Tyrosine metabolism: identification of a key residue in the acquisition of prephenate aminotransferase activity by 1ß aspartate aminotransferase.

Alternative routes for the post-chorismate branch of the biosynthetic pathway leading to tyrosine exist, the 4-hydroxyphenylpyruvate or the arogenate route. The arogenate route involves the transamination of prephenate into arogenate. In a previous study, we found that, depending on the microorganisms possessing the arogenate route, three different aminotransferases evolved to perform prephenate transamination, that is, 1ß aspartate aminotransferase (1ß AAT), N-succinyl-l,l-diaminopimelate aminotransferase, and branched-chain aminotransferase. The present work aimed at identifying molecular determinant(s) of 1ß AAT prephenate aminotransferase (PAT) activity. To that purpose, we conducted X-ray crystal structure analysis of two PAT competent 1ß AAT from Arabidopsis thaliana and Rhizobium meliloti and one PAT incompetent 1ß AAT from R. meliloti. This structural analysis supported by site-directed mutagenesis, modeling, and molecular dynamics simulations allowed us to identify a molecular determinant of PAT activity in the flexible N-terminal loop of 1ß AAT. Our data reveal that a Lys/Arg/Gln residue in position 12 in the sequence (numbering according to Thermus thermophilus 1ß AAT), present only in PAT competent enzymes, could interact with the 4-hydroxyl group of the prephenate substrate, and thus may have a central role in the acquisition of PAT activity by 1ß AAT.

Lysine catabolism in Streptomyces spp. is primarily through cadaverine: beta-lactam producers also make alpha-aminoadipate.

Genetic and biochemical evidence was obtained for lysine catabolism via cadaverine and delta-aminovalerate in both the beta-lactam producer Streptomyces clavuligerus and the nonproducer Streptomyces lividans. This pathway is used when lysine is supplied as the sole source of nitrogen for the organism. A second pathway for lysine catabolism is present in S. clavuligerus but not in S. lividans. It leads to alpha-aminoadipate, a precursor for beta-lactam biosynthesis. Since it does not allow S. clavuligerus to grow on lysine as the sole nitrogen source, this pathway may be used exclusively to provide a precursor for beta-lactam biosynthesis. beta-Lactam producers were unable to grow well on alpha-aminoadipate as the only nitrogen source, whereas three of seven species not known to produce beta-lactam grew well under the same conditions. Lysine epsilon-aminotransferase, the initial enzyme in the alpha-aminoadipate pathway for lysine catabolism, was detected in cell extracts only from the beta-lactam producers. These results suggest that synthesis of alpha-aminoadipate is exclusively a secondary metabolic trait, present or expressed only in beta-lactam producers, while genes governing the catabolism of alpha-aminoadipate are present or fully expressed only in beta-lactam nonproducers.

A simple method for the preparation of D-alpha-aminoadipic acid.

High quantity (1 g and more) of racemically and chromatographically pure D-alpha-aminoadipic acid was prepared by selective metabolism of the L-isomer of the commercially available DL-alpha-aminoadipate by Pseudomonas putida. The overall yield of this preparation averaged 40%. The final product has [a]25D value of -25 degrees. This procedure can be useful in the synthesis of high purity D-alpha-amino-adipate, a compound shown recently to be a useful tool in the study of neurotransmission mechanism mediating synaptic excitation.

Biosynthesis of stizolobinic acid and stizolobic acid in higher plants. An enzyme system(s) catalyzing the conversion of dihydroxyphenylalanine into stizolobinic acid and stizolobic acid from etiolated seedlings of Stizolobium hassjoo.

It was demonstrated that an enzyme system(s) extracted from etiolated seedlings of Stizolobium hassjoo catalyzed the conversion of L-dihydroxyphenylalanine into stizolobinic acid, alpha-amino-6-carboxy-2-oxo-2H-pyran-3-propionic acid, and stizolobic acid, alpha-amino-6-carboxy-2-oxo-2H-pyran-4-propionic acid, in the presence of NADP+ or NAD+ under aerobic conditions. Enzymically synthesized radioactive stizolobinic acid and stizolobic acid isolated from the reaction mixtures were purified and confirmed to have constant specific radioactivities by cocrystallization with authentic samples. Maximal activity of the enzyme preparation was obtained by using an insoluble polyphenol adsorbent (Polyclar AT) and a reducing agent (araboascorbic acid) in the extraction medium and by subsequent fractionation of the extract with ammonium sulfate followed by Sephadex G-25 gel filtration. Catalytic activity of the enzyme preparation was more unstable under aerobic condition than anaerobic. Attempts to stabilise the enzyme activity were made by the use of many substances which are known to stabilise other enzymes or expected to arrest the inactivation. Evidence is provided in this paper that the previously proposed biosynthetic pathways of stizolobinic acid and stizolobic acid from dihydroxyphenylalanine proceeded in the cell-free system from etiolated seedlings of S. hassjoo.