Biosynthesis of ketomycin.

The antibiotic ketomycin is formed from shikimic acid via chorismic acid and prephenic acid. Phenylalanine and 2',5'-dihydrophenylalanine are not intermediates in the biosynthesis. Degradation of ketomycin derived from [1,6-14C]shikimic acid showed that prephenic acid is converted into ketomycin with stereospecific discrimination between the two enantiotopic edges of the ring, the pro-S-R edge giving rise to the C-2', C-3' side of the cyclohexene ring of ketomycin.

Application of high-performance liquid chromatography to the determination of glyoxylate synthesis in chick embryo liver.

The isolation and identification of three major alpha-keto end products (glyoxylate, pyruvate, alpha-ketoglutarate) of the isocitrate lyase reaction in 18-day chick embryo liver have been described. This was accomplished by the separation of these alpha-keto acids as their 2,4-dinitrophenylhydrazones (DNPHs) by high-performance liquid chromatography (HPLC). The DNPHs of alpha-keto acids were eluted with an isocratic solvent system of methanol-water-acetic acid (60:38.5:1.5) containing 5 mM tetrabutylammonium phosphate from a reversed-phase ultrasphere C18 (IP) and from a radial compression C18 column. The separation can be completed on the radial compression column within 15-20 min as compared to 30-40 min with a conventional reversed-phase column. Retention times and peak areas were integrated for both the assay samples and reference compounds. A relative measure of alpha-keto acid in the peak was calculated by comparison with the standard. The identification of each peak was done on the basis of retention time matching, co-chromatography with authentic compounds, and stopped flow UV-VIS scanning between 240 and 440 nm. Glyoxylate represented 5% of the total product of the isocitrate lyase reaction. Day 18 parallels the peak period of embryonic hepatic glycogenesis which occurs at a time when the original egg glucose reserve has been depleted.

In-vivo studies on C2 organic acids in the tissues of rats injected with xylitol and glucose.

Oxalic, glyoxylic, and glycollic acid were determined in rat liver and kidney after injection with [U-14C]-xylitol or [U-14C]-glucose. Neither glucose nor xylitol led to the formation of oxalic and glyoxylic acid, yet glycollic acid was found in both tissues after injection with xylitol. Possible pathways leading from xylitol to glycollic acid are discussed.

Enzymes of glycollate formation and oxidation in two members of the rhodospirillacae (purple non-sulphur bacteria).

1. Phototrophic cultures of Rhodomicrobium vanielii do not excrete glycollate when gassed anaerobically with nitrogen plus carbon dioxide, although the addition of alpha-hydroxy-2-pyridine methanesulphonate (HPMS) results in the excretion of a trace amount of glycollate. The inclucion of low amounts of oxygen in this gas mixture results in marked glycollate excretion, higher rates occurring in the presence of HPMS. 2. Cell extracts of Rhodomicrobium vannielii, and also of Rhodospirillum rubrum, which excretes glycollate only under aerobic conditions in the light, catalyze the formation of glycollate from phosphoglycollate and also the oxidation of glycollate to glyoxylate.

Phenotypic suppression of phosphofructokinase mutations in Escherichia coli by constitutive expression of the glyoxylate shunt.

Fructose-6-phosphate kinase (pfkA) mutants have impaired growth on carbon sources which enter glycolysis at or above the level of fructose-6-phosphate, but the degree of impairment depends on the carbon source (e.g., growth on glucose is very much slower than growth on glucose-6-phosphate). The present report contains considerable data on this complicated growth phenotype and derives mainly from the finding of a class of partial revertants which grow as fast on glucose as on glucose-6-phosphate; the reversion mutation is shown to be constitutivity of the glyoxylate shunt (iclR(c)). iclR(c) does not increase the fructose-6-phosphate kinase level in the mutants, and the exact mechanism of the partial phenotypic suppression is not understood. However, iclR(c) was already known to suppress some mutations which affected phosphoenolpyruvate levels, and H. L. Kornberg and J. Smith have suggested (1970) that the growth phenotype of pfkA mutants might be related to pathways of phosphoenolpyruvate formation. Surprisingly, the hexose-monophosphate shunt is not necessary for the suppression, which therefore must act to restore metabolism via the residual phosphofructokinase activity present in all pfkA mutants. A mutant totally lacking phosphofructokinase activity was not suppressed.

Cytochemical localization of malate synthase in glyoxysomes.

Cytochemical staining techniques for microbodies (peroxisomes) are limited at present to the enzymes catalase and alpha-hydroxy acid oxidase, and neither technique can distinguish glyoxysomes from other microbodies. Described here is a procedure using ferricyanide for the cytochemical demonstration by light and electron microscopy of malate synthase activity in glyoxysomes of cotyledons from fat-storing cucumber and sunflower seedlings. Malate synthase, a key enzyme of the glyoxylate cycle, catalyzes the condensation of acetyl CoA with glyoxylate to form malate and release free coenzyme A. Localization of the enzyme activity is based on the reduction by free CoA of ferricyanide to ferrocyanide, and the visualization of the latter as an insoluble, electron-opaque deposit of copper ferrocyanide (Hatchett's brown). The conditions and optimal concentrations for the cytochemical reaction mixture were determined in preliminary studies using a colorimetric assay developed to measure disappearance of ferricyanide at 420 nm. Ultrastructural observation of treated tissue reveals electron-opaque material deposited uniformly throughout the matrix portion of the glyoxysomes, with little background deposition elsewhere in the cell. The reaction product is easily visualized in plastic sections by phase microscopy without poststaining. Although the method has been applied thus far only to cotyledons of fat-storing seedlings, it is anticipated that the technique will be useful in localizing and studying glyoxylate cycle activity in a variety of tissues from both plants and animals.

Accumulation of methylglyoxal in a mutant of Escherichia coli constitutive for gluconate catabolism.

A culture of a mutant of Escherichia coli, derepressed for gluconate catabolism, is killed by the addition of gluconate to the culture. The product responsible for this bactericidal effect was identified as methylglyoxal. Two types of mutants resistant to gluconate were isolated. One of them showed increased activity of glyoxalase I.