Phenylalanine and tyrosine metabolism in the facultative methylotroph Nocardia sp. 239

Nocardia sp. 239 is able to use l-tyrosine and both d- and l-phenylalanine as carbon-, energy- and nitrogen sources for growth. The catabolism of these compounds is by way of (4-hydroxy)phenylpyruvate and (4-hydroxy)-phenylacetate as intermediates and the pathways merge at the level of homogentisate. The conversion of the amino acids into (4-hydroxy)phenylpyruvate is catalyzed by an inducible NAD-dependent phenylalanine dehydrogenase and l-tyrosine aminotransferase, respectively. Incubation of the organism in media with l-phenylalanine plus phenyl-pyruvate resulted in diauxic growth, with phenylpyruvate used first. Phenylalanine dehydrogenase activity cold only be detected after depletion of phenylpyruvate, in the ensuing second growth phase on l-phenylalanine. During growth on phenylalanine plus methanol, low levels of phenylalanine dehydrogenase were detected and this resulted in simultaneous utilization of the two substrates. Following diepoxyoctane treatment, mutants of Nocardia sp. 239 affected in phenylalanine and phenylpyruvate degradation were isolated. Double mutants blocked in both phenylalanine dehydrogenase and phenylpyruvate decarboxylase completely failed to catabolize phenylalanine. The absence of these enzymes did not affect growth on tyrosine.Abbreviations RuMP ribulose monophosphate - EMS ethylmethanesulphonate - NTG N-methyl-N-nitro-N-nitrosoguanidine     

Anaerobic metabolism of l-phenylalanine via benzoyl-CoA in the denitrifying bacterium Thauera aromatica

The anaerobic metabolism of phenylalanine was studied in the denitrifying bacterium Thauera aromatica, a member of the β-subclass of the Proteobacteria. Phenylalanine was completely oxidized and served as the sole source of cell carbon. Evidence is presented that degradation proceeds via benzoyl-CoA as the central aromatic intermediate; the aromatic ring-reducing enzyme benzoyl-CoA reductase was present in cells grown on phenylalanine. Intermediates in phenylalanine oxidation to benzoyl-CoA were phenylpyruvate, phenylacetaldehyde, phenylacetate, phenylacetyl-CoA, and phenylglyoxylate. The required enzymes were detected in extracts of cells grown with phenylalanine and nitrate. Oxidation of phenylalanine to benzoyl-CoA was catalyzed by phenylalanine transaminase, phenylpyruvate decarboxylase, phenylacetaldehyde dehydrogenase (NAD⁺), phenylacetate-CoA ligase (AMP-forming), enzyme(s) oxidizing phenylacetyl-CoA to phenylglyoxylate with nitrate, and phenylglyoxylate:acceptor oxidoreductase. The capacity for phenylalanine oxidation to phenylacetate was induced during growth with phenylalanine. Evidence is provided that α-oxidation of phenylacetyl-CoA is catalyzed by a membrane-bound enzyme. This is the first report on the complete anaerobic degradation of an aromatic amino acid and the regulation of this process. Received: 6 March 1997 / Accepted: 16 May 1997     

Effect of aromatic acids on protein synthesis in subcellular preparations from the rat brain.

The incorporation of [3H]phenylalanine, [3H]tyrosine, and [3H]tryptophan into protein and amino acyl-tRNA was studied in cell-free preparations from rat brain. Tyrosine and tryptophan inhibited the incorporation of phenylalanine into protein, and tyrosine inhibited the incorporation of phenylalanine and tryptophan into amino acyl-tRNAs. In most cases, homogentisate, phenylpyruvate, and phenyllactate inhibited the incorporation of phenylalanine, tyrosine, and tryptophan into protein and amino acyl-tRNAs, and the incorporation of phenylalanine into polyphenylalanine. All other protein amino acids, and phenylacetate, salicylate, and benzoate were wholly ineffectual. The results suggest that the formation of amino acyl-tRNAs may have been the step which was affected most by the inhibitors. The incorporation data at different concentrations of the aromatic amino acids were fitted to the simple Michaelis equation. Homogentisate and phenylpyruvate generally tended to reduce both Km and V in the incorporation of aromatic amino acids into protein and amino acyl-tRNAs, even if V decreased more than Km.     

Reduced Function of a Phenylacetate-Oxidizing Cytochrome P450 Caused Strong Genetic Improvement in Early Phylogeny of Penicillin-Producing Strains

The single-copy pahA gene from Penicillium chrysogenum encodes a phenylacetate 2-hydroxylase that catalyzes the first step of phenylacetate catabolism, an oxidative route that decreases the precursor availability for penicillin G biosynthesis. PahA protein is homologous to cytochrome P450 monooxygenases involved in the detoxification of xenobiotic compounds, with 84% identity to the Aspergillus nidulans homologue PhacA. Expression level of pahA displays an inverse correlation with the penicillin productivity of the strain and is subject to induction by phenylacetic acid. Gene expression studies have revealed a reduced oxidative activity of the protein encoded by pahA genes from penicillin-overproducing strains of P. chrysogenum compared to the activity conferred by phacA of A. nidulans. Sequencing and expression of wild-type pahA from P. chrysogenum NRRL 1951 revealed that an L181F mutation was responsible for the reduced function in present industrial strains. The mutation has been tracked down to Wisconsin 49-133, a mutant obtained at the Department of Botany of the University of Wisconsin in 1949, at the beginning of the development of the Wisconsin family of strains.     

Alginate oligosaccharides as enhancers of penicillin production in cultures of penicillium chrysogenum

Oligosaccharide fragments were prepared by partial acid hydrolysis of sodium alginate and consisted of oligomannuronate (OM) and oligoguluronate (OG) blocks. Effects of the OM and OG blocks on penicillin G production by P. chrysogenum were investigated. The oligosaccharides were found to cause significant increases in penicillin G yields. OM blocks at concentrations 10 to 100 mug/mL were used to further evaluate the effects of the oligosaccharides, and were found to enhance the production of penicillin G in shaken flask cultures of P. chrysogenum P2 (high penicillin producer) and NRRL 1951 (low penicillin producer) at the test concentrations. There was an approximately 50% maximum increase in penicillin G yield from biomass in P. chrysogenum P2 cultures and 150% in P. chrysogenum NRRL 1951 cultures, when compared to control cultures without the oligosaccharides. (c) 1997 John Wiley & Sons, Inc.     

Biosynthesis of beta-phenethyl alcohol in Candida guilliermondii.

$\text{Candida guilliermondii}$ produced β-phenethyl alcohol and β-phenyllactic acid when grown in a synthetic medium containing L-phenylalanine as sole source of nitrogen. The cell-free preparations from these cells showed the following enzymes: phenylalanine aminotransferase, phenylpyruvate decarboxylase, phenylpyruvate reductase and phenylacetaldehyde reductase. The cell-free preparations of $\text{C. guilliermondii}$ grown in medium with ammonium sulfate, lacked these enzyme activities, indicating the inducible nature of these enzymes. The results indicate the role of β-phenylpyruvate as a key intermediate in the pathway of biosynthesis of β-phenethyl alcohol and β-phenyllactic acid from L-phenylalanine.     

Autophagy deficiency promotes beta-lactam production in Penicillium chrysogenum

We have investigated the significance of autophagy in the production of the β-lactam antibiotic penicillin (PEN) by the filamentous fungus Penicillium chrysogenum. In this fungus PEN production is compartmentalized in the cytosol and in peroxisomes. We demonstrate that under PEN-producing conditions significant amounts of cytosolic and peroxisomal proteins are degraded via autophagy. Morphological analysis, based on electron and fluorescence microscopy, revealed that this phenomenon might contribute to progressive deterioration of late subapical cells. We show that deletion of the P. chrysogenum ortholog of Saccharomyces cerevisiae serine-threonine kinase atg1 results in impairment of autophagy. In P. chrysogenum atg1 cells, a distinct delay in cell degeneration is observed relative to wild-type cells. This phenomenon is associated with an increase in the enzyme levels of the PEN biosynthetic pathway and enhanced production levels of this antibacterial compound.     

Selection of Pseudomonas melanvgenum KY 3987 as a New Ampicillin-producing Bacteria

Further selection for a better strain capable of producing D(?)-α-aminobenzylpenicillin (APc) from 6-aminopenicillanic acid (6–APA) was carried out. Pseudomonas melanogenum KY 3987 was consequently selected as a new strain possessing an APc-specific penicillin acylase.

The acylase could synthesize APc in good yields from 6–APA and phenylglycine ester and form 6–APA only from APc, not from other common penicillins. Since the Pseudomonas acylase was found incapable of forming penicillin G (Pc–G) from 6–APA and phenylacetic acid, in contrast with E. coli and Kluyvera citrophila enzymes, the enzymatic hydrolysate of Pc–G, for example by K. citrophila cells, which contained 6–APA and phenylacetate, became employed as a source of 6–APA instead of purified 6–APA to synthesize APc by the cells of P. melanogenum.     

Effect of aromatic acids on protein synthesis in subcellular preparations from the rat brain

The incorporation of [³H]phenylalanine, [³H]tyrosine, and [³H]tryptophan into protein and amino acyl–tRNA was studied in cell-free preparations from rat brain. Tyrosine and tryptophan inhibited the incorporation of phenylalanine into protein, and tyrosine inhibited the incorporation of phenylalanine and tryptophan into amino acyl–tRNAs. In most cases, homogentisate, phenylpyruvate, and phenyllactate inhibited the incorporation of phenylalanine, tyrosine, and tryptophan into protein and amino acyl–tRNAs, and the incorporation of phenylalanine into polyphenylalanine. All other protein amino acids, and phenylacetate, salicylate, and benzoate were wholly ineffectual. The results suggest that the formation of amino acyl–tRNAs may have been the step which was affected most by the inhibitors. The incorporation data at different concentrations of the aromatic amino acids were fitted to the simple Michaelis equation. Homogentisate and phenylpyruvate generally tended to reduce both K_m and V in the incorporation of aromatic amino acids into protein and amino acyl-tRNAs, even if V decreased more than K_m.     

Effect of phenylalanine derivatives on the main regulatory enzymes of hepatic cholesterogenesis

Phenylalanine, phenylpyruvate and phenylacetate produced a considerable inhibition of chick liver mevalonate 5-pyrophosphate decarboxylase while mevalonate kinase and mevalonate 5-phosphate kinase were not significantly affected. Phenolic derivatives of phenylalanine produced a similar inhibition of decarboxylase activity than that found in the presence of phenyl metabolites. The degree of inhibition was progressive with increasing concentrations of inhibitors (1.25–5.00 mM). Simultaneous supplementation of different metabolites in conditions similar to those in experimental phenylketonuria (0.25 mM each) produced a clear inhibition of liver decarboxylase and 3-hydroxy-3-methylglutaryl-CoA reductase. To our knowledge, this is the first report on the in vitro inhibition of both liver regulatory enzymes of cholesterogenesis in phenylketonuria-like conditions. Our results show a lower inhibition of decarboxylase than that of reductase but suggest an important regulatory role of decarboxylase in cholesterol synthesis.     

Simultaneous Involvement of a Tungsten-Containing Aldehyde:Ferredoxin Oxidoreductase and a Phenylacetaldehyde Dehydrogenase in Anaerobic Phenylalanine Metabolism

Anaerobic phenylalanine metabolism in the denitrifying betaproteobacterium Aromatoleum aromaticum is initiated by conversion of phenylalanine to phenylacetate, which is further metabolized via benzoyl-coenzyme A (CoA). The formation of phenylacetate is catalyzed by phenylalanine transaminase, phenylpyruvate decarboxylase, and a phenylacetaldehyde-oxidizing enzyme. The presence of these enzymes was detected in extracts of cells grown with phenylalanine and nitrate. We found that two distinct enzymes are involved in the oxidation of phenylacetaldehyde to phenylacetate, an aldehyde:ferredoxin oxidoreductase (AOR) and a phenylacetaldehyde dehydrogenase (PDH). Based on sequence comparison, growth studies with various tungstate concentrations, and metal analysis of the enriched enzyme, AOR was shown to be a tungsten-containing enzyme, necessitating specific cofactor biosynthetic pathways for molybdenum- and tungsten-dependent enzymes simultaneously. We predict from the genome sequence that most enzymes of molybdopterin biosynthesis are shared, while the molybdate/tungstate uptake systems are duplicated and specialized paralogs of the sulfur-inserting MoaD and the metal-inserting MoeA proteins seem to be involved in dedicating biosynthesis toward molybdenum or tungsten cofactors. We also characterized PDH biochemically and identified both NAD⁺ and NADP⁺ as electron acceptors. We identified the gene coding for the enzyme and purified a recombinant Strep-tagged PDH variant. The homotetrameric enzyme is highly specific for phenylacetaldehyde, has cooperative kinetics toward the substrate, and shows considerable substrate inhibition. Our data suggest that A. aromaticum utilizes PDH as the primary enzyme during anaerobic phenylalanine degradation, whereas AOR is not essential for the metabolic pathway. We hypothesize a function as a detoxifying enzyme if high aldehyde concentrations accumulate in the cytoplasm, which would lead to substrate inhibition of PDH.     

Why did the Fleming strain fail in penicillin industry?

Penicillin, discovered 75 years ago by Sir Alexander Fleming in Penicillium notatum, laid the foundations of modern antibiotic chemotherapy. Early work was carried out on the original Fleming strain, but it was later replaced by overproducing strains of Penicillium chrysogenum, which became the industrial penicillin producers. We show how a C(1357)-->T (A394V) change in the gene encoding PahA in P. chrysogenum may help to explain the drawback of P. notatum. PahA is a cytochrome P450 enzyme involved in the catabolism of phenylacetic acid (PA; a precursor of penicillin G). We expressed the pahA gene from P. notatum in P. chrysogenum obtaining transformants able to metabolize PA (P. chrysogenum does not), and observing penicillin production levels about fivefold lower than that of the parental strain. Our data thus show that a loss of function in P. chrysogenum PahA is directly related to penicillin overproduction, and support the historic choice of P. chrysogenum as the industrial producer of penicillin.     

THE EFFECTS OF PHENYLKETONURIC AND OTHER METABOLITES ON SULFATED GALACTOCEREBROSIDE SYNTHESIS IN VIVO AND IN CULTURE

Abstract— Sulfated galactocerebroside synthesis was examined in vitro in mouse spinal cord cultures. This system permitted the study of the effects of phenylketonuric metabolites upon synthesis of a specific myelin component, sulfatide, formed early in postnatal development in mice. A significant reduction of Na₂³⁵SO₄ incorporation into myelin sulfatide was observed when spinal cord cultures were grown in the presence of 1000 μm -l -phenylalanine and 500 μm -phenylpyruvate (51 and 700%, respectively). No reduction was observed with β-phenyllactate (300 μm and) phenylacetate (250 μm ). Light microscopy indicated that the phenylpyruvate and phenylalanine treated cultures were less extensively myelinated compared to control and β-phenyllactate or phenylacetate treated cultures. The reduction of sulfatide synthesis by phenylpyruvate was shown to be reversible. Intracerebral bilateral injections (8 μg) of l -phenylalanine, phenylpyruvate, α-ketobutyrate, α-ketoisocaproate, α-ketoisovalerate, β-phenyllactate, and phenylacetate in mice 8–15 days old, followed by i.p. administration of radioactive sulfate, resulted in significantly reduced incorporation (all P < 0.05) of sulfate into brain sulfatides with all compounds tested with the exception of β-phenyllactate and phenylacetate. In adult mouse, phenylpyruvate treatment also resulted in a significant decrease in labelling of brain sulfatide. The effects of phenylpyruvate and other metabolites upon pyruvate oxidation in mouse brain homogenates were examined by measuring ¹⁴CO₂ release from [1-¹⁴C]pyruvate. Both phenylpyruvate and α-ketoisocaproate at 1 × 10^-3 resulted in a decrease in ¹⁴CO₂ produced, while phenylacetate and β-phenyllactate had no effect. Sulfate incorporation into sulfatide was reduced by α-ketoisocaproate and phenylpyruvate, and to a lesser extent by phenylalanine, α-ketobutyrate, and α-ketoisovalerate. Phenyllactate and phenylacetate had no effect, either in vivo, or in culture. This order of effectiveness may be related in part to the effects of these compounds on pyruvate oxidation.