Catabolism of dl-α-phenylhydracrylic,phenylacetic and 3- and 4-hydroxyphenylacetic acid via homogentisic acid in a Flavobacterium sp.

A degradation pathway for dl--phenylhydracrylic, phenylacetic, 3- and 4-hydroxyphenylacetic acid by a Flavobacterium is presented. Experiments with washed cells and enzyme studies revealed that dl--phenylhydracrylic acid in an initial reaction was oxidatively decarboxylated to phenylacetaldehyde. Whole cells oxidized both stereoisomers of phenylhydracrylic acid at different rates. The product phenylacetaldehyde in turn was oxidized to phenylacetic acid. No hydroxylation of phenylacetic acid was detected in cell extracts, but on the basis of experiments with washed cells it is assumed that phenylacetic acid is mainly metabolized via 3-hydroxyphenylacetic acid. This latter product was subsequently hydroxylated yielding the ring-cleavage substrate homogentisate. 4-Hydroxyphenylacetic acid was also degraded via homogentisate. Ringcleavage of homogentisate gave maleylacetoacetate which was further degraded through a glutathione-dependent pathway. Homoprotocatechuate was not an intermediate in the metabolism of dl-phenylhydracrylic acid, phenylacetic, 3- and 4-hydroxyphenylacetic acid metabolism, but it could be hydroxylated aspecifically to 2,4,5-trihydroxyphenylacetic acid by the action of the 3-hydroxyphenylacetic acid-6-hydroxylase.Abbreviations HPLC high-performance liquid chromatography - PHA phenylhydracrylic acid - PA phenylacetic acid - HPA hyxdroxyphenylacetic acid - PMS phenazine methosulphate - PMA phenylmalonic acid - GSH glutathione     

Regulation of the 4-hydroxyphenylacetic acid meta-cleavage pathway in an Acinetobacter sp

Lactate-grown cultures of $\text{Acinetobacter}$ sp. strain 3B-1 synthesize constitutively all enzymes except the 4-hydroxyphenylacetic acid-3-hydroxylase. All enzymes are further synthesized when strain 3B-1 is grown with 4-hydroxyphenylacetic acid. Induction studies with two mutant strains, one defective in the 3-hydroxylase, and the other defective in the dehydrogenase, indicate that 4-hydroxyphenylacetic acid induces the 3-hydroxylase only, and the second metabolite 3,4-dihydroxyphenylacetic acid appears to induce 3,4-dihydroxyphenylacetic acid-2,3-dioxygenase and subsequent enzymes. Thus, the enzymes of the 4-hydroxyphenylacetic acid $\text{meta}$-cleavage pathway are synthesized following at least two sequential inductive events.     

Studies on Metabolic Products of Hypochnus sasakii Shirai

A considerable amount of p-hydroxyphenylacetic acid was isolated from the culture filtrate of Hypochnus sasakii.

p-Hydroxyphenylacetic acid induced wilt of soybean plant and retarded the growth of rice seedlings at concentrations up to 1:20,000. The O₂ uptake in roots decreased in proportion to reduction of dry weight of rice seedlings.     

Metabolism of lunularic acid in liverworts

The presence, in Conocephalum conicum, of the enzymes phenylalanine ammonia lyase, trans-cinnamic acid-4-hydroxylase and lunularic acid decarbo     

Purification and properties of two succinic semialdehyde dehydrogenases from Klebsiella pneumoniae

Two forms of succinic semialdehyde dehydrogenase have been isolated in Klebsiella pneumoniae M5a1. The two enzymes could be separated by filtration on Sephacryl S-300 and their apparent molecular weights were approx. 275,000 and 300,000. The large enzyme is specific for NADP. The smaller enzyme, which is induced by growth on 3-hydroxyphenylacetic acid, 4-hydroxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid and gamma-aminobutyrate, has been purified to 96% homogeneity by affinity chromatography. The NAD-linked succinic semialdehyde dehydrogenase was able to use NADP as cofactor. Its induction is coordinated with 3- and 4-hydroxylase, the enzymes which initiate degradation of 3- and 4-hydroxyphenylacetic acid. The NAD-linked form is also induced by exogenous succinic semialdehyde. The large enzyme is specific for NADP and has been isolated from a defective mutant which lacked the activity of the NAD-linked succinic semialdehyde dehydrogenase. Activity and stability conditions and true K m values for substrates and cosubstrates of the two enzymes were determined. Some aspects of the induction of the NAD-linked enzyme participating in the metabolism of 4-hydroxyphenylacetic and gamma-aminobutyrate were studied.     

Separation of acetanilide and its hydroxylated metabolites and quantitative determination of "acetanilide 4-hydroxylase activity" by high-pressure liquid chromatography.

A simple and very sensitive method for the separation of 4-hydroxyacetanilide, 3-hydroxyacetanilide, 2-hydroxyacetanilide, and acetanilide was developed with the use of high-pressure liquid chromagraphy. Each of these phenolic derivatives can be separated completely from acetanilide and from one another. A simple assay for “acetanilide 4-hydroxylase activity” is thus described. The limit of sensitivity for cytochrome P-450-mediated acetanilide 4-hydroxylase activity is estimated to be 1.0 pmol/min/mg microsomal protein, thereby allowing this assay to be useful in detecting monooxygenase activity in “low level” nonhepatic tissues. Hepatic acetanilide 4-hydroxylase activity is induced about fourfold in $\text{C57BL}\text{6N}$ mice by 3-methylcholanthrene. Although acetanilide 2-hydroxylase activity is about seven times lower than the 4-hydroxylase activity, the 2-hydroxylase is also induced about three- or fourfold in $\text{C57BL}\text{6N}$ mice by 3-methylcholanthrene. The “2-hydroxylase activity” cannot, however, be strictly quantitated under the conditions described herein. The K_m values of both the 3-methylcholanthrene-induced and control 4-hydroxylase activity are about 0.55 mm; V_max values for 3-methylcholanthrene-treated and control mice, respectively, are 4.9 ± 1.1 and 1.1 ± 0.31 nmol/min/mg microsomal protein. The 4-hydroxylase in the liver of both 3-methylcholanthrene-treated and control mice appears to represent two or more catalytic activities, i.e., two or more forms of P-450 having widely differing affinities for the substrate acetanilide.     

Characterization of 4-Hydroxyphenylacetate 3-Hydroxylase (HpaB) of Escherichia coli as a Reduced Flavin Adenine Dinucleotide-Utilizing Monooxygenase

4-Hydroxyphenylacetate 3-hydroxylase (HpaB and HpaC) of Escherichia coli W has been reported as a two-component flavin adenine dinucleotide (FAD)-dependent monooxygenase that attacks a broad spectrum of phenolic compounds. However, the function of each component in catalysis is unclear. The large component (HpaB) was demonstrated here to be a reduced FAD (FADH₂)-utilizing monooxygenase. When an E. coli flavin reductase (Fre) having no apparent homology with HpaC was used to generate FADH₂ in vitro, HpaB was able to use FADH₂ and O₂ for the oxidation of 4-hydroxyphenylacetate. HpaB also used chemically produced FADH₂ for 4-hydroxyphenylacetate oxidation, further demonstrating that HpaB is an FADH₂-utilizing monooxygenase. FADH₂ generated by Fre was rapidly oxidized by O₂ to form H₂O₂ in the absence of HpaB. When HpaB was included in the reaction mixture without 4-hydroxyphenylacetate, HpaB bound FADH₂ and transitorily protected it from rapid autoxidation by O₂. When 4-hydroxyphenylacetate was also present, HpaB effectively competed with O₂ for FADH₂ utilization, leading to 4-hydroxyphenylacetate oxidation. With sufficient amounts of HpaB in the reaction mixture, FADH₂ produced by Fre was mainly used by HpaB for the oxidation of 4-hydroxyphenylacetate. At low HpaB concentrations, most FADH₂ was autoxidized by O₂, causing uncoupling. However, the coupling of the two enzymes' activities was increased by lowering FAD concentrations in the reaction mixture. A database search revealed that HpaB had sequence similarities to several proteins and gene products involved in biosynthesis and biodegradation in both bacteria and archaea. This is the first report of an FADH₂-utilizing monooxygenase that uses FADH₂ as a substrate rather than as a cofactor.     

Properties of a Mixed Function Oxygenase Catalyzing Ipomeamarone 15-Hydroxylation in Microsomes from Cut-Injured and Ceratocystis fimbriata-Infected Sweet Potato Root Tissues

Ipomeamarone 15-hydroxylase activity was found in a microsomal fraction from cut-injured and Ceratocystis fimbriata-infected sweet potato (Ipomoea batatas Lam. cv. Norin No. 1) root tissues and its optimum pH was 8.0. The enzyme reaction required O₂ and NADPH. The K_m values calculated for ipomeamarone and NADH were approximately 60 and 2 micromolar, respectively. NADPH alone had little effect on enzyme activity but activated the reaction in the presence of low concentrations of NADPH. Ipomeamarone 15-hydroxylase activity was strongly inhibited by p-chloromercuribenzoic acid and markedly suppressed by cytochrome c and p-benzoquinone. KCN was an activator rather than an inhibitor for the reaction. CO inhibited the activity strongly and its inhibition was partially reversed by light. CO difference spectra of the reduced microsomal fraction showed two absorption maxima at 423 and 453 nm; the latter maximum may be due to a cytochrome P-450. These results suggest that ipomeamarone 15-hydroxylase is a cytochrome P-450-dependent, mixed-function oxygenase.

Ipomeamarone 15-hydroxylase activity was not found in fresh tissue of sweet potato roots. However, the activity appeared and increased markedly in response to cut-injury or infection by Ceratocystis fimbriata, and reached a maximum after 24 to 36 hours of incubation. The increase in activity in the latter case was 3- to 5-fold higher than in the former. The time course patterns of development and successive decline in ipomeamarone hydroxylase activities were similar to those for cinnamic acid 4-hydroxylase activity, which had been described as a cytochrome P-450-dependent, mixed-function oxygenase. However, little substrate competition was found between ipomeamarone 15-hydroxylase and cinnamic acid 4-hydroxylase in our preparations.

     

Effect of Δ9-stearoyl-ACP-desaturase-C mutants in a high oleic background on soybean seed oil composition

Key message

Two new sources of elevated seed stearic acid were identified and the feasibility of an elevated stearic acid, high oleic acid germplasm was studied.

Abstract

Soybean [Glycine max (L.) Merr.] oil typically contains 2–4 % stearic acid. Oil with at least 20 % stearic acid is desirable because of its improved baking properties and health profile. This study identifies two new sources of high stearic acid and evaluates the interaction of high stearic and oleic acid alleles. TCHM08-1087 and TCHM08-755, high stearic acid ‘Holladay’ mutants, were crossed to FAM94-41-3, a line containing a point mutation in a seed-specific isoform of a Δ9-stearoyl-acyl carrier protein-desaturase (SACPD-C). F₂-derived lines were evaluated for fatty acid content in four field environments. Sequencing of SACPDs in TCHM08-1087 and TCHM08-755 revealed distinct deletions of at least one megabase encompassing SACPD-C in both lines. After genotyping, the additive effect for stearic acid was estimated at +1.8 % for the SACPD-C point mutation and +4.1 % for the SACPD-C deletions. Average stearic acid in lines homozygous for the deletions was 12.2 %. A FAM94-41-3-derived line and TCHM08-1087-11, a selection from TCHM08-1087, were crossed to S09-2902-145, a line containing missense mutations in two fatty acid desaturases (FAD2-1A and FAD2-1B). F₁ plants were grown in a greenhouse and individual F₂ seed were genotyped and phenotyped. No interaction was observed between either FAD2-1A or FAD2-1B and any of the SACPD-C mutant alleles. Seed homozygous mutant for SACPD-C/FAD2-1A/FAD2-1B contained 12.7 % stearic acid and 65.5 % oleic acid while seed homozygous for the SACPD-C deletion and mutant for FAD2-1A and FAD2-1B averaged 10.4 % stearic acid and 75.9 % oleic acid.     

Biotransformation of Phenylacetonitrile to 2-Hydroxyphenylacetic Acid by Marine Fungi

Marine fungi belonging to the genera Aspergillus, Penicillium, Cladosporium, and Bionectria catalyzed the biotransformation of phenylacetonitrile to 2-hydroxyphenylacetic acid. Eight marine fungi, selected and cultured with phenylacetonitrile in liquid mineral medium, catalyzed it quantitative biotransformation to 2-hydroxyphenylacetic acid. In this study, the nitrile group was firstly hydrolysed, and then, the aromatic ring was hydroxylated, producing 2-hydroxyphenylacetic acid with 51 % yield isolated. In addition, the 4-fluorophenylacetonitrile was exclusively biotransformed to 4-fluorophenylacetic acid by Aspergillus sydowii Ce19 (yield?=?51 %). The enzymatic biotransformation of nitriles is not trivial, and here, we describe an efficient method for production of phenylacetic acids in mild conditions.     

Stereospecificity of plant microsomal cinnamic acid 4-hydroxylase

Cinnamic acid 4-hydroxylase from parsley cell suspension cultures is specific for trans-cinnamic acid as substrate. cis-Cinnamic acid is a competitive inhibitor of the enzyme with a K_i of approximately 0.34 mm. Hydrocinnamic acid is neither a substrate nor an inhibitor.     

Metabolic and bioprocess engineering of the yeast Candida famata for FAD production

Flavins in the form of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) play an important role in metabolism as cofactors for oxidoreductases and other enzymes. Flavin nucleotides have applications in the food industry and medicine; FAD supplements have been efficiently used for treatment of some inheritable diseases. FAD is produced biotechnologically; however, this compound is much more expensive than riboflavin. Flavinogenic yeast Candida famata synthesizes FAD from FMN and ATP in the reaction catalyzed by FAD synthetase, a product of the FAD1 gene. Expression of FAD1 from the strong constitutive promoter TEF1 resulted in 7- to 15-fold increase in FAD synthetase activity, FAD overproduction, and secretion to the culture medium. The effectiveness of FAD production under different growth conditions by one of these recombinant strains, C. famata T-FD-FM 27, was evaluated. First, the two-level Plackett–Burman design was performed to screen medium components that significantly influence FAD production. Second, central composite design was adopted to investigate the optimum value of the selected factors for achieving maximum FAD yield. FAD production varied most significantly in response to concentrations of adenine, KH₂PO₄, glycine, and (NH₄)₂SO₄. Implementation of these optimization strategies resulted in 65-fold increase in FAD production when compared to the non-optimized control conditions. Recombinant strain that has been cultivated for 40 h under optimized conditions achieved a FAD accumulation of 451 mg/l. So, for the first time yeast strains overproducing FAD were obtained, and the growth media composition for maximum production of this nucleotide was designed.     

Development of low-linolenic acid Brassica oleracea lines through seed mutagenesis and molecular characterization of mutants

Designing the fatty acid composition of Brassica napus L. seed oil for specific applications would extend the value of this crop. A mutation in Fatty Acid Desaturase 3 (FAD3), which encodes the desaturase responsible for catalyzing the formation of α-linolenic acid (ALA; 18:3 ^cisΔ9,12,15), in a diploid Brassica species would potentially result in useful germplasm for creating an amphidiploid displaying low ALA content in the seed oil. For this, seeds of B. oleracea (CC), one of the progenitor species of B. napus, were treated with ethyl-methane-sulfonate to induce mutations in genes encoding enzymes involved in fatty acid biosynthesis. Seeds from 1,430 M₂ plants were analyzed, from which M₃ seed families with 5.7–6.9 % ALA were obtained. Progeny testing and selection for low ALA content were carried out in M₃–M₇ generations, from which mutant lines with <2.0 % ALA were obtained. Molecular analysis revealed that the mutation was due to a single nucleotide substitution from G to A in exon 3 of FAD3, which corresponds to an amino acid residue substitution from glutamic acid to lysine. No obvious differences in the expression of the FAD3 gene were detected between wild type and mutant lines; however, evaluation of the performance of recombinant Δ-15 desaturase from mutant lines in yeast indicated reduced production of ALA. The novelty of this mutation can be inferred from the position of the point mutation in the C-genome FAD3 gene when compared to the position of mutations reported previously by other researchers. This B. oleracea mutant line has the potential to be used for the development of low-ALA B. napus and B. carinata oilseed crops.     

Production of Skatole and para-Cresol by a Rumen Lactobacillus sp.

The objective of this study was to examine the substrate specificity of several ruminal strains of a Lactobacillus sp. which previously was shown to produce skatole (3-methylindole) by the decarboxylation of indoleacetic acid. A total of 13 compounds were tested for decarboxylase activity. The Lactobacillus strains produced p-cresol (4-methylphenol) by the decarboxylation of p-hydroxyphenylacetic acid, but did not produce either o-cresol or m-cresol from the corresponding hydroxyphenylacetic acid isomers. These strains also decarboxylated 5-hydroxyindoleacetic acid to 5-hydroxyskatole and 3,4-dihydroxyphenylacetic acid to methylcatechol. Skatole and p-cresol were produced in a 0.5:1 ratio, when indoleacetic acid and p-hydroxyphenylacetic acid were combined in equimolar concentrations. Competition studies with indoleacetic acid and p-hydroxyphenylacetic acid suggested that two different decarboxylating enzymes are involved in the production of skatole and p-cresol by these strains. This is the first demonstration of both skatole production and p-cresol production by a single bacterium.     

Activity of enzymes involved in lignin biosynthesis in swede root disks

The activity of nine enzymes involved in the biosynthesis of lignin precursors has been studied during the ageing of swede root disks in the presence and absence of ethylene. Peroxidase, aromatic alcohol dehydrogenase and phenylalanine transaminase show very little change in activity during ageing under either ageing condition. O-methyl transferase, shikimate dehydrogenase and ferulyl CoA reductase show only a 2–3 fold increase on ageing and are relatively insensitive to ethylene treatment. A third group (comprising phenylalanine ammonia lyase, cinnamic acid-4-hydroxylase and hydroxycinnamate CoA ligase) show 20–30 fold increase on ageing and are most sensitive to ethylene treatment. Phenylalanine ammonia lyase and cinnamic acid-4-hydroxylase behave very similarly in respect of their time course of ageing and in their responses to metabolic inhibitors such as cycloheximide, puromycin and actinomycin D. In addition the properties of the O-methyl transferase of swede root tissue are described.     

Cytochrome P-450-Dependent omega-Hydroxylation of Lauric Acid by Microsomes from Pea Seedlings

Microsomes from apical buds of pea (Pisum sativum L. var. Téléphone à rames) seedlings hydroxylate lauric acid at the ω-position. This oxidation is catalyzed by a cytochrome P-450 enzyme which differs from laurate hydroxylases previously described in microorganisms and mammals by its strict substrate specificity and the ability of low NADH concentrations to support unusually high oxidation rates. The apparent K_m for lauric acid was 20 micromolar. NADPH- and NADH-dependent laurate hydroxylation followed non-Michaelian kinetics with apparent K_m values ranging from 0.2 to 28 micromolar for NADPH, and 0.2 to 318 micromolar for NADH. When induced by the photomorphogenic photoreceptor phytochrome, the time course for the enhancement of laurate ω-hydroxylase was totally different from that of the cinnamic acid 4-hydroxylase, providing evidence for the existence of multiple cytochrome P-450 species in pea microsomes.     

Degradation of 4-chlorophenylacetic acid by a Pseudomonas species.

Pseudomonas sp. strain CBS3 was able to utilize 4-chlorophenylacetic acid as the sole source of carbon and energy. When this strain was grown with 4-chlorophenylacetic acid, homoprotocatechuic acid was found to be an intermediate which was further metabolized by the meta-cleavage pathway. Furthermore, three isomers of chlorohydroxyphenylacetic acid, two of them identified as 3-chloro-4-hydroxyphenylacetic acid and 4-chloro-3-hydroxyphenylacetic acid, were isolated from the culture medium. 4-Hydroxyphenylacetic acid was catabolized in a different manner by the glutathione-dependent homogentisate pathway. Degradation enzymes of both of these pathways were inducible.     

A rapid assay for prolyl 3-hydroxylase activity.

An assay is reported for prolyl 3-hydroxylase activity. The method is based on the release of tritiated water (THO) during 3-hydroxylation of a 2,3-T-l-proline-labeled (T = tritium) polypeptide substrate in which all prolyl residues recognized by prolyl 4-hydroxylase have been converted to 4-hydroxyprolyl residues. The formation of THO was essentially linear with enzyme concentration and time, and the K_m for the polypeptide substrate was about 3.4 × 10^?8m. A linear correlation was found between THO release and the synthesis of 3-hydroxyproline, the latter being analyzed by amino acid analyzer. The assay is simple, rapid, sensitive, and reproducible, and it is specific even in tissue samples containing a large excess of prolyl 4-hydroxylase activity.     

Functional Characterization of Flax Fatty Acid Desaturase FAD2 and FAD3 Isoforms Expressed in Yeast Reveals a Broad Diversity in Activity

With 45 % or more oil content that contains more than 55 % alpha linolenic (LIN) acid, linseed (Linum usitatissimum L.) is one of the richest plant sources of this essential fatty acid. Fatty acid desaturases 2 (FAD2) and 3 (FAD3) are the main enzymes responsible for the Δ12 and Δ15 desaturation in planta. In linseed, the oilseed morphotype of flax, two paralogous copies, and several alleles exist for each gene. Here, we cloned three alleles of FAD2A, four of FAD2B, six of FAD3A, and seven of FAD3B into a pYES vector and transformed all 20 constructs and an empty construct in yeast. The transformants were induced in the presence of oleic (OLE) acid substrate for FAD2 constructs and linoleic (LIO) acid for FAD3. Conversion rates of OLE acid into LIO acid and LIO acid into LIN acid were measured by gas chromatography. Conversion rate of FAD2 exceeded that of FAD3 enzymes with FAD2B having a conversion rate approximately 10 % higher than FAD2A. All FAD2 isoforms were active, but significant differences existed between isoforms of both FAD2 enzymes. Two FAD3A and three FAD3B isoforms were not functional. Some nonfunctional enzymes resulted from the presence of nonsense mutations causing premature stop codons, but FAD3B-C and FAD3B-F seem to be associated with single amino acid changes. The activity of FAD3A-C was more than fivefold greater than the most common isoform FAD3A-A, while FAD3A-F was fourfold greater. Such isoforms could be incorporated into breeding lines to possibly further increase the proportion of LIN acid in linseed.