Abstract
The aromatic amino acids phenylalanine and tyrosine represent essential sources of high value natural aromatic compounds for human health and industry. Depending on the organism, alternative routes exist for their synthesis. Phenylalanine and tyrosine are synthesized either via phenylpyruvate/4-hydroxyphenylpyruvate or via arogenate. In arogenate-competent microorganisms, an aminotransferase is required for the transamination of prephenate into arogenate, but the identity of the genes is still unknown. We present here the first identification of prephenate aminotransferases (PATs) in seven arogenate-competent microorganisms and the discovery that PAT activity is provided by three different classes of aminotransferase, which belong to two different fold types of pyridoxal phosphate enzymes: an aspartate aminotransferase subgroup 1β in tested α- and β-proteobacteria, a branched-chain aminotransferase in tested cyanobacteria, and an N-succinyldiaminopimelate aminotransferase in tested actinobacteria and in the β-proteobacterium Nitrosomonas europaea. Recombinant PAT enzymes exhibit high activity toward prephenate, indicating that the corresponding genes encode bona fide PAT. PAT functionality was acquired without other modification of substrate specificity and is not a general catalytic property of the three classes of aminotransferases.
Highlights
The genes encoding prephenate aminotransferase in arogenate-competent microorganisms for tyrosine synthesis are still unknown
We present here the first identification of prephenate aminotransferases (PATs) in seven arogenate-competent microorganisms and the discovery that PAT activity is provided by three different classes of aminotransferase, which belong to two different fold types of pyridoxal phosphate enzymes: an aspartate aminotransferase subgroup 1 in tested ␣- and -proteobacteria, a branched-chain aminotransferase in tested cyanobacteria, and an N-succinyldiaminopimelate aminotransferase in tested actinobacteria and in the -proteobacterium Nitrosomonas europaea
Identification of Prephenate Aminotransferase in R. meliloti, Synechocystis, and S. avermitilis—To identify the protein carrying PAT activity in bacteria known to use the arogenate route for tyrosine biosynthesis, PAT activity was partially purified from cellular extracts of the ␣-proteobacterium R. meliloti, the cyanobacterium Synechocystis sp. (PCC 6803), and the actinobacterium S. avermitilis in a four-step process
Summary
The genes encoding prephenate aminotransferase in arogenate-competent microorganisms for tyrosine synthesis are still unknown. We present here the first identification of prephenate aminotransferases (PATs) in seven arogenate-competent microorganisms and the discovery that PAT activity is provided by three different classes of aminotransferase, which belong to two different fold types of pyridoxal phosphate enzymes: an aspartate aminotransferase subgroup 1 in tested ␣- and -proteobacteria, a branched-chain aminotransferase in tested cyanobacteria, and an N-succinyldiaminopimelate aminotransferase in tested actinobacteria and in the -proteobacterium Nitrosomonas europaea. Biochemical analyses and mass spectrometry identification were combined to identify the genes responsible for arogenate synthesis in arogenate-competent microorganisms (cyanobacteria, ␣- and -proteobacteria, and actinobacteria). It appeared that bona fide PAT activity can be hosted by three different aminotransferases, which belong to two different fold types of pyridoxal phosphate (PLP)-dependent enzymes.
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