Bacterial Metabolism of 3-Hydroxy-3-Methylglutaric Acid

An organism belonging to Pseudomonadaceae and capable of utilizing 3-hydroxy-3-methylglutarate as sole carbon source has been isolated from soil. Whole-cell preparations catalyze the oxidation of acetoacetate, acetate, glyoxylate, and citric acid cycle intermediates. Cell-free extracts of 3-hydroxy-3-methylglutarate-grown cells show an adenosine triphosphate, coenzyme A (CoA), and Mg(2+)-dependent conversion of 3-hydroxy-3-methylglutarate to 3-hydroxy-3-methylglutaryl-CoA. Succinyl-CoA-generating system has no effect on the activation and catabolism of 3-hydroxy-3-methylglutarate.     

Metabolism of Indole-3-acetic Acid: IV. Biological Properties of Amino Acid Conjugates

The biological activity of 20 l-alpha-amino acid conjugates of indole-3-acetic acid (IAA) to stimulate cell elongation of Avena sativa coleoptile sections and to stimulate growth of soybean cotyledon tissue cultures has been examined at concentrations of 10(-4) to 10(-7)m. In the Avena coleoptile test, most of the amino acid conjugates stimulated elongation. Several of the conjugates stimulated as much elongation as IAA but their half-maximum concentrations tended to be higher. Some of the more active conjugates were alanine, glycine, lysine, serine, aspartic acid, cystine, cysteine, methionine, and glutamic acid.In the soybean cotyledon tissue culture test, all of the l-alpha-amino acid conjugates of IAA stimulated growth except for the phenylalanine, histidine, and arginine conjugates. Most of the conjugates produced responses at least as great as that caused by IAA. Conjugates with half-maximum concentrations lower than IAA included cysteine, cystine, methionine, and alanine. These conjugates exceed the IAA-induced callus growth at all tested concentrations. Other conjugates significantly better than IAA at 10(-6)m were serine, glycine, leucine, proline, and threonine.     

Metabolism of Indole-3-Acetic Acid in Arabidopsis

The metabolism of indole-3-acetic acid (IAA) was investigated in 14-d-old Arabidopsis plants grown in liquid culture. After ruling out metabolites formed as an effect of nonsterile conditions, high-level feeding, and spontaneous interconversions, a simple metabolic pattern emerged. Oxindole-3-acetic acid (OxIAA), OxIAA conjugated to a hexose moiety via the carboxyl group, and the conjugates indole-3-acetyl aspartic acid (IAAsp) and indole-3-acetyl glutamate (IAGlu) were identified by mass spectrometry as primary products of IAA fed to the plants. Refeeding experiments demonstrated that none of these conjugates could be hydrolyzed back to IAA to any measurable extent at this developmental stage. IAAsp was further oxidized, especially when high levels of IAA were fed into the system, yielding OxIAAsp and OH-IAAsp. This contrasted with the metabolic fate of IAGlu, since that conjugate was not further metabolized. At IAA concentrations below 0.5 μm, most of the supplied IAA was metabolized via the OxIAA pathway, whereas only a minor portion was conjugated. However, increasing the IAA concentrations to 5 μm drastically altered the metabolic pattern, with marked induction of conjugation to IAAsp and IAGlu. This investigation used concentrations for feeding experiments that were near endogenous levels, showing that the metabolic pathways controlling the IAA pool size in Arabidopsis are limited and, therefore, make good targets for mutant screens provided that precautions are taken to avoid inducing artificial metabolism.The plant hormone IAA is an important signal molecule in the regulation of plant development. Its central role as a growth regulator makes it necessary for the plant to have mechanisms that strictly control its concentration. The hormone is believed to be active primarily as the free acid, and endogenous levels are controlled in vivo by processes such as synthesis, oxidation, and conjugation. IAA has been shown to form conjugates with sugars, amino acids, and small peptides. Conjugates are believed to be involved in IAA transport, in the storage of IAA for subsequent use, in the homeostatic control of the pool of the free hormone, and as a first step in the catabolic pathways (Cohen and Bandurski, 1978; Nowacki and Bandurski, 1980; Tuominen et al., 1994; Östin et al., 1995; Normanly, 1997). It is generally accepted that in some species conjugated IAA is the major source of free IAA during the initial stages of seed germination (Ueda and Bandurski, 1969; Sandberg et al., 1987; Bialek and Cohen, 1989), and there is also evidence that in some plants (but not all; see Bialek et al., 1992), the young seedling is entirely dependent on the release of free IAA from conjugated pools until the plant itself is capable of de novo synthesis (Epstein et al., 1980; Sandberg et al., 1987).The function of conjugated IAA during vegetative growth is somewhat less clear. It has been shown that conjugated IAA constitutes as much as 90% of the total IAA in the plant during vegetative growth (Normanly, 1997). However, the role of the IAA conjugates at this stage of the plant''s life cycle remains unknown. Analysis of endogenous IAA conjugates in vegetative tissues has revealed the presence of a variety of different compounds, including indole-3-acetyl-inositol, indole-3-acetyl-Ala, IAAsp, and IAGlu (Anderson and Sandberg, 1982; Cohen and Baldi, 1983; Chisnell, 1984; Cohen and Ernstsen, 1991; Östin et al., 1992). Studies of vegetative tissues have indicated that IAAsp, one of the major conjugates in many plants, is the first intermediate in an irreversible deactivation pathway (Tsurumi and Wada, 1986; Tuominen et al., 1994; Östin, 1995). Another mechanism that is believed to be involved in the homeostatic control of the IAA pool is catabolism by direct oxidation of IAA to OxIAA, which has been shown to occur in several plant species (Reinecke and Bandurski, 1983; Ernstsen et al., 1987).One area in the study of IAA metabolism in which our knowledge is increasing is the analysis of the homeostatic controls of IAA levels in plants. It has been possible, for instance, to increase the levels of IAA in transgenic plants expressing iaaM and iaaH genes from Agrobacterium tumefaciens. Analysis of these transgenic plants has indicated that plants have several pathways that can compensate for the increased production of IAA (Klee et al., 1987; Sitbon, 1992). It is expected that future studies using now-available genes will provide further insight into IAA metabolism. For example, a gene in maize encoding IAA-Glc synthetase has been identified, and several genes (including ILR1, which may be involved in hydrolysis of the indole-3-acetyl-Leu conjugate) have been cloned from Arabidopsis (Szerszen et al., 1994; Bartel and Fink, 1995). Furthermore, Chou et al. (1996) identified a gene that hydrolyzes the conjugate IAAsp to free IAA in the bacterium Enterobacter aggloremans.Because of its small genome size, rapid life cycle, and the ease of obtaining mutants, Arabidopsis is increasingly used as a genetic model system to investigate various aspects of plant growth and development. IAA signal transduction is also being investigated intensively in Arabidopsis in many laboratories (Leyser, 1997). Mutants with altered responses to externally added auxins or IAA conjugates have been identified in Arabidopsis. The identified mutants are either signal transduction mutants such as axr1-4 (Lincoln et al., 1990), or have mutations in genes involved in auxin uptake or transport, such as aux1 and pin1 (Okada et al., 1991; Bennett et al., 1996). A few mutants that are unable to regulate IAA levels or are unable to hydrolyze IAA conjugates, sur1-2 and ilr1, respectively, have also been identified (Bartel and Fink, 1995; Boerjan et al., 1995). To our knowledge, no mutant that is auxotrophic for IAA has been identified to date, which may reflect the redundancy in IAA biosynthetic pathways or the lethality of such mutants.In spite of the work reported thus far, many aspects of the metabolism of IAA in Arabidopsis require further investigation, because few details of the processes involved in IAA regulation are known. This lack of knowledge puts severe constraints on genetic analysis of IAA metabolism in Arabidopsis. For example, it is essential to have prior knowledge of IAA metabolism to devise novel and relevant screens with which to identify mutants of IAA metabolism. We have sought to address this issue by identifying the metabolic pathways involved in catabolism and conjugation under conditions that minimally perturb physiological processes. In this investigation we studied the conjugation and catabolic pattern of IAA by supplying relatively low levels of labeled IAA and identifying the catabolites and conjugates by MS. Different feeding systems were tested to optimize the application of IAA and to avoid irregularities in metabolism attributable to culturing, feeding conditions, or microbial activity. It is well documented that IAA metabolism is altered according to the amount of exogenous auxin applied; therefore, we placed special emphasis on distinguishing between catabolic routes that occur at near-physiological concentrations and those that occur at the high auxin concentrations commonly used in mutant screens.     

The Metabolism of 3-Indolylacetic Acid in Plant Tissues3

The nature of metabolic products of 3–indolylacetic acid(IAA) extracted from potato tuber disks treated with aeratedIAA solution has been investigated. Two major products, knownat first as ‘V’ and ‘P’ in these studieshave been isolated and ‘V’ has been identified as3-indolylacetylaspartic acid (IacAsp). The rapid uptake of IAA is inhibitited by metabolic poisonssuch as 10^–3 M. cyanide. The maximum mean internal concentrationexceeds the external concentration well–aerated cultures.The mean internal concentration, however only remains for aperiod and then falls off rapidly as a result of extrusion ofabsorbed IAA into the external solution. This extrusion is notinhibited by 10^-3 cyanide; when the mean internal IAA concentrationis 150 µ mol/ml. and the localized IAA concentration musttherefore exceed this value. We conclude therefore that theIAA concentration in the sites where it has accumulated exceedsthe concentration of IAA outside. Uptake of IAA and also its further conversion are inhibitedby indolylacetonitrile and promoted by aspartate, but this promotionis not associated with any gain in amount of indolylacetylaspartate(IacAsp). The data suggest that IacAsp may be formed in tissue from ‘boundIAA’ rather then free IAA. The ‘accelerator ’ found in potato and beans whichhas similar R_F to IAcAsp has been shown definity to be someother substance or substances and not IAcAsp as was at firstthought possible.     

Crassulacean Acid Metabolism and Crassulacean Acid Metabolism Modifications in Peperomia camptotricha

Peperomia camptotricha, a tropical epiphyte from Mexico, shows variable forms of Crassulacean acid metabolism (CAM). Young leaves exhibit CAM-cycling, while mature leaves show an intermediate type of metabolism, between CAM and CAM-cycling, having approximately the same amount of nighttime gas exchange as daytime. Metabolism of young leaves appears independent of daylength, but mature leaves have a tendency toward more CAM-like metabolism under short days (8 hours). Large differences in the physical appearance of plants were found between those grown under short daylengths and those grown under long daylengths (14 hours). Some anatomical differences were also detected in the leaves. Water stress caused a switch to CAM in young and mature leaves, and as water stress increased, they shifted to CAM-idling.     

Anaerobic Amino Acid Metabolism

Anoxic stress induces a strong change in sugar, protein, and amino acid metabolism in higher plants. Sugars are rapidly consumed through the anaerobic glycolysis to sustain energy production. Protein degradation under anoxia is a mechanism to release free amino acids contributing in this way to maintaining the osmotic potential of the tissue under stress. Among free amino acids, a particular role is played by glutamic acid, being a precursor of some characteristic compounds of the anaerobic metabolism (alanine, -aminobutyric acid, and putrescine). The glutamine synthetase/glutamate synthase cycle contributes to ammonia reassimilation and primary assimilation of nitrate, and resynthesizes constantly glutamate for the synthesis of other compounds. Some polypeptides involved in these pathways are expressed under anoxia. The importance of amino acid metabolism for the response to anaerobic stress is discussed.     

Investigations on the Mechanism of the Brassinosteroid Response: I. Indole-3-acetic Acid Metabolism and Transport

A brassinosteroid treatment of light-grown first internode sections of Phaseolus vulgaris results in an increased bending response following unilateral indole-3-acetic acid (IAA) application. Reverse isotope dilution analysis shows that this increased response is not due to an increase in the concentration of applied IAA in the tissue or a change in the amount of IAA conjugated. Treatment with the brassinosteroid also does not affect the rate of IAA transport as measured using the agar block method. These results indicate that even though brassinosteroid potentiates auxin action, it does not have a direct effect on IAA uptake, metabolism, or cell to cell transport.     

Studies on Kojic Acid Metabolism by Microorganisms

An enzymatic oxidation of kojic acid to comenic aldehyde was found in the decomposition process of kojic acid by Arthrobacter ureafaciens strain (K-l), a kojic acid decomposing bacteria.

This enzyme was (probable a new type of non-heme iron protein) is assumed to catalyze the dehydrogenation of kojic acid, while the ferric ion contained in the enzyme is considered to serve as an acceptor of hydrogen released from kojic acid. The resulted ferrous ions are oxidized either by molecular oxygen under aerobic conditions or by NAD under anaerobic conditions, accompanying hydrogen peroxide in the former and reduced NAD in the latter. The enzyme was partially purified by using ammonium sulfate precipitation, gel filtration on Sephadex G-200 column and column chromatography with DEAE-Sephadex A-50. The activity increased to 85 fold, compared with crude extracts and the recovery of the activity was 33.9%. The optimum pH of the reaction was 7.75. The enzyme was inactivated by PCMB, and unstable upon heat treatment. A loss of about 50% of the activity was caused by heating at 35%C for 5 min, but some reducing agents protected the enzyme from PCMB inhibition and the heat inactivation. Not only kojic acid, but also benzyl kojic acid or 5-methoxy kojic acid may be substrates. Km value for kojic acid was 1.43 × 10^?5m. The molecular weight of the enzyme was estimated to be about 55,000 and the enzyme contained about two atoms of iron in one molecule. The reaction mechanism for kojic acid oxidase is discussed.     

Studies on Kojic Acid Metabolism by Microorganisms

An enzyme, comenic aldehyde dehydrogenase, which catalyzes the oxidation of comenic aldehyde to comenic acid was partially purified from cell extract of Arthrobacter ureafaciens K-1.

The enzyme was purified 31-fold at Sephadex G-100 filtration step, 112-fold at DEAE-Sephadex A-50 fractionation step, and recovery of the activity was 73.3% and 38.5% respectively.

NADP and magnesium ion were essential for the oxidation. The enzyme shows optimum activity at pH 7.8. Enzyme activity was extremely sensitive to sulfhydryl reagents such as p-chloromercuribenzoate and monoiodoacetate. l-Cysteine or dithiothreitol protected the enzyme from p-chloromercuribenzoate inhibition. Carbonyl reagents, such as hydroxylamine and semicarbazide, inhibit the enzyme reaction by formation of addition compounds between carbonyl reagents and aldehyde group of the substrate. The enzyme was completely inactivated after heating for 5 min at 40°C The Km for 5-methoxy comenic aldehyde is 2.5×10^?6 m, and for NADP is 0.4×1O^?6 m. The reaction product, 5-methoxy comenic acid was identified by paperchromatography. The characterization of the enzyme has been carried out by using 5-methoxy comenic aldehyde as the substrate in stead of comenic aldehyde.     

Bacterial Metabolism of Mevalonic Acid

Soluble cell-free extracts of actinomycete S4 grown on media containing mevalonate catalyze acetoacetate formation from mevalonate, mevaldate, and β-hydroxy-β-methylglutaryl-coenzyme A (CoA). Conversion of mevalonate to acetoacetate involves formation of free β-hydroxy-β-methylglutaryl-CoA, but not free mevaldate. The reaction favors mevalonate oxidation, and nicotinamide adenine dinucleotide, rather than nicotinamide adenine dinucleotide phosphate, acts as oxidant.     

Fatty Acid Metabolism in Microorganisms

The production of pimelic acid from azelaic acid by microorganisms was studied. About 100 strains of bacteria which were able to utilize azelaic acid as a sole carbon source were isolated from soil and other natural materials. Among these bacteria, several strains produced a large quantity of an organic acid (pimelic acid) from azelaic acid in their culture fluids during the cultivation. The acid was isolated from the culture fluid of strain A133 in crystalline form. The crystal was identified as pimelic acid by physicochemical and biological methods.

From the results of investigations on the morphological and physiological characters, the bacterial strain A133 was assumed to be Micrococcus sp.     

Amino Acid Metabolism in Microorganisms

Certain strains of Streptomyces were found to convert l-methionine into 3-methylthio-propylamine (MTPA), but not d-methionine. Now, optical resolution of DL-methionine was attempted using this phenomenon. Streptomyces sp. K37 was cultured in a medium containing DL-methionine (10 mg/ml). The culture filtrate was applied to a column of Diaion SA-21A (OH form). MTPA was recovered from the effluent by ether exraction. The Diaion SA-21A was eluted with 1N HCl and the eluate was applied to a column of Diaion SK-1 (H form). d-Methionine was eluted from the column with 1N NH₄OH and recovered after concentration, decolorization with active carbon, and precipitation with ethanol. The yields of MTPA and d-methionine from the broth were 69.5% and 89.5%, respectively.