Studies on the Enzymatic Production of l-Aspartic Acid from Maleic Acid

The enzymatic production of l-aspartic acid from maleic acid with cell suspensions of Alcaligenes faecalis 5-24, isolated from solid by the authors, was investigated.

The optimum conditions of this reaction and some cultural conditions which influenced on the ability of the cells to catalyze the above reaction were mainly studied.

The cells grown on maleic acid as a sole source of carbon showed exclusively the strong ability. The cells grown on a carbon source other than maleic acid showed no activity of this reaction.

It was concluded that an inducibles enzyme whose formation was stimulated by the presence of maleic acid might be involved in the reaction for the production of l-aspartic acid from maleic acid.

It was found that malonic acid was replaceable for maleic acid which played an inductive role for the formation of the enzyme system concerned with the reaction of l-aspartic acid production from maleic acid.

The cells grown in the medium containing malonic acid showed a stronger activity of the above reaction than the cells grown on maleic acid. The induction effect of malonic acid was remarkable when the organism was cultured in an acid medium. Whereas, consumption of C¹⁴-malonic acid in the medium by the organism was not observed at all in any pH milieu even where the formation of the enzyme system essential for the reaction was fully conducted. It indicated that malonic acid penetrated preferentially in acid milieu into the cells was a non-metabolic inducer like thiomethyl-β-d-galactoside in β-galactosidase system and that permeability barrier might exist in the organism.

The formation of cis-trans isomerase which catalyzed the conversion of maleic acid to fumaric acid was much stimulated by the addition of either malonic acid or maleic acid. From these results, it was concluded that l-aspartic acid was produced from maleic acid and ammonium ion by both actions of the inducible cis-trans isomerase and the constitutive aspartase.     

Accumulation of Diphosphorylated Butirosin A Derivatives by Phosphatase-negative Mutants of Bacillus vitellinus

It has been reported that Bacillus vitellinus, a butirosin A-producing bacterium, has two enzymes: butirosin A-3′-phosphotransferase, catalyzing phosphorylation of butirosin A, and phosphatase, catalyzing dephosphorylation of butirosin A-3′-phosphate.

A phosphatase-negative mutant, P-15, was derived from a potent butirosin A producer, BA-44, by NTG treatment. The mutant, P-15, was found to produce a butirosin A derivative when it was grown in a medium containing a relatively high concentration of inorganic phosphate. This compound was isolated and confirmed to be butirosin A-6′-N-diphosphate by physico-chemical analysis and ¹³C-NMR spectrometry.

Furthermore, a mutant strain, AP-165, was derived from the phosphatase-negative mutant, P-15. This strain, AP-165, was isolated as a nonproducer on an agar piece of bouillon medium, but was found to accumulate a major product, 6′-deamino-6′-hydroxybutirosin A-6′-O-diphosphate, and a minor one, 6′-deamino-6′-hydroxybutirosin A-6′-O-monophosphate.

It seems likely that these phosphorylated compounds are possible intermediates of butirosin A biosynthesis in B. vitellinus.     

Biosynthesis of Enduracidin: Origin of Enduracididine and Other Amino Acids

The biosynthetic origin of the amino acid moieties of enduracidin was investigated by feeding experiments with labeled compounds. Results of the incorporation and the distribution of radioactivity into the antibiotic revealed that glycine, l-serine, l-threonine, l-alanine, L-aspartic acid, l-ornithine and l-citrulline were incorporated into the corresponding amino acid moieties. Unique amino acids, enduracididine and its isomer with an imidazolidine ring, were derived from l-arginine, but not histidine. K₁ (4-hydroxyphenylglycine) and K₂ (3,5-dichloro-K₁) moieties were derived from l-tyrosine. ³⁶Cl-Sodium chloride was incorporated into the antibiotic in the early stage of fermentation.     

2′-N-Acetylation of Butirosin A by Mutants of Streptomyces fradiae

To reveal the mechanism of reducing sugar-induced polymerization of proteins, monomeric lysozymes were isolated at various stages of storage from whole lysozyme (WL) being kept with glucose at 75% relative humidity and 50°C for up to 30 days, and their chemical properties were investigated and compared with the corresponding WL. The impairment of Lys, Arg, and Trp residues was observed in all the isolated monomeric lysozymes (IM) as well as in the WL.

When the IM were stored for another 10 days without glucose, they polymerized and had an additional impairment of Arg and Lys but not Trp residues. All IM exhibited almost the same polymerization rate, but the sum of additional losses of Lys and Arg residues varied. The IM was also found to cross-link untreated lysozymes even in the absence of glucose.

On basis of the results obtained hitherto, it is suggested that the glucose-induced polymerization of lysozymes proceeds through the following paths. At the first step, some bifunctional agents (BF), probably α-dicarbonyl compounds, generated from the reaction between ?-amino groups of lysine residues and glucose, attach to Arg, Lys, and Trp residues through one of their two functional sites. At the second step, some of those proteins with BF attached polymerize by binding of the other unoccupied functional site with the remaining Lys and Arg (not Trp) residues of the other protein molecules. The other of the proteins with BF attached polymerize through the combination between the other unoccupied functional sites themselves with no loss of amino acid residues.     

A new class of aggregation inhibitor of amyloid-β peptide based on an O-acyl isopeptide

Inhibition of amyloid β peptide (Aβ) aggregation is a potential therapeutic approach to treat Alzheimer’s disease. We report that an O-acyl isopeptide of Aβ1–42 (1) containing an ester bond at the Gly²⁵-Ser²⁶ moiety inhibits Aβ1–42 fibril formation at equimolar ratio. Inhibitory activity was retained by an N-Me-β-Ala²⁶ derivative (2), in which the ester of 1 was replaced with N-methyl amide to improve chemical stability at physiological pH. Inhibition was verified by fluorescence anisotropy, Western blot, and atomic force microscopy. This report suggests a new class of Aβ aggregation inhibitor based on modification of Aβ1–42 at Gly²⁵-Ser²⁶.     

Structural analysis and reaction mechanism of the disproportionating enzyme (D‐enzyme) from potato

Starch produced by plants is a stored form of energy and is an important dietary source of calories for humans and domestic animals. Disproportionating enzyme (D‐enzyme) catalyzes intramolecular and intermolecular transglycosylation reactions of α‐1, 4‐glucan. D‐enzyme is essential in starch metabolism in the potato. We present the crystal structures of potato D‐enzyme, including two different types of complex structures: a primary Michaelis complex (substrate binding mode) for 26‐meric cycloamylose (CA26) and a covalent intermediate for acarbose. Our study revealed that the acarbose and CA26 reactions catalyzed by potato D‐enzyme involve the formation of a covalent intermediate with the donor substrate. HPAEC of reaction substrates and products revealed the activity of the potato D‐enzyme on acarbose and CA26 as donor substrates. The structural and chromatography analyses provide insight into the mechanism of the coupling reaction of CA and glucose catalyzed by the potato D‐enzyme. The enzymatic reaction mechanism does not involve residual hydrolysis. This could be particularly useful in preventing unnecessary starch degradation leading to reduced crop productivity. Optimization of this mechanism would be important for improvements of starch storage and productivity in crops.     

Biochemical characterization of the <Emphasis Type="Italic">Arabidopsis</Emphasis> KS-type dehydrin protein,whose gene expression is constitutively abundant rather than stress dependent

Dehydrins are known as plant stress-responsive genes. Arabidopsis thaliana has 10 dehydrin genes. Among them, one of the highly expressed genes is a KS-type dehydrin (At1g54410). However, the gene product, which is a histidine-rich dehydrin whose molecular mass is 11 kDa (AtHIRD11), has not been studied. Thus, we report the biochemical characterization of the AtHIRD11 protein. Although the AtHIRD11 protein was detected in all organs of Arabidopsis, the bolting stem and the flower showed higher accumulation than the other organs, with the AtHIRD11 protein detected in the cambial zone of the stem vasculature. Most of the AtHIRD11 protein was found to be a bound form. The bound AtHIRD11 was solubilized by 1 M NaCl solution. The extracted AtHIRD11 was retained in immobilized metal-affinity chromatography, and eluted by an imidazole gradient. The native AtHIRD11 prepared from Arabidopsis was partially phosphorylated, but further phosphorylated by casein kinase 2 in vitro. Metal-binding assays indicated that Zn²⁺ may be the best metal for AtHIRD11 binding. These results suggest that AtHIRD11 is a metal-binding dehydrin that shows a house-keeping expression in Arabidopsis.