Walter G. Niehaus

A proposed role of superoxide anion as a biological nucleophile in the deesterification of phospholipids

Abstract Potassium superoxide dissolved in dry dimethyl sulfoxide effects rapid deesterification of ethyl hexadecanoate and of dilauroyl phosphatidyl choline. The reaction with ethyl hexadecanoate is reversible, having an apparent equilibrium constant of 0.4. It is proposed that some of the deleterious effects on biological membranes which have been atributed to oxidation by superoxide may actually be the result of deesterification by superoxide acting as a nucleophile.

Purification and kinetic characterization of mannitol-1-phosphate dehydrogenase from Aspergillus niger☆

Abstract Mannitol-1-phosphate dehydrogenase from Aspergillus niger catalyzes the NAD-dependent oxidation of mannitol-1-phosphate to fructose-6-phosphate. The enzyme, purified 800-fold to homogeneity has a molecular weight of 40,000 and appears to contain two subunits of equal molecular weight. The enzyme is highly specific for the substrates mannitol-1-phosphate, fructose-6-phosphate, NAD, and NADH. The kinetic mechanism is random Bi-Bi with two dead-end complexes. From the variation of kinetic parameters with pH it is concluded that the enzyme contains one amino acid residue with pKa 9–10 which is involved in binding of fructose-6-phosphate, and another amino acid residue with pKa about 8.2 which is involved in catalysis.

Regulation of Zn2+ uptake and versicolorin A synthesis in a mutant strain ofAspergillus parasiticus

Abstract Supplementation of cultures ofAspergillus parasiticus with Zn2+ stimulates the synthesis of versicolorin A only if the supplemental Zn2+ is present between 20 and 30 h postinoculation, during early vegetative growth. Cultures which are grown with minimal Zn2+ avidly internalize Zn2+ which is added late in the growth phase or in early stationary phase. A 15-min exposure to 10 μM Zn2+ during the responsive period, 20–30 h postinoculation, prevents this later uptake of Zn2+. The Zn2+ content of the mycelia at the end of the responsive period, 30 h postinoculation, in some way determines subsequent metabolism of the organism. Versicolorin synthesis, which begins about 50 h postinoculation, is directly proportional to the Zn2+ content of the mycelia at 30 h. The uptake of Zn2+, measured late in the growth phase or in early stationary phase, is inversely proportional to the Zn2+ content of the mycelia at 30 h.

Effect of zinc on versicolorin production by a mutant strain ofAspergillus parasiticus

A rapid method for quantification of versicolorin, based upon the red shift in the visible absorbance spectrum upon ionization of the phenolic hydroxyl groups, has been developed. The production of versicolorin requires zinc concentrations in excess of those required for growth. Maximal growth occurs with 2 μM zinc; maximal versicolorin production requires 5 μM zinc in the medium. Maximum production of versicolorin occurs after cessation of rapid growth, but does not absolutely depend upon exhaustion of the carbon source.

Effect of nitrogen supply, Zn2+, and salt concentration on kojic acid and versicolorin biosynthesis byAspergillus parasiticus

Abstract We have compared the effects of variation of culture conditions on the biosynthesis of kojic acid and versicolorin A by Aspergillus parasiticus (ATCC 36537). Under permissive conditions, both of these secondary metabolites appear in the culture between 40 and 50 h after conidial inoculation. Kojic acid synthesis is maximal with 0.2% peptone and is depressed by additional peptone or ammonia, with 1% peptone or 50 m M NH 4 + almost completely eliminating synthesis. These concentrations of nitrogen source cause a much smaller depression of versicolorin synthesis. Versicolorin production is completely dependent on Zn 2+ whereas kojic acid synthesis is unaffected by changes in the concentration of Zn 2+ or Fe 3+ . Certain salts inhibit production of both kojic acid and versicolorin, with nearly complete inhibition by 0.8 M NaCl or by 0.05 M NaOAc.

Mannitol metabolism in the mycorrhizal fungus,Piloderma croceum

Abstract NADP-specific mannitol dehydrogenase, mannitol-1-phosphatase, and hexokinase were detected in, and partly purified from mycelia of, the mycorrhizal fungus, Piloderma croceum . Mannitol-1-phosphate dehydrogenase activity was low or absent under the growth conditions used. Substrate specificity for mannitol-1-phosphatase was limited to mannitol 1-phosphate, with an activity of ca. 20% or less with the other sugar phosphates tested. The K m (fructose) for mannitol dehydrogenase and hexokinase favored the forward reaction of mannitol ⇄ fructose ⇄ fructose 6-phosphate with net transformation of mannitol to fructose 6-phosphate. Zinc ion was a competitive inhibitor for mannitol dehydrogenase with respect to mannitol, with a K i of 5.5 μ M . The properties of mannitol-1-phosphatase, mannitol dehydrogenase, and hexokinase suggest that mannitol dehydrogenase is used for mannitol utilization and the evidence taken together suggests that mannitol is synthesized by the action of mannitol-1-phosphatase and an inducible mannitol-1-phosphate dehydrogenase. However, direct evidence for an inducible mannitol-1-phosphate dehydrogenase is still missing and our failure to detect mannitol in our cultures indicates that the proper condition for such an induction was not present.

Purification and characterization of glucose-6-phosphate dehydrogenase from Aspergillus parasiticus

Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was purified from mycelium of Aspergillus parasiticus (1-11-105 Whl). The enzyme had a molecular weight of 1.8 X 10(5) and was composed of four subunits of apparently equal size. The substrate was very strict, only glucose 6-phosphate and glucose being oxidized by NADP or thio-NADP. Zinc ion was a powerful inhibitor of the enzyme, inhibition being competitive with respect to glucose 6-phosphate, with Ki about 2.5 microM. Other divalent metal ions which also serve as inhibitors are nickel, cadmium, and cobalt. It is proposed that the stimulation of polyketide synthesis by zinc ion may be mediated in part by inhibition. of glucose-6-phosphate dehydrogenase.

Stimulation of alternariol biosynthesis by zinc and manganese ions

Abstract Production of the polyketides alternariol and alternariol methyl ether by mycelia ofAlternaria alternata is absolutely dependent on an adequate concentration of Zn2+ in the culture medium. Minimal alternariol synthesis occurs with 1.0 μM Zn2+, and concentrations between 10 and 100 μM stimulate alternariol synthesis maximally. Mn2+ acts synergistically with Zn2+ to provide a further threefold stimulation of alternariol synthesis. The relationship between Zn2+ stimulation and light inhibition of alternariol synthesis is discussed.

Cadmium ion stimulation of growth and versicolorin synthesis in a mutant strain ofAspergillus parasiticus

Abstract Cultures ofAspergillus parasiticus produce the polyketide versicolorin A in response to elevation of the Zn2+ content of the growth medium. With suboptimal Zn2+ (0.8 μM) mycelial growth is about half maximal, and versicolorin synthesis is essentially zero. Inclusion of Cd2+ (1–100 μM) in the Zn2+-limiting growth medium allows optimal growth and stimulates full versicolorin synthesis. Cd2+, like Zn2+, will stimulate versicolorin sysnthesis only when added within the first 30 h after conidial inoculation. The transport system for Cd2+ uptake may be the same as that for Zn2+, as judged byin vivo competition studies. Cd2+ is a competitive inhibitor of Zn2+ uptake, with Ki = 20 μM.

Versicolorin synthesis by Aspergillus parasiticus: Regulation by temperature and zinc

Abstract A mutant strain of Aspergillus parasiticus (ATCC 36537) was grown in shaken liquid culture at 23 or 37°C. The polyketide secondary metabolite versicolorin A was produced at the lower but not the higher temperature. Temperature upshift (23 → 37°C) arrested versicolorin synthesis; temperature downshift (37 → 23°C) allowed versicolorin synthesis to occur after a 6-h lag. Versicolorin synthesis after temperature downshift was blocked by addition of cycloheximide or actinomycin D at the time of temperature shift. Zn 2+ is known to be required for versicolorin synthesis, and Zn 2+ uptake must occur before temperature downshift. We suggest that a transcriptional event required for versicolorin synthesis occurs only in the presence of Zn 2+ and at temperatures below 37°C.