Enzymatic conversion of norsolorinic acid to averufin in aflatoxin biosynthesis

5'-Hydroxyaverantin (HAVN) was isolated from a mold, Emericella heterothallica IFO 30842. Aspergillus parasiticus NIAH-26, a UV-irradiated mutant of A. parasiticus SYS-4, produced neither aflatoxins nor precursors in yeast extract-sucrose (YES) medium. When the postmicrosome (cytosol) fraction of NIAH-26, which had been prepared from the culture in YES medium, was incubated with norsolorinic acid (NA) in the presence of NADH or NADPH, averantin (AVN) was produced. The reverse reaction from AVN to NA was promoted by the addition of NAD or NADP (dehydrogenase reaction). When the microsome fraction of NIAH-26 was incubated with AVN, HAVN was produced in the presence of NADPH (monooxygenase reaction). HAVN was, furthermore, oxidized to averufin (AVR) by the cytosol fraction of NIAH-26 in the presence of NAD or NADP (dehydrogenase reaction). In the feeding experiments with A. parasiticus NIAH-26, aflatoxins were produced from AVN, HAVN, NA, and AVR but not from averufanin or averythrin. These results indicate that the reaction sequence NA in equilibrium AVN----HAVN----AVR is involved in the biosynthetic pathway of aflatoxins. The enzyme activities described here were dependent on the culture medium, and no enzyme activities were observed in the nonaflatoxigenic strain A. oryzae SYS-2 (IFO 4251).     

Enzymatic conversion of norsolorinic acid to averufin in aflatoxin biosynthesis.

5'-Hydroxyaverantin (HAVN) was isolated from a mold, Emericella heterothallica IFO 30842. Aspergillus parasiticus NIAH-26, a UV-irradiated mutant of A. parasiticus SYS-4, produced neither aflatoxins nor precursors in yeast extract-sucrose (YES) medium. When the postmicrosome (cytosol) fraction of NIAH-26, which had been prepared from the culture in YES medium, was incubated with norsolorinic acid (NA) in the presence of NADH or NADPH, averantin (AVN) was produced. The reverse reaction from AVN to NA was promoted by the addition of NAD or NADP (dehydrogenase reaction). When the microsome fraction of NIAH-26 was incubated with AVN, HAVN was produced in the presence of NADPH (monooxygenase reaction). HAVN was, furthermore, oxidized to averufin (AVR) by the cytosol fraction of NIAH-26 in the presence of NAD or NADP (dehydrogenase reaction). In the feeding experiments with A. parasiticus NIAH-26, aflatoxins were produced from AVN, HAVN, NA, and AVR but not from averufanin or averythrin. These results indicate that the reaction sequence NA in equilibrium AVN----HAVN----AVR is involved in the biosynthetic pathway of aflatoxins. The enzyme activities described here were dependent on the culture medium, and no enzyme activities were observed in the nonaflatoxigenic strain A. oryzae SYS-2 (IFO 4251).     

Enzymatic conversion of benzylpenicillin to 6-aminopenicillanic acid in concentrated ultrafiltered broths

Benzylpenicillin filtered broths purified by ultrafiltration and fermented broths clarified by ultrafiltration and afterwards concentrated by reverse osmosis were used directly for enzymatic conversion of benzylpenicillin to 6-aminopenicillanic acid and phenylacetic acid by immobilised penicillin G acylase or amidase. It was concluded that, when the ultrafiltration operation retained 100% of protein, the concentrates from reverse osmosis could be successfully directly fed to the enzymatic reactor, giving high enzymatic conversion yield of benzylpenicillin to 6-aminopenicillanic acid.     

Enzymatic conversion of glutamate to delta-aminolevulinic acid in soluble extracts of Euglena gracilis

Glutamate was converted to the chlorophyll and heme precursor delta-aminolevulinic acid in soluble extracts of Euglena gracilis. delta-Aminolevulinic acid-forming activity depended on the presence of native enzyme, glutamate, ATP, Mg2+, NADPH or NADH, and RNA. The requirement for reduced pyridine nucleotide was observed only if, prior to incubation, the enzyme extract was filtered through activated carbon to remove firmly bound reductant. Dithiothreitol was also required for activity after carbon treatment. delta-Aminolevulinic acid formation was stimulated by RNA from various plant tissues and algal cells, including greening barley leaves and members of the algal groups Chlorophyta (Chlorella vulgaris, Chlamydomonas reinhardtii), Rhodophyta (Cyanidium caldarium), Cyanophyta (Anacystis nidulans, Synechocystis sp. PCC 6803), and Prochlorophyta (Prochlorothrix hollandica), but not by RNA derived from Escherichia coli, yeast, wheat germ, bovine liver, and Methanobacterium thermoautotrophicum. E. coli glutamate-specific tRNA was inhibitory. Several of the RNAs that did not stimulate delta-aminolevulinic acid formation nevertheless became acylated when incubated with glutamate in the presence of Euglena enzyme extract. RNA extracted from nongreen dark-grown wild-type Euglena cells was about half as stimulatory as that from chlorophyllous light-grown cells, and RNA from aplastidic mutant cells stimulated only slightly. delta-Aminolevulinic acid-forming enzyme activity was present in extracts of light-grown wild-type cells, but undetectable in extracts of aplastidic mutant and dark-grown wild-type cells. Gabaculine inhibited delta-aminolevulinic acid formation at submicromolar concentration. Heme inhibited 50% at 25 microM, but protoporphyrin IX, Mg-protoporphyrin IX, and protochlorophyllide inhibited only slightly at this concentration.     

Site-directed mutagenesis of rat liver S-adenosylhomocysteinase. Effect of conversion of aspartic acid 244 to glutamic acid on coenzyme binding

Aspartic acid 244 that occurs at the putative NAD(+)-binding site of rat liver S-adenosylhomocysteinase was replaced by glutamic acid by oligonucleotide-directed mutagenesis. The mutant enzyme was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel permeation chromatography showed that the purified mutant enzyme was a tetramer as is the wild-type enzyme. In contrast to the wild-type enzyme, which possesses 1 mol of tightly bound NAD+ per mol of enzyme subunit, the mutant enzyme had only 0.05 mol of NAD+ but contained about 0.6 mol each of NADH and adenine per mol of subunit. The mutant enzyme, after removal of the bound compounds by acid-ammonium sulfate treatment, exhibited S-adenosylhomocysteinase activity when assayed in the presence of NAD+. From the appearance of activity as a function of NAD+ concentration, the enzyme was shown to bind NAD+ with a Kd of 23.0 microM at 25 degrees C, a value greater than 280-fold greater than that of the wild-type enzyme. In the presence of a saturating concentration of NAD+, the mutant enzyme showed apparent Km values for substrates similar to those of the wild-type enzyme. Moderate decreases of 8- and 15-fold were observed in Vmax values for the synthetic and hydrolytic directions, respectively. These results indicate the importance of Asp-244 in binding NAD+, and are consistent with the idea that the region of S-adenosylhomocysteinase from residues 213 to 244 is part of the NAD+ binding site. This region has structural features characteristic of the dinucleotide-binding domains of NAD(+)- and FAD-binding proteins (Ogawa, H., Gomi, T., Mueckler, M. M., Fujioka, M., Backlund, P.S., Jr., Aksamit, R.R., Unson, C.G., and Cantoni, G.L. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 719-723).     

Enzymatic conversion of O-carbamyl-L-serine to pyruvate and ammonia

Treatment of partially hepatectomized male rats with urethan 6 hr after operation resulted in 50–55% inhibition of the incorporation of orotic acid-5-³H into nuclear ribosomal RNA and heterogeneous RNA 18 hr later. Neither partially hepatectomized female rats similarly treated with urethan nor operated male animals treated with an equitoxic dose of butyl carbamate presented evidence of an impairment of nuclear RNA synthesis.     

Engineering of the active site of human lysozyme: conversion of aspartic acid 53 to glutamic acid and tyrosine 63 to tryptophan or phenylalanine

Three human lysozymes containing a mutation either at Asp-53 to Glu or at Tyr-63 to Trp or Phe were synthesized and examined for their immunological and enzymatical activities in comparison with the native one. All mutants were immunologically indistinguishable from native human lysozyme. The [Trp63] and [Phe63] mutants catalysed the hydrolysis of Micrococcus lysodeikticus cell wall and glycol chitin effectively, while the [Glu53] mutant displayed very low activity toward M. lysodeikticus cells and no detectable activity toward glycol chitin.