Thymine 7-hydroxylase from Neurospora crassa. Substrate specificity studies.

A partially purified preparation of thymine 7-hydroxylase (thymine, 2-oxoglutarate : oxygen oxidoreductase (7-hydroxylating), EC 1.14.11.6) from Neurospora crassa was incubated with a number of pyrimidines chemically related to tyymine. 1. Pyrimidines with oxygen or sulfur substituents on atoms Nos. 2 and 4 as well as an alkyl group on atom Nos. 1 or 5 were substrates. 2. Km values were determined for 1-methyluracil, 1-ethyluracil, thymine, 6-azathymine, 1-methylthymine, 1-ethylthymine, 5-formyluracil and 5-hydroxymethyluracil. 3. Uracil was identified as one of the metabolites after incubation with 1-methyluracil. The one-carbon metabolite has not been characterized. 4. Several pyrimidines with polar groups on atoms Nos. 2 and 4 were inhibitory. 5. Addition of 1-methyluracil, 1-methylthymine, 1-ethylthymine or 5-hydroxymethyluracil to incubations with thymine and 2-oxo[1-14C1]glutarate did not result in additional formation of 14CO2, indicating that the same enzyme acts on the different compounds. It has previously been found (Bankel, L., Holme, E., Lindstedt, G. and Lindstedt, S. (1972) FEBS Lett. 21, 135-138) that a mutant strain of N. crassa which is devoid of thymine 7-hydroxylase activity also lacks ability to perform the coupled oxygenation of 2-oxoglutarate and 1-methyluracil, 5-hydroxymethyluracil and 5-formyluracil, respectively. It is concluded that one and the same oxygenase is responsible for the activities studied.     

Uracil's uncoupling of the decarboxylation of alpha-ketoglutarate in the thymine 7-hydroxylase reaction of Neurospora crassa

Highly purified preparations of thymine 7-hydroxylase from Neurospora crassa catalyzed the decarboxylation of alpha-ketoglutarate but yielded no hydroxylated product when uracil was substituted for thymine in the standard incubation mixture. Although the uracil-dependent decarboxylation was much slower than the coupled reaction, both reactions were similar with respect to the requirement for molecular oxygen, the stoichiometric formation of succinate, and the stimulations effected by Fe2+, ascorbate, and catalase. That the same enzyme catalyzed both reactions was indicated by the parallel loss of the uracil- and thymine-dependent activities upon heat denaturation, their copurification, and the lower level of both activities in a mutant strain deficient in the 7-hydroxylase. These data are consonant with molecular oxygen initially attacking alpha-ketoglutarate in the thymine 7-hydroxylase reaction.     

A rapid and simple radioassay for thymine 7-hydroxylase

This work describes a simple and convenient procedure for measuring the activity of thymine 7-hydroxylase. The principle of the procedure depends upon the conversion of tritiated thymine by the enzyme to 5-hydroxymethyluracil. This reaction simultaneously invokes the loss of a tritium atom and the formation of tritiated water. The quantity of tritiated water formed is stoichiometrically proportional to the amount of 5-hydroxymethyluracil produced.The sensitivity of this procedure was markedly improved when both catalase and BSA were included in the reaction mixture.     

Calmodulin from neurospora crassa. General properties and conformational changes

Calmodulin from Neurospora crassa has been purified to electrophoretic homogeneity. Equilibrium gel filtration experiments suggest that its Ca-binding properties are indistinguishable from those of vertebrate calmodulins. The isoelectric point of 4.04 and electrophoretic behavior under nondenaturing conditions indicate that N. crassa calmodulin is slightly less acidic than its vertebrate counterpart. The amino acid composition is typical of plant calmodulins with the exception that trimethyllysine is absent and that the content of Ser is unusually high. The tryptic peptide map of N. crassa calmodulin reveals an important number of point mutations as compared to vertebrate calmodulin. Differences in primary structure may explain why N. crassa calmodulin is less potent in the activation of myosin light chain kinase than calmodulins from higher organisms. The far UV circular dichroic spectra of the Ca-, Mg-, and metal-free forms of N. crassa calmodulin are similar to those of vertebrate calmodulin; in contrast, the near UV circular dichroic spectra are very different, apparently due to the differences in Tyr content. The single Tyr residue of N. crassa calmodulin, presumably located in position 138, undergoes an inversion of optical chirality upon addition of Ca2+, but not of Mg2+, to the metal-free protein.     

Cryobiology of neurospora crassa. I. Freeze response of Neurospora crassa conidia

Neurospora crassa conidia were frozen and thawed in water suspensions at various rates and with different minimum temperatures. Colony counts of the experimental conidia were compared with those of controls, which were taken as 100% survival. The data revealed that (1) survivals were near 100% after fast thaw (400 °/min) regardless of the freeze rate, (2) percentage of survival was inversely related to freeze rate when combined with slow thaw, (3) slow thaw (0.5 °/min) was damaging, and (3) the rates of freeze-thaw affected the system only in the −5 to −20 ° interval. The damaging freeze conditions were those which favor ice crystal growth. It is suggested that rupture of the membrane by ice crystals seems to be the plausible mechanism of damage in freezing and thawing N. crassa conidia.     

Catalysis of three sequential dioxygenase reactions by thymine 7-hydroxylase

A purification scheme has been developed for an enzyme, thymine 7-hydroxylase, which appears to catalyze three sequential dioxygenase reactions, i.e., thymine → 5-hydroxymethyluracil → formyluracil → uracil-5-carboxylic acid. The enzyme was purified 1,300-fold from Neurospora crassa and had specific activities of approximately 1200, 600, and 250 U/mg for the respective reactions. Evidence that a single protein catalyzes the three reactions includes: the parallel purification of the three activities throughout the purification scheme, the inhibition of each reaction by the substrates of the other two, the inhibition of the three reactions by uracil, the parallel loss of the three activities upon heat denaturation, and considerations of a mechanism which suggest that a single active site may be involved.     

Separation and characterization of pyrimidine deoxyribonucleoside 2'-hydroxylase and thymine 7-hydroxylase from Aspergillus nidulans

Partially purified preparations from Aspergillus nidulans were shown to catalyze two alpha-ketoglutarate dependent dioxygenase reactions: the pyrimidine deoxyribonucleoside 2'-hydroxylase (EC 1.14.11.3) and the thymine 7-hydroxylase (EC 1.14.11.6) reactions. These reactions showed an absolute requirement for alpha-ketoglutarate and molecular oxygen and were stimulated by Fe(II), ascorbate and catalase. Both reactions demonstrated a stoichiometry such that for each mole of substrate (deoxyribonucleoside or pyrimidine) hydroxylated one mole of CO2 was produced from alpha-ketoglutarate. These two activities were separated using DEAE-Sephacel chromatography.     

Some kinetic properties of the tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Neurospora crassa.

The steady-state kinetic properties of purified tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Neurospora crassa were examined. The results suggest that the enzyme obeys a Rapid-Equilibrium Ordered mechanism, in which phosphoenolpyruvate is the first substrate to bind and 3-deoxy-D-arabino-heptulosonate 7-phosphate is the second product to be released, rather than a Ping Pong mechanism as has been reported previously. The inhibition by tryptophan was found to be parabolic competitive with respect to D-erythrose 4-phosphate and parabolic non-competitive with respect to phosphoenolpyruvate. The enzyme was inactivated by EDTA, and could be protected against this inactivation by phosphoenolpyruvate or 3-deoxy-D-arabino-heptulosonate 7-phosphate but not by D-erythrose 4-phosphate, tryptophan or Pi. This suggests that the enzyme may be a metalloenzyme.     

Substitution of nucleoside triphosphates for ascorbate in the thymine 7-hydroxylase reaction of Rhodotorula glutinis

Nucleoside di- and triphosphates substituted for ascorbate in the thymine 7-hydroxylase reaction in studies carried out with purified preparations from Rhodotorula glutinis. The stimulations brought about by ascorbate and ATP were found not to be additive. Studies with analogues of ATP indicated that hydrolysis may not need to occur in order for the nucleotide effect to be expressed. The stoichiometry of the production of 5-hydroxymethyluracil and CO2 was not changed by the substitution of ATP for ascorbate. The 7-hydroxylase was found to have considerable thermal stability, and inactivation at 98 degrees C resulted in a parallel loss of the activities effected by ascorbate and ATP. This and the retention of the nucleotide effect upon purification suggest the effect is not mediated through another protein co-purified with the 7-hydroxylase.     

Determination of intra- and intermolecular tritium isotope effects in the reaction of thymine 7-hydroxylase

Different [7-3H]thymine preparations have been used to determine the inter- and intramolecular isotope effects of the 2-oxoglutarate-dependent thymine hydroxylation, catalyzed by thymine 7-hydroxylase (thymine, 2-oxoglutarate:oxygen oxidoreductase, EC 1.14.11.6). Specific activity ratios of products, viz., 3H2O and 5-hydroxymethyluracil, and remaining substrate to initial substrate have been determined. The influence on these ratios of intra- and intermolecular isotope effects at different degrees of tritium substitution has been analyzed. An intramolecular isotope effect with a kH/kT of about 6.5 has been found. No intermolecular isotope effect of TV/K could be detected when oxygen concentration was varied from 0.4 to 0.01 mM. This agrees with a mechanism in which 2-oxoglutarate is irreversibly changed before the bond-breaking in thymine takes place.     

Studies on the partial reaction of thymine 7-hydroxylase in the presence of 5-fluorouracil

The uncoupling of 2-oxoglutarate decarboxylation from hydroxylation in the reaction catalyzed by thymine 7-hydroxylase (thymine, 2-oxoglutarate:oxygen oxidoreductase (7-hydroxylating), EC 1.14.11.6) in the presence of 5-fluorouracil has been studied. In the complete reaction no external reductant is formally needed. The uncoupled reaction is almost negligible in the absence of ascorbate and the optimal ascorbate concentration is 5-times higher than in the presence of a hydroxylatable substrate. This indicates that ascorbate acts as the external reductant that is formally needed in the catalytic cycle. The complete reaction follows the steady-state kinetics of an ordered ter reactant mechanism where 2-oxoglutarate and thymine have to be bound to the enzyme before oxygen (E. Holme (1975) Biochemistry 14, 4999-5003). The uncoupled reaction follows the same kinetic pattern as the complete reaction, and in accordance with this no decarboxylation of 2-oxoglutarate occurs in the absence of a substrate analogue even at elevated oxygen tension. There is a good agreement between Kia values for 2-oxoglutarate of the two reactions, but there is at least a 6-fold increase in KO2 where a minimum value of 25% O2 in the gas phase was found for the partial reaction. The high KO2 found means that the reaction rate could increase considerably at elevated oxygen tension.     

Dissociation of mitochondrial ribosomes of neurospora crassa by a bacterial dissociation factor

From ribosomes of Escherichia coli a protein factor can be obtained that promotes dissociation of bacterial ribosomes into subunits. Incubation of mitochondrial ribosomes from Neurospora crassa with the bacterial dissociation factor also leads to the formation of subunits. Under the same conditions no dissociation of cytoplasmic ribosomes from Neurospora crassa was observed.     

Markedly different ascorbate dependencies of the sequential alpha-ketoglutarate dioxygenase reactions catalyzed by an essentially homogeneous thymine 7-hydroxylase from Rhodotorula glutinis

The alpha-ketoglutarate dioxygenase, thymine 7-hydroxylase (EC 1.14.11.6), has been purified from cultures of Rhodotorula glutinis grown with thymine as a nitrogen source. The purification scheme developed yielded essentially homogeneous preparations of the 7-hydroxylase and also purified another alpha-ketoglutarate dioxygenase, pyrimidine deoxyribonucleoside 2'-hydroxylase (EC 1.14.11.3). The purity of the 7-hydroxylase was determined with analytical disc gel electrophoresis in which runs were varied with respect to pH, extent of cross-linking, and the presence of sodium dodecyl sulfate-mercaptoethanol. The 7-hydroxylase apparently exists as a monomer since its molecular weight was 42,700 when determined by molecular gel filtration chromatography and was 40,300 when determined by analytical disc gel electrophoresis under denaturing conditions. Gel filtration chromatography under nondenaturing conditions was used to show that the 2'-hydroxylase has a molecular weight of 64,600. The essentially homogeneous preparations of the 7-hydroxylase were shown to catalyze the thymine-, 5-hydroxymethyluracil-, and 5-formyluracil-dependent oxygenations that are coupled to the decarboxylation of alpha-ketoglutarate, as well as a putative uncoupled decarboxylation which is dependent on uracil. Furthermore, these enzyme preparations were used to show that ATP stimulated the 7-hydroxylase reaction in the absence of ascorbate. Even though it is attractive to consider the four pyrimidine-dependent reactions as being catalyzed by the same active site, they were shown to differ markedly in their dependencies on ascorbate or ATP. The effects of ascorbate and ATP on these reactions, and on the 2'-hydroxylase reaction, are discussed in terms of the possible roles of ascorbate and ATP.