrophosphate in bacteria. In contrast, no intermediates of this reaction sequence could be detected using cell-free extracts of Zymomonas mobilis and Escherichia coli. These results indicate that at least two different pathways for the biosynthesis of isopentenylpyrophosphate are present in bacteria.

Acetyl Coenzyme A↗
PubMed1992

Expression cloning in Escherichia coli and preparative isolation of the reductase coacting with chalcone synthase during the key step in the biosynthesis of soybean phytoalexins.

The cDNA for the reductase involved in the biosynthesis of 6'-deoxychalcone (4,2',4'-trihydroxychalcone), the first specific intermediate in the pathway to soybean phytoalexins, was cloned into the expression vector pKK233-2 and transformed into Escherichia coli. Using this source, about 5 mg of homogeneous reductase was isolated from 45 g of cells. The protein purification protocol differs completely from the scheme applied to soybean cell cultures. Size, N-terminal and specific enzyme activities were identical for the plant and E. coli protein. The pure protein is fairly stable, retaining 70% of initial activity after storage at 5 degrees C during 4 weeks. This protein is used for crystallization and in the study of its protein-protein interaction with chalcone synthase.

Acyltransferases↗
PubMed1991

Induced plant responses to pathogen attack. Analysis and heterologous expression of the key enzyme in the biosynthesis of phytoalexins in soybean (Glycine max L. Merr. cv. Harosoy 63).

In soybean (Glycine max L.), pathogen attack induces the formation of glyceollin-type phytoalexins. The biosynthetic key enzyme is a reductase which synthesizes 4,2', 4'-trihydroxychalcone in co-action with chalcone synthase. Screening of a soybean cDNA library from elicitor-induced RNA in lambda gt11 yielded two classes of reductase-specific clones. The deduced proteins match to 100% and 95%, respectively, with 229 amino acids sequenced in the purified plant protein. Four clones of class A were expressed in Escherichia coli, and the proteins were tested for enzyme activity in extracts supplemented with chalcone synthase. All were active in 4,2',4'-trihydroxychalcone formation, and the quantification showed that shorter lengths of the cDNAs at the 5' end correlated with progressively decreasing enzyme activities. Genomic blots with DNA from plants capable of 4,2',4'-trihydroxychalcone synthesis revealed related sequences in bean (Phaseolus vulgaris L.) and peanut (Arachis hypogaea L.), but not in pea (Pisum sativum L.). No hybridization was observed with parsley (Petroselinum crispum) and carrot (Daucus carota) which synthesize other phytoalexins. The reductase protein contains a leucine-zipper motif and reveals a marked similarity with other oxidoreductases most of which are involved in carbohydrate metabolism.

Acyltransferases↗
PubMed1989

Phytoalexin synthesis in soybean cells: elicitor induction of reductase involved in biosynthesis of 6'-deoxychalcone.

Chromatofocusing on Mono P proved to be an efficient purification procedure for the NADPH-dependent reductase from soybean (Glycine max L.) cell cultures which acts together with chalcone synthase in the biosynthesis of 2',4',4-trihydroxychalcone (6'-deoxychalcone). By isoelectric focusing the pI of reductase was determined to be 6.3. Addition of pure soybean reductase to cell-free extracts from stimulated cell cultures of parsley and bean (Phaseolus vulgaris) and from young flowers of Dahlia variabilis caused in each case synthesis of 6'-deoxychalcone. When 4-coumaroyl-CoA was replaced by caffeoyl-CoA in the reductase assay, formation of 2',4',3,4-tetrahydrochalcone (butein) was observed. A polyclonal antireductase antiserum was raised in rabbits and proved to be specific in Ouchterlony diffusion experiments, Western blots and immunotitration. The reductase antiserum showed no cross-reactivity with soybean chalcone synthase (CHS). A biotin/[125I]streptavidin system provided a quantitative Western blot for the reductase. Changes in the activities, amounts of protein, and mRNA activities of reductase and CHS were determined after challenge of soybean cell cultures by elicitor (from Phytophthora megasperma f.sp. glycinea or yeast). For both enzymes a pronounced and parallel increase in activity and amounts of protein was observed after elicitor addition with a maximum at about 16 h after challenge. Parallel increases in mRNA activities occurred earlier. The results indicate a parallel induction of de novo synthesis of reductase and CHS which coact in synthesis of 6'-deoxychalcone.

Acyltransferases↗
PubMed1988

Induction of phytoalexin synthesis in soybean: enzymatic cyclization of prenylated pterocarpans to glyceollin isomers.

A microsome preparation from elicitor-challenged soybean cell suspension cultures catalyzed an NADPH-dependent and oxygen-dependent cyclization of a mixture of 2- and 4-dimethylallylglycinols to the glyceollin isomers I-III. This is the last committed step in glyceollin biosynthesis. The cyclase was inhibited in a light-reversible manner by carbon monoxide in the presence of oxygen. Cyclase was also inhibited by cytochrome c, NADP+, and a number of inhibitors of cytochrome P-450 enzymes. NADH in the presence of low concentrations of NADPH had a synergistic effect. On a Percoll gradient, the position of cyclase coincided with marker enzymes for the endoplasmic reticulum. These properties identify the cyclase as a cytochrome P-450-dependent monooxygenase. Unstimulated soybean cell culture did not contain detectable cyclase activity. Challenge with either a glucan elicitor from Phytophthora megasperma f.sp. glycinea or with yeast extract caused strong stimulation of cyclase activity with a maximum at about 24 h after elicitor addition.

Benzopyrans↗