F. Gibson

THE BIOSYNTHESIS OF PHENYLALANINE AND TYROSINE; ENZYMES CONVERTING CHORISMIC ACID INTO PREPHENIC ACID AND THEIR RELATIONSHIPS TO PREPHENATE DEHYDRATASE AND PREPHENATE DEHYDROGENASE.

Abstract Cell-free extracts of Aerobacter aerogenes and Escherichia coli were chromatographed on DEAE-cellulose and the eluates examined for some of the enzymes concerned in the conversion of chorismic acid into phenylalanine and tyrosine. Enzymes which are able to convert chorismate into prephenate (chorismate mutase) are eluted in two peaks of activity. The effects of phenylalanine and tyrosine on enzyme levels and activity showed that one of these enzymes (chorismate mutase P) is probably related to phenylalanine biosynthesis and the other (chorismate mutase T) to tyrosine biosynthesis. Chorismate mutase T travelled on DEAE-cellulose with prephenate dehydrogenase, (prephenate; NAD oxidoreductase (decarboxylating) the subsequent enzymic activity on the tyrosine biosynthetic pathway. The two activities were also affected simultaneously by mutation. Chorismate mutase P travelled on columns with the next enzymic activity on the pathway of phenylalanine biosynthesis, prephenate dehydratase, (prephenate hydro-lyase (decarboxylating)) and both activities were affected following mutation. In extracts of the A. aerogenes strain examined, but not in E. coli extracts, a second peak of prephenate dehydratase activity was eluted in the early column fractions. This activity, unlike the prephenate dehydratase travelling with chorismate mutase P is not inhibited by phenylalanine. It is suggested that, in the biosynthesis of phenylalanine and tyrosine, there are two enzymes or enzyme complexes metabolising chorismate, one leading through prephenate to phenylpyruvate and the other leading through prephenate to 4-hydroxyphenylpyruvate.

Regions of variant histone His2AvD required for Drosophila development

One way in which a distinct chromosomal domain could be established to carry out a specialized function is by the localized incorporation of specific histone variants into nucleosomes. H2AZ, one such variant of the histone protein H2A, is required for the survival of Drosophila melanogaster, Tetrahymena thermophila and mice (R. Faast et al., in preparation). To search for the unique features of Drosophila H2AZ (His2AvD, also referred to as H2AvD) that are required for its essential function, we have performed amino-acid swap experiments in which residues unique to Drosophila His2AvD were replaced with equivalently positioned Drosophila H2A.1 residues. Mutated His2AvD genes encoding modified versions of this histone were transformed into Drosophila and tested for their ability to rescue null-mutant lethality. We show that the unique feature of His2AvD does not reside in its histone fold but in its carboxy-terminal domain. This C-terminal region maps to a short α-helix in H2A that is buried deep inside the nucleosome core.

Phenolic compounds accumulated by washed cell suspensions of a tryptophan auxotroph of Aerobacter aerogenes.

Abstract Washed cell suspensions of Aerobacter aerogenes NC 3 , an auxotroph growing with trypophan, indole or anthranilic acid, accumulate a number of phenolic compounds when incubated in phosphate buffer with glucose, ammonium and magnesium ions. 2,3-Dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, catechol and 4-hydroxybenzoic acid have been identified after 6 h incubation. After longer incubation (20 h) 3,4-dihydroxybenzoic is not detected. An as yet unidentified compound with certain characteristics of a phenol and properties suggesting the presence of o -dihydroxy, amino and carboxylic acid grouping in the molecule has also been detected.

The source of the nitrogen atom for the biosynthesis of anthranilic acid

Abstract Chorismic acid was converted into anthranilic acid using cell-free extracts of Aerobacter aerogenes and l -[amide-15N]glutamine as nitrogen source. Mass spectroscopy of the anthranilic acid showed that the amide-nitrogen of glutamine was incorporated. NH4+ ions were also found effective as a nitrogen source. Anthranilic acid synthesis with NH4+, unlike that with glutamine, was not inhibited by 6-diazo-5-oxo- l -nor-leucine. End-product inhibition of the conversion of chorismic acid into anthranilic acid by l -tryptophan was effective with either nitrogen source.

Methionine as the source of methyl groups for ubiquinone and vitamin K: A study using nuclear magnetic resonance and mass spectrometry

Abstract 1. 1. [ Me - 2 H]Methione was used as a tracer to determine the origin of the methyl groups in vitamin K 2 (MK-8) and ubiquinone (Q-8) in Escherichia coli . 2. 2. The methyl group of methionine is the source of the ring-methyl group of vitamin K 2 and the C -methyl and O -methyl groups of the ring of ubiquinone. The 2 H was not incorporated elsewhere in the quinones. 3. 3. The use of the techniques of nuclear magnetic resonance (NMR) and mass spectrometry to give easily interpretable results on small amounts of material without the need for chemical degradation, is described.

A possible relationship between the formation of o-dihydric phenols and tryptophan biosynthesis byAerobacter aerogenes

Abstract A number of auxotrophs ofAerobacter aerogenes requiring aromatic amino acids for growth have been shown to excrete o-dihydric phenols when incubated with a mixture of glucose, phosphate, magnesium and ammonium ions. Auxotrophs requiring histidine, methionine, cysteine or leucine do not excrete these substances under similar conditions. An auxotroph requiring tyrosine, phenylalanine and tryptophan for growth but unable to grow on shikimic acid accumulates a mixture of 3,4-dihydroxy-benzoic acid and catechol. A tryptophan auxotroph able to utilise anthranilic acid for growth accumulates 2,3-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid and catechol. A tyrosine auxotroph able to utilise 4-hydroxyphenylpyruvic acid has been shown to accumulate 2,3-dihydroxybenzoic acid and catechol. Catechol may arise consequent on 3,4-dihydroxybenzoic acid accumulation since suspensions of wild typeA. aerogenes form catechol from 3,4-dihydroxybenzoic acid. Such suspensions also metabolise 3,4-dihydroxybenzoic acid and catechol to compounds not possessing the o-dihydric phenol structure when aerated in the absence of glucose. l -Tryptophan inhibits the accumulation of o-dihydric phenols by three different tryptophan auxotrophs ofA. aerogenes but has no effect on the accumulation of these compounds by a tyrosine auxotroph or an auxotroph requiring tyrosine, phenylalanine and tryptophan for growth. The possible relationship of these o-dihydric phenols to the biosynthesis of aromatic compounds is discussed.

The formation of 4-hydroxyphenylpyruvic acid and phenylpyruvic acid by tryptophan auxotrophs and wild-type Aerobacter aerogenes considered in relation to the general aromatic pathway

Abstract 4 -Hydroxyphenylpyruvic acid and phenylpyruvic acid are accumulated by cell suspensions of tryptophan auxotrophs and a wild-type strain of Aerobacter aerogenes when these are transferred from conditions of adequate nitrogen to conditions of nitrogen starvation. A mutant strain which requires the full supplement of five aromatic compounds does not form hydroxyphenylpyruvic acid or phenylpyruvic acid. The compounds 4 -hydroxybenzaldehyde and 3,4 -dihydroxybenzoic acid may also be detected in supernatants after the cells are incubated with substrates lacking nitrogen. The effects of nitrogen starvation are discussed in relation to the critical specific steps of tryptophan biosynthesis and the problem of the branch point in aromatic biosynthesis. It is suggested that nitrogen is introduced in tryptophan biosynthesis at the first step specific to this pathway.

The use of a ubiquinone-deficient mutant in the study of malate oxidation in Escherichia coli

Abstract 1. Malate oxidation catalyzed by sub-cellular fractions of a normal strain of Escherichia coli and a mutant strain unable to form ubiquinone has been compared. 2. The system catalyzing the aerobic oxidation of malate was localized in a membranous small particle fraction separated by (NH4)2SO4 fractionation following disruption of the cells in a French pressure cell. 3. Comparison of malate oxidation catalyzed by particles from normal and mutant cells indicates that ubiquinone is concerned in malate oxidation. Malate oxidation proceeds at about half the normal rate in particles from cells lacking ubiquinone. 4. Malate oxidation catalyzed by small particles from cells lacking ubiquinone was insensitive to the low concentrations of dicoumarol which inhibited oxidation catalyzed by particles from normal cells. Malate oxidation catalyzed by particles from cells lacking vitamin K was even more sensitive to dicoumarol than that catalyzed by particles from normal cells. Therefore dicoumarol at low concentrations is not acting as a vitamin K antagonist.

A mutational alteration of the tryptophan synthetase of Escherichia coli.

SUMMARY: A tryptophan auxotroph of Escherichia coli produced an altered tryptophan synthetase which could not convert indole to tryptophan but converted indole-3-glycerol phosphate to indole. As distinct from the normal tryptophan synthetase, which also catalysed this reaction, both pyridoxal phosphate and serine stimulated the activity of the mutant enzyme system. Fractionation and chromatography of the mutant tryptophan synthetase separated it into its two protein components, A and B. Examinations of the separated components showed that the A protein was normal, while the B protein was altered. Studies of the effect of serine on the pH-activity response of mutant preparations in the indole-3-glycerol phosphate → indole reaction demonstrated that different pH-activity responses were obtained, respectively, in the presence and absence of serine. The curves obtained were characteristic of the serine-requiring and serine-non-requiring reactions, respectively, of normal tryptophan synthetase. The saturation curves of the mutant component B by normal component A, with and without serine added, suggest that one role of serine and pyridoxal phosphate in the stimulation of the indole-3-glycerol phosphate → indole reaction is to bind together the A and B proteins in a catalytically effective complex.