The enzymes involved in the synthesis of phytic acid in Lemna gibba (studies on the biosynthesis of cyclitols, XL.(1)).

The biosynthesis of phytic acid is known to be catalyzed by enzymes causing a stepwise phosphorylation of myo-inositol or 1L-myo-inositol 1-phosphate with adenosine triphosphate as phosphate donor. The kinase responsible for these phosphorylations in Lemna gibba were purified by affinity chromatography on a Sepharose gel carrying myo-inositol 2-phosphate at the binding site. Three fractions with enzymatic activity could be identified; in the first one, we find myo-inositol kinase (EC 2.7.1.64) phosphorylating myo-inositol to 1L-myo-inositol 1-phosphate; the second one brings about the phosphorylation of myo-inositol trisphosphate to phytic acid; the third one phosphorylates myo-inositol 1-phosphate to a myo-inositol trisphosphate. An enzyme oxidizing 1L-myo-inositol 1-phosphate to an uronic acid derivative is found in the first two fractions. In the presence of ATP, Mg2+, Mn2+, and the second and the third enzyme fractions in an appropraite mixture, 1L-myo-inositol 1-phosphate can be phosphorylated to phytic acid. The structure of the trisphosphate acting as an intermediate is not yet known.

Preparation of homogeneous crystals of myo-inositol 1-phosphate synthase from rat testicles--further data on the chemical and catalytic properties of the enzyme (studies on the biosynthesis cyclitols, XXXIX).

Pre-purified preparations of myoinositol-1-phosphate synthase (E.C. 5.5.1.4) from rat testes can be purified to homogeneity by first crystallizing the enzyme according to JAKOBY and then recrystallizing it at a pH value close to the isoelectric point while slowly increasing the temperature from 0 to 15 degrees C. This method gives a much yield of homogeneous enzyme than the previously used purification by affinity chromatography. It was further found that the pure enzyme contains close to 2 mol NAD+ per mol enzyme; it does not contain any metal. At substrate saturation the enzyme binds close to 1 mol substrate per mol enzyme, as determined by using radioactively labelled substrate and binding it to the enzyme by reduction with NaBH4. The reaction catalyzed by the enzyme is irreversible.

Myo-inositol-1-phosphate synthase from streptomyces griseus (studies on the biosynthesis of cyclitols, XXXVIII).

It could be shown that Streptomyces griseus, the microorganism producing the antibiotic streptomycin and also mutant strains of this species that cannot synthesize streptomycin, possess myo-inositol-1-phosphate synthase (EC 5.5.1.4), the enzyme cyclizing D-glucose 6-phosphate. The enzyme isolated from that organism is extremely instable, its molecular weight is approximately 260,000, and it requires a divalent metal ion for its activity. This is the first instance that an enzyme of this specificity has been found in a prokaryotic organism.

myo-Inositol oxygenase from rat kidneys. I: Purification by affinity chromatography; physical and catalytic properties.

Using the technique of affinity chromatography on a myo-inositol-substituted Sepharose, the myo-inositol oxygenase from rat kidneys was purified to homogeneity. The active enzyme contains iron, most probably in its divalent form. Electrophoresis on polyacrylamide gel containing sodium dodecylsulphate causes the cleavage of the enzyme protein into apparently identical subunits with a molecular weight of approximately 17,000. The smallest active unit consists of 4 subunits, and is in a pH-dependent equilibium with species consisting of 8, 12, and 16 subunits, respectively, which all show the same specific enzyme activity. In the presence of oxygen the enzyme is highly unstable; at the early stages of inactivation it can be reactivated by reducing agents like NaBH4. Under anaerobic conditions or under the influence of Fe2-chelating agents, the enzyme is also inactivated; this inactivation is caused by the loss of iron and concomitant cleavage into the subunits. It can be reversed by incubation with FeSO4 in the presence of air. If myo-inositol and FeSO4 are present, the reactivation involves an oligomerization to the species with 16 subunits with the uptake of 8 gram-atoms of iron per mole of this species. The enzyme reaction follows Michaelis-Menten kinetics; the Michaelis constants are 4.5 x 10(-2)M for myo-inositol and 9.5 x 10(-6)M for oxygen.

Studies on the biosynthesis of cyclitols, XXXVII. On mechanism and function of Schiff's base formation as an intermediary reaction step of myo-inositol-1-phosphate synthase from rat testicles.

It could be shown that the formation of a Schiff's base by myo-inositol-1-phosphate synthase of rat testicles occurs by binding the aldehyde group of the open form of its substrate, D-glucose 6-phosphate, to a lysyl residue of one or both smaller subunits of the enzyme. The participation of the Schiff's base formation in the catalytic process is supported by the observations that (a) no Schiff's base is formed if NAD is removed from the enzyme, and (b) in the presence of NAD, the dehydrogenation step involved in the catalytic mechanism apparently takes place rapidly after the formation of the Schiff's base.

Studies on the biosynthesis of cyclitols, XXXVI. Purification of myo-inositol-1-phosphate synthase of the duckweed, Lemna gibba, to homogeneity by affinity chromatography on NAD-Sepharose molecular and catalytic properties of the enzyme.

Using the technique of affinity chromatography on NAD-Sepharose the myo-inositol-1-phosphate synthase of Lemna gibba was purified to homogeneity. The molecular and catalytic properties of this enzyme differ very much from those of myo-inositol-1-phosphate synthase from animal sources. Thus the specific activity of the duckweed enzyme is more than two orders of magnitude higher than that of the enzyme from rat testes. It is inhibitied by EDTA and can be reactivated by Mn2. Its molecular weight (135000 +/- 5000), its subunit composition (3 subunits with identical electrophoretic behaviour) and its isoelectric point (pH 7.7) are also very different from the corresponding parameters for the animal enzyme.

Studies on the biosynthesis of cyclitols, XXXV. On the mechanism of action of myo-inositol-1-phosphate synthase from rat testicles.

The animal myo-inositol-1-phosphate synthase is competitively inhibited by pyridoxal phosphate and trinitrobenzensulphonate, both compounds known to prevent Schiff's base formation. When incubated with labelled substrate and then treated with NaBH4, label can be recovered in the enzyme protein. In analogous experiments with tritiated NaBH4 the enzyme protein also becomes labelled; after hydrolysis of such protein only one labelled compound, derived from lysine and D-glucose 6-phosphate, could be isolated. Its exact structure is not yet known. From these results it can be concluded that during its action myo-inositol-1-phosphate synthase forms a Schiff's base with its substrate, in analogy to the class I aldolases.

Studies on the biosynthesis of cyclitols, XXXIV[l] Purification of myo-inositol 3-methyltransferase from Pisum sativum and of myo-inositol 1-methyltransferase from Vinca minor to homogeneity by affinity chromatography.

With the help of affinity chromatography on an agarose gel containing epi-inositol as the group exhibiting affinity towards enzymes acting on myo-inositol, the two methyltransferases from higher plant materials transforming myo-inositol to D-bornesitol and L-bornesitol, respectively, were purified to homogeneity. The two enzymes show certain similarities as far as pH optima, isoelectric points and specific activities are concerned, but differ significantly in the molecular weight and in their affinity towards the methyl donor, S-adenosyl-L-methionine.

Myo-inositol oxygenase from oat seedlings.

Enzyme preparations from oat seedlings showing the activity of myo-inositol oxygenase (E.C.1.13.99.1) have been described previously. In contrast to myo-inositol oxygenase preparations from other sources, e.g. rat kidney or yeast, the oat enzyme seemed to exhibit a somewhat less stringent activity, acting on other inositols and inositol methyl ethers as well as on myo-inositol. By purification of the enzyme present in the extract from oat seedlings with the help of an affinity gel specific for enzymes acting on myo-inositol a homogeneous enzyme preparation was obtained, which shows the same strict specificity as the myo-inositol oxygenase from other sources. It has a molecular weight of 62,000 and tends to aggregate to oligomers (up to tetramers) under physiological pH-values; in more alkaline media dissociation to monomers is observed. The action on the other inositols and inositol methyl ethers is apparently due to one or more other enzymes, which are also adsorbed on the affinity gel, but can be separated from the myo-inositol oxygenase by elution with increasing concentrations of myo-inositol.