Nitrogen use efficiency. 2. Amino acid metabolism

In a previous article, we highlighted the latest developments in the isolation and characterisation of genes involved in the uptake of nitrogen from the soil, which might be used to improve the nitrogen use efficiency (NUE) of crop plants. In this article, we have concentrated on the genes controlling the enzymes of amino acid metabolism that may be involved in transferring nitrogen to the protein in the grain. Evidence is now accumulating from the use of knockout mutants, of the role of individual isoenzymes involved in amino acid metabolism, which are encoded by specific genes that are often members of a multigene family. In addition, a significant number of overexpressing plant lines have been obtained, which have increased activities of cytosol located, glutamine synthetase, asparagine synthetase and alanine aminotransferase that appear to have improved NUE.     

Polynucleotides. L. Synthesis and properties of poly (2''-chloro-2''-deoxyadenylic acid) and poly (2''-bromo-2''-deoxyadenylic acid).

Poly (2'-chloro-2'-deoxyadenylic acid) and poly (2'-bromo-2'-deoxyadenylic acid) were synthesized from the corresponding diphosphates with the aid of polynucleotide phosphorylase from E. coli. UV, CD, acid titration and mixing with poly (U) were investigated. Comparing these properties with those of poly (A) and poly (2'-azido-2'-deoxyadenylic acid), it was found that 2'substituents exert significant effects on the thermal stability of these polynucleotides, though the overall conformational structure was not greatly changed.     

Polynucleotides. XL. Synthesis and properties of poly 2''-azido-2''-deoxyadenylic acid.

Poly 2'-azido-2'-deoxyadenylic acid (Poly Az) was synthesized from 2'-azido-2'-deoxyadenosine diphosphate by polynucleotide phosphorylase. Poly (Az) has U.V. absorption properties similar to poly (A) and hypochromicity of 40% at 0.1 M Na+ and neutrality. CD curve also resembled to that of poly (A), but has smaller ellipticity. Titration of poly (Az) with HCl gave a transition at pH 5.5, but exact structure of the acid-form complex was not elucidated. Upon mixing with poly (U), poly (Az) forms a 1:1 and 1:2 complexes having Tm's somewhat higher than that of poly (A)- poly (U) complex in the same condition.     

Effect of 2-allyl-2-isopropylacetamide on poly(adenylic acid)-containing ribonucleic acid.

A single administration of 2-allyl-2-isopropylacetamide, a porphyrinogenic drug, enhanced the 32P-labelling of nucleoplasmic as well as cytoplasmic poly(A)-containing RNA in rat liver. The synthesis of total microsomal RNA is only marginally increased under these conditions. The drug enhances the labelling of a variety of cytoplasmic poly(A)-containing RNA species, and this effect is counteracted by the simultaneous administration of haemin. 2-Allyl-2-isopropylacetamide also enhanced the release of RNA from the nucleus to the cytoplasm.     

Fluorescent 2-aza-epsilon-adenosine derivatives of polyadenylic acid, polyuridylic acid, and polyinosinic acid.

A new fluorescent analog of adenosine, 1,N⁶-etheno-2-aza-adenosine, has been incorporated into polynucleotides by polynucleotide phosphorylase polymerization of 1,N⁶-etheno-2-aza-adenosine-5′-diphosphate and adenosine-5′-diphosphate, uridine-5′-diphosphate, or inosine-5′-diphosphate. These new oligonucletides possess high fluorescence when excited at 358 nm and emit at 495 nm. The ratio of the fluorescent and nonfluorescent portions of the copolymer can be controlled by the initial composition of the 2-aza-ε-adenosine-diphosphate and the corresponding nucleoside diphosphate. Fluorescent copolymers with a ratio varying from 1.6 to 35 have thus been synthesized. The physicochemical study of copolymers containing less than 10% of the 1,N⁶-etheno-2-aza-adenosine moiety showed that they are similar to poly(A), poly(U), or poly(I). Therefore, fluorescence and polarization study of the 1,N⁶-etheno-2-aza-adenosine residues that have been incorporated into the copolymer provides a sensitive indicator for the structure of the copolymer. Potentially these new copolymers may provide unique roles in probing the structure of poly(C) and poly(A) in cellular mRNA.     

2-O-methyl-D-glucuronic acid, a new hexuronic acid of biological origin.

Acid hydrolysis of the extracellular polysaccharide of Porphyridium cruentum (a unicellular red alga) produced a mixture of aldobiuronic acids and free hexuronic acids. Fractionation of this mixture on an ion-exchange column yielded a hexuronic acid characterized as the title compound. Its identity was confirmed by chromatographic comparisons with the authentic compound, by reduction to the corresponding methylated aldose, by resistance to controlled lead tetra-acetate oxidation and by chemical-ionization mass spectrometry. Complete spectra have been deposited as Supplementary Publication SUP50062 (7 pages) with the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies may be obtained under the terms given in Biochem. J. (1976) 153, 5.     

Solid-liquid phase behavior of binary fatty acid mixtures. 2. Mixtures of oleic acid with lauric acid, myristic acid, and palmitic acid

Solid-liquid phase behavior was investigated for binary fatty acid mixtures composed of oleic acid (OA; cis-9-octadecenoic acid) and saturated fatty acids, lauric acid (LA; dodecanoic acid), myristic acid (MA; tetradecanoic acid), and palmitic acid (PA; hexadecanoic acid), by means of differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FT-IR). When the mixture was heated immediately after the solidification from the melt, the heat effect due to the gamma-to-alpha transformation of OA varied depending on the composition of the mixture. However, the mixture subjected to an annealing at the temperature slightly below the melting temperature provided the transformation at constant temperature which corresponds to the gamma-to-alpha transformation temperature of pure OA. This suggests that a solid phase formed by cooling of the melt of the mixture is not in an equilibrium state, but it relaxes to a stable solid during the annealing process. The T-X phase diagrams of these mixtures constructed from the DSC measurements demonstrate that the two fatty acid species are completely immiscible in a solid phase regardless of the type of polymorphs of OA, alpha- or gamma-form. According to a thermodynamic analysis of liquidus line basing on the regular solution model for the melt, the non-ideality of mixing tends to increase with the decrease in the acyl chain length of the saturated fatty acid, although the mixing is rather close to ideal.     

2-Amino-2-deoxygalacturonic acid in lipopolysaccharides from Pseudomonas aerugimosa.

2-Amino-2-deoxygalacturonic acid was identified as a component of the lipopolysaccharide from Pseudomonas aeruginosa N.C.T.C. 8505. The compound probably occurs in the region of polysaccharide responsible for O-antigenic specificity.     

Degradation of 2-methylbenzoic acid by Pseudomonas cepacia MB2.

We report the isolation of Pseudomonas cepacia MB2, believed to be the first microorganism to utilize 2-methylbenzoic acid as the sole carbon source. Its growth range included all mono- and dimethylbenzoates (with the exception of 2,5- and 2,6-dimethylbenzoates) and 3-chloro-2-methylbenzoate (but not 4- or 5-chloro-2-methylbenzoate) but not chlorobenzoates lacking a methyl group. 2-Chlorobenzoate, 3-chlorobenzoate, and 2,3-, 2,4-, and 3,4-dichlorobenzoates inhibited growth of MB2 on 2-methylbenzoate as a result of cometabolism to the corresponding chlorinated catechols which blocked the key enzyme catechol 2,3-dioxygenase. A metapyrocatechase-negative mutant, MB2-G5, showed accumulation of dimethylcatechols from 2,3- and 3,4-dimethylbenzoates, and phenols were detected in resting-cell transformation extracts bearing the same substitution pattern as the original substrate, presumably following thermal degradation of the intermediate dihydrodiol. 2-Methylphenol was also found in extracts of the mutant cells with 2-methylbenzoate. These observations suggested a major route of methylbenzoate metabolism to be dioxygenation to a carboxy-hydrodiol which then forms a catechol derivative. In addition, the methyl group of 2-methylbenzoate was oxidized to isobenzofuranone (by cells of MB2-G5) and to phthalate (by cells of a separate mutant that could not utilize phthalate, MB2-D2). This pathway also generated a chlorinated isobenzofuranone from 3-chloro-2-methylbenzoate.     

Degradation of 2-methylbenzoic acid by Pseudomonas cepacia MB2.

We report the isolation of Pseudomonas cepacia MB2, believed to be the first microorganism to utilize 2-methylbenzoic acid as the sole carbon source. Its growth range included all mono- and dimethylbenzoates (with the exception of 2,5- and 2,6-dimethylbenzoates) and 3-chloro-2-methylbenzoate (but not 4- or 5-chloro-2-methylbenzoate) but not chlorobenzoates lacking a methyl group. 2-Chlorobenzoate, 3-chlorobenzoate, and 2,3-, 2,4-, and 3,4-dichlorobenzoates inhibited growth of MB2 on 2-methylbenzoate as a result of cometabolism to the corresponding chlorinated catechols which blocked the key enzyme catechol 2,3-dioxygenase. A metapyrocatechase-negative mutant, MB2-G5, showed accumulation of dimethylcatechols from 2,3- and 3,4-dimethylbenzoates, and phenols were detected in resting-cell transformation extracts bearing the same substitution pattern as the original substrate, presumably following thermal degradation of the intermediate dihydrodiol. 2-Methylphenol was also found in extracts of the mutant cells with 2-methylbenzoate. These observations suggested a major route of methylbenzoate metabolism to be dioxygenation to a carboxy-hydrodiol which then forms a catechol derivative. In addition, the methyl group of 2-methylbenzoate was oxidized to isobenzofuranone (by cells of MB2-G5) and to phthalate (by cells of a separate mutant that could not utilize phthalate, MB2-D2). This pathway also generated a chlorinated isobenzofuranone from 3-chloro-2-methylbenzoate.     

Polynucleotides. XLIV. Synthesis and properties of poly (2-azaadenylic acid) and poly(2-azainosinic acid).

Chemically synthesized 2-azaadenosine 5'-diphosphate (n2ADP) and 2-azainosine 5'-diphosphate (n2IDP) were polymerized to yield poly(2-azaadenylic acid), poly(n2A), and poly(2-azainosinic acid), poly(n2I), using Escherichia coli polynucleotide phosphorylase. In neutral solution, poly(n2A) and poly(n2I) had hypochromicities of 32 and 5.5%, respectively. Poly(n2A) formed an ordered structure, which had a melting temperature (Rm) of 20 degrees C at 0.15 M salt concentration. Upon mixing with poly(U), poly(n2A) formed a 1 : 2 complex with Tm of 41 degrees C at 0.15 M salt concentration. Poly(n2A) and poly(n2I) formed three-stranded complexes with poly(I), and poly(A), respectively. Poly(n2A) . 2poly(I), poly(A) . 2poly(n2I), and poly(n2A) . 2poly(n2I) complexes had Tm values of 23, 48, and 31 degrees C at 0.15 M salt concentration, respectively. Poly(n2I) formed a double-stranded complex with poly(C), but its Tm was very low.     

L-ascorbic acid 2-sulphate. A substrate for mammalian arylsulphatases.

Ascorbic acid 2-sulphate has a stability in acid comparable to that of phenyl sulphate and is rather more acid-labile than simple carbohydrate sulphates. At its optimum pH of 4.8 sulphatase A(aryl-sulphate sulphohydrolase EC 3.1.6.1.) hydrolyses ascorbic acid sulphate with a specific activity of 90 mumol/mg per min (150 mumol/mg per min with nitrocatechol sulphate at pH 5.6). At pH 4.8 the kinetics are non-Michaelis. At pH 5.6 Michaelis kinetics are obeyed and Km 12 21 mM ascorbic acid 2-sulphate. K2SO4 is a competitive inhibitor with a Ki of 0.2 and 0.6 mM at pH 4.8 and 5.6, respectively. Sulphatase A is converted into a substrate-modified form during its hydrolysis of ascorbic acid sulphate. Sulphatase B also hydrolyses ascorbic acid 2-sulphate. At pH 4.8 and in the presence of 0.15 M NaCl the specific activity is 0.92 mumol/mg per min (90 mumol/mg per min for nitrocatechol sulphate at pH 5.6). In the absence of NaCl the activity is greatly decreased. Km is 8 mM. K2SO4 is a competitive inhibitor with a Ki of 0.1 mM. Ascorbic acid is not hydrolysed at a detectable rate by the arylsulphatases of the mollusc Dicathais orbita or of Aerobacter aerogenes.?     

2-Benzyl-2-methylsuccinic acid as inhibitor for carboxypeptidase A. synthesis and evaluation.

Recently, Asante-Appiah et al. (Asante-Appiah, E.; Seetharaman, J.; Sicheri, F.; Yang, D. S.-C.; Chan, W. W.-C. Biochemistry 1997, 36, 8710 8715) reported that 2-ethyl-2-methylsuccinic acid is a highly potent inhibitor for carboxypeptidase A (CPA), a prototypic zinc protease. The X-ray crystal structure of the complex of the enzyme formed with 2-ethyl-2-methylsuccinic acid revealed that at the active site of CPA there is present a small cavity which accommodates the methyl group of the inhibitor. These investigators postulated that incorporation of a methyl group at the alpha-position to the carboxylate of existing inhibitors of CPA would improve the inhibitory potency. We have synthesized racemic and optically active 2-benzyl-2-methylsuccinic acids and evaluated their inhibitory activities for CPA to find the K(i) values to be 0.28, 0.15, and 17microM for racemic form, (R)-, and (S)-enantiomer, respectively. Contrary to the expectation, the effect on the binding affinity by the incorporation of the methyl group is minimal. The validity of the proposition that the small cavity may be utilized for the improvement of the inhibitory potency appears questionable.     

2-Oxo acid dehydrogenase complexes in redox regulation.

A number of cellular systems cooperate in redox regulation, providing metabolic responses according to changes in the oxidation (or reduction) of the redox active components of a cell. Key systems of central metabolism, such as the 2-oxo acid dehydrogenase complexes, are important participants in redox regulation, because their function is controlled by the NADH/NAD+ ratio and the complex-bound dihydrolipoate/lipoate ratio. Redox state of the complex-bound lipoate is an indicator of the availability of the reaction substrates (2-oxo acid, CoA and NAD+) and thiol-disulfide status of the medium. Accumulation of the dihydrolipoate intermediate causes inactivation of the first enzyme of the complexes. With the mammalian pyruvate dehydrogenase, the phosphorylation system is involved in the lipoate-dependent regulation, whereas mammalian 2-oxoglutarate dehydrogenase exhibits a higher sensitivity to direct regulation by the complex-bound dihydrolipoate/lipoate and external SH/S-S, including mitochondrial thioredoxin. Thioredoxin efficiently protects the complexes from self-inactivation during catalysis at low NAD+. As a result, 2-oxoglutarate dehydrogenase complex may provide succinyl-CoA for phosphorylation of GDP and ADP under conditions of restricted NAD+ availability. This may be essential upon accumulation of NADH and exhaustion of the pyridine nucleotide pool. Concomitantly, thioredoxin stimulates the complex-bound dihydrolipoate-dependent production of reactive oxygen species. It is suggested that this side-effect of the 2-oxo acid oxidation at low NAD+in vivo would be overcome by cooperation of mitochondrial thioredoxin and the thioredoxin-dependent peroxidase, SP-22.