ON OF METABOL

ON OF METABOL. ic lesions, the scientists were succeeded in the isolation and characterization of a large number of auxotrophic mutants, covering virtually every step in aromatic amino acid biosynthesis (Euverink et al 1995, O.V.Mosin, 1998). Characterization of these mutants revealed that synthesis of L-phenylalanine and L-tyrosine proceeds via single pathways, involving phenylpyruvate and L-arogenate as intermediates, respectively.

Dehydroquinate (DHQ) dehydratase mutants invariably were also blocked in DHQ synthase, suggesting common control elements or gene clustering (Euverink et al 1992). No mutants were obtained in 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase and prephenate aminotransferase, suggesting the presence of isoenzymes, as has been reported for various other organisms. This was subsequently confirmed in biochemical studies (G.J.W. Euverink). L-Phenylalanine aminotransferase (aroAT) catalyzes the last step in L-phenylalanine biosynthesis.

Enzyme purification studies showed the presence of a minor aroATI activity, coeluting with branchd chain aminotransferase, and a major aroATII activity using both L-phenylalanine and L-tyrosine as substrates (Abou- Zeid et al 1995). L-phenylalanine auxotroph, strain GH141, was subsequently identified as deficient in the aroATII enzyme.

In this strain the minor aroATI activity is responsible for L-phenylalanine biosynthesis; its low specific activity explains the leaky phenotype.

Interestingly, strain GH141 also had lost the ability to grow on L-tyrosine as carbon source. Apparently the aroATII protein of A. methanollca is functioning in both L-phenylalanine biosynthesis and in L-tyrosine catabolism (Abou-Zeid et al 1995; Euverink et al 1995b). Three species of prephenate aminotransferase (PpaATI-III), the first enzyme of L-tyrosine biosynthesis, were chromatographically resolved. PpaATI and PpaATII coeluted with aroATI (branched chain aminotransferase) and with aspartate aminotransferase, respectively.

PpaATIII appeared highly specific for prephenate and thus appears to be the main in vivo PpaAT activity (Abou-Zeid et al 1995). The presence of these three isoenzymes with PpaAT activity explains the failure to isolate L-tyrosine auxotrophic mutants in this step. The product of the PpaAT activity with prephenate, arogenate, is subsequently converted into L-tyrosine by an NAD(P)-dependent L-arogenate dehydrogenase.

Numerous mutants blocked in this step have been isolated, confirming the presence of a single pathway from prephenate towards L-tyrosine in A. methanolica (Abou-Zeid et al 1995; Euverink et al 1995b). The single DAHP synthase enzyme species that can be detected in wild type A, methanolica is sensitive to cumulative feedback inhibition by all three aromatic amino acids.

Partially purified enzyme showed apparent K values of 3, 160 and 180 mM for L-tryptophan, L-phenylalanine and L-tyrosine, respectively. The aromatic amino acids displayed competitive inhibition with respect to E4P. L-Tryptophan and E4P showed uncompetitive and competitive inhibition towards PEP, with apparent K, values of 11 and 530 mM, respectively (de Boer et al 1989). Chorismate mutase functions in L-phenylalanine and L-tyrosine biosynthesis.

The activity of the single chorismate mutase detectable in extracts of the wild type organism was inhibited by both L-phenylalanine and L-tyrosine (apparent K values of 60 and 35 mМ, respectively) (de Boer et al, 1989). Prephenate dehydratase, an enzyme specifically involved in L-phenylalanine biosynthesis, was purified to homogeneity and characterized as a 150 kDa homotetrameric protein with a subunit size of 34 kDa (L. Dijkhuizen, 1996). Kinetic studies showed that this enzyme is allosterically inhibited by L-phenylalanine and activated by L-tyrosine (Euverink et al 1995a). L-Phenylalanine caused an increase in the S for prephenate and a decrease in the F. L-Tyrosine caused a decrease in the S for prephenate and an increase in the V (Euverink et al 1995a). Anthranilate synthase, the first enzyme in the L-tryptophan specific branch, was strongly inhibited by L-tryptophan (L. Dijkhuizen). Addition of the aromatic amino acids, either separately or in combinations, did not result in significant repression of the synthesis of these enzymes (de Boer et al 1989). o-Fluoro- and p-fluorophenylalanine inhibited the activities of chorismate mutase and prephenate dehydratase in vitro (de Boer et al 1990b). Many analog-resistant mutant methylotrophic strains had become unable to grow on L-phenylalanine as carbon source and most likely had lost phenylalanine(analog) transport systems. Several mutants were found to possess either a chorismate mutase or a prephenate dehydratase enzyme which had become completely insensitive to L-phenyalanine(analog) inhibition.

Some prephenate dehydratase mutants were still activated by tyrosine, while others had become insensitive to both phenylalanine and tyrosine (Euverink et al 1995). Gene cloning systems for A. methanolica A. methanolica was found to possess a conjugative plasmid (рМЕАЗОО) which is able to integrate into the chromosome at a specific site (Vrijbbed et al 1994). Recently scientists have completed the nucleotide sequence analysis of рМЕА3ОО-plasmide, revealing a total of twenty open reading frames with relatively little untranslated intervening sequences (L. Dijkhuizen, 1996). The overall G+C content of рМЕАЗОО iwas found to be 69%. This high value is characteristic for actinornycetes. Suitable рМЕАЗОО derivatives were used by scientists as a basis for the construction of E. coli - A. methanollca shuttle vectors, based on the pHSS6 coffil replicon (L. Dijkhuizen, 2000). It was reported by L. Dijkhuizen, that cloning of genes involved in glucose, methanol utilization, and in aromatic amino acid biosynthesis.

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