Phenylpropanoid compounds encompass a wide range of structural classes and biological functions. Limiting discussion to stress-induced phenylpropanoids eliminates few of the structural classes, because many compounds thst are constitutive in one plant species or tissue can be induced by various stresses in another species or in another tissue of the same plant (Beggs et al., 1987; Christie et al., 1994).

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Stress-Induced Phenylpropanoid Metabolism.

Studies of the molecular genetics, mechanisms of activation and functional connections between general phenylpropanoid metabolism and certain branch pathways in parsley, bean, potato plants and cell cultures

Physiology and Molecular Biology of Phenylpropanoid Metabolism

We present a Protein Competition Model (PCM) for predicting total phenolic allocation and concentration in leaves of terrestrial higher plants. In contrast to predictions based on the carbon composition of end products, the PCM is based on metabolic origins of pathway constituents, alternative fates of pathway precursors, and biochemical regulatory mechanisms. Protein and phenolic synthesis compete for the common, limiting resource phenylalanine, so protein and phenolic allocation are inversely correlated. Phenolic allocation can be predicted from the effects of development, inherent growth rate and environment on leaf functions that create competing demands for proteins or phenolics. We present the model general principles. We predict phenolic concentrations as leaves develop; in inherently fast versus slow growing species; and in response to the environment (nitrogen, light, phosphorus, heat shock, herbivore and pathogen injury, and carbon dioxide). Because predictions generally fit observed patterns, we argue that, for phenylalanine-derived phenolics, the mechanistically distinctive PCM complements the Growth Differentiation and Resource Availability Hypotheses, and is a viable, testable alternative to the Carbon Nutrient Balance Hypothesis.

A protein competition model of phenolic allocation

Phenylalanine ammonia lyase (PAL; E.C.4.3.1.5), which catalyses the biotransformation of l-phenylalanine to trans-cinnamic acid and ammonia, was first described in 1961 by Koukol and Conn. Since its discovery, much knowledge has been gathered with reference to the enzyme’s catabolic role in microorganisms and its importance in the phenyl propanoid pathway of plants. The 3-dimensional structure of the enzyme has been characterized using X-ray crystallography. This has led to a greater understanding of the mechanism of PAL-catalyzed reactions, including the discovery of a recently described cofactor, 3,5-dihydro-5-methyldiene-4H-imidazol-4-one. In the past 3 decades, PAL has gained considerable significance in several clinical, industrial, and biotechnological applications. The reversal of the normal physiological reaction can be effectively employed in the production of optically pure l-phenylalanine, which is a precursor of the noncalorific sweetener aspartame (l-phenylalanyl-l-aspartyl methyl ester). The...

A modern view of phenylalanine ammonia lyase

Because of its key role in secondary phenylpropanoid metabolism, Phe ammonia-lyase is one of the most extensively studied plant enzymes. To provide a basis for detailed structure–function studies, the enzyme from parsley (Petroselinum crispum) was crystallized, and the structure was elucidated at 1.7-A resolution. It contains the unusual electrophilic 4-methylidene-imidazole-5-one group, which is derived from a tripeptide segment in two autocatalytic dehydration reactions. The enzyme resembles His ammonia-lyase from the general His degradation pathway but contains 207 additional residues, mainly in an N-terminal extension rigidifying a domain interface and in an inserted α-helical domain restricting the access to the active center. Presumably, Phe ammonia-lyase developed from His ammonia-lyase when fungi and plants diverged from the other kingdoms. A pathway of the catalyzed reaction is proposed in agreement with established biochemical data. The inactivation of the enzyme by a nucleophile is described in detail.

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Structural Basis for the Entrance into the Phenylpropanoid Metabolism Catalyzed by Phenylalanine Ammonia-Lyase

In order to provide technical information necessary for the production of L-phenylalanine ammonia-lyase (PAL) by utilizing genetic engineering techniques, the structural gene for PAL and the amino acid sequence of PAL have been elucidated in Rhodosporidium toruloides , and novel recombinant DNA plasmids (e.g., pSW101, pYtrp6 and pKY201) have been created by inserting a DNA strand coding for the PAL gene between the 3ʹ-terminus of the promoter region and the 5ʹ-terminus of the terminator region. Moreover, transformants having such a novel recombinant DNA plasmid have been created, and a process for the production of PAL by growing such a novel transformant so as to cause PAL to be produced and accumulated in the culture has been established. Furthermore, there has been established a novel technique for the production of L-phenylalanine by reacting an ammonia donor with cinnamic acid in the presence of the PAL prepared by the aforesaid novel process.

L-phenylalanine ammonia lyase

We found the occurrence of thermophilic reversible γ-resorcylate decarboxylase (γ-RDC) in the cell extract of a bacterium isolated from natural water, Rhizobium sp. strain MTP-10005, and purified the enzyme to homogeneity. The molecular mass of the enzyme was determined to be about 151 kDa by gel filtration, and that of the subunit was 37.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; in other words, the enzyme was a homotetramer. The enzyme was induced specifically by the addition of γ-resorcylate to the medium. The enzyme required no coenzyme and did not act on 2,4-dihydroxybenzoate, 2,5-dihydroxybenzoate, 3,4-dihydroxybenzoate, 3,5-dihydroxybenzoate, 2-hydroxybenzoate, or 3-hydroxybenzoate. It was relatively thermostable to heat treatment, and its half-life at 50°C was estimated to be 122 min; furthermore, it catalyzed the reverse carboxylation of resorcinol. The values of kcat/Km (mΜ−1 · s−1) for γ-resorcylate and resorcinol at 30°C and pH 7 were 13.4 and 0.098, respectively. The enzyme contains 327 amino acid residues, and sequence identities were found with those of hypothetical protein AGR C 4595p from Agrobacterium tumefaciens strain C58 (96% identity), 5-carboxyvanillate decarboxylase from Sphingomonas paucimobilis (32%), and 2-amino-3-carboxymuconate-6-semialdehyde decarboxylases from Bacillus cereus ATCC 10987 (26%), Rattus norvegicus (26%), and Homo sapiens (25%). The genes (graA [1,230 bp], graB [888 bp], and graC [1,056 bp]) that are homologous to those in the resorcinol pathway also exist upstream and downstream of the γ-RDC gene. Judging from these results, the resorcinol pathway also exists in Rhizobium sp. strain MTP-10005, and γ-RDC probably catalyzes a reaction just before the hydroxylase in it does.

Thermophilic, Reversible γ-Resorcylate Decarboxylase from Rhizobium sp. Strain MTP-10005: Purification, Molecular Characterization, and Expression

Escherichia coil carrying a hybrid plasmid having been constructed by inserting a desired foreign gene into an expression vector so as to permitting expression of said desired foreign gene therein was cultured at a temperature 40° C. or over so that the expression of said desired foreign gene was suppressed. This E. coil (i.e., transformant) was cultured at 40° C. or over in a first process to suppress the expression of the foreign gene and to support sufficient cell growth and thereafter below 40° C. in a second process to release the suppression of the expression so as to permit effective production of the foreign gene product, which resulted in high concentration of the foreign gene product in the final culture.

Method of regulating expression of a foreign gene by controlling culture temperature and a process of producing a foreign gene product thereby

(57) [Problem] To find a microorganism having nitrile hydrolysis activity, which is useful as a catalyst for producing β-alanine. SOLUTION: The microorganism is alkaligenes sp (Al) Caligenes sp. ) OMT-MY14 (Life Science Deposit No. FERM P-15694) or Aminobacter a belonging to ATCC 23314 and 3 -A method for producing β-alanine, in which a culture solution, cells, processed cells, cell extracts and the like of a microorganism having an activity of hydrolyzing aminopropionitrile are allowed to act on 3-aminopropionitrile.

Production of beta-alanine by microorganism

PURPOSE: To suppress the activity of serine decomposition enzyme and to improve the yield of L-tryptophan, by heat-treating cells of bacteria belonging to Escherichia coli and containing tryptophan synthetase at a specific temperature. CONSTITUTION: The cells of the bacteria belonging to Escherichia coli and containing tryptophan synthetase which is an enzyme catalyzing the synthesis of L- tryptophan from indole and L-serine, e.g. cultured liquid of Escherichia coli MN 10232 (FERM BP-19), Escherichia coli MT 10242 (FERM BP-20) or wet bacterial cells collected from the cultured liquid, are heat-treated at 40W60°C to effect the remarkable suppression of the activity of the serine decomposition enzyme keeping the activity of the tryptophan synthetase. Accordingly, the reaction yield of the L-tryptophan based on serine can be improved without addition of chemical substances to the reaction liquid, and the industrial productivity of the L-tryptophan can be improved. COPYRIGHT: (C)1983,JPO&Japio

Nobuhiro Fukuhara

Papers

L-phenylalanine ammonia lyase

Thermophilic, Reversible γ-Resorcylate Decarboxylase from Rhizobium sp. Strain MTP-10005: Purification, Molecular Characterization, and Expression

Method of regulating expression of a foreign gene by controlling culture temperature and a process of producing a foreign gene product thereby

Production of beta-alanine by microorganism

Method for suppressing activity of serine decomposing enzyme in bacterial cell