Three Different Classes of Aminotransferases Evolved Prephenate Aminotransferase Functionality in Arogenate-competent Microorganisms

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.

Phylobiochemical Characterization of Class-Ib Aspartate/Prephenate Aminotransferases Reveals Evolution of the Plant Arogenate Phenylalanine Pathway

“…coli and yeast, two extensively studied model microbes, use exclusively the phenylpyruvate/4-hydroxyphenylpyruvate pathways for Phe/Tyr biosynthesis and do not have PPA-AT genes and activity (Gelfand and Steinberg, 1977;Urrestarazu et al, 1998). However, our analysis and the most recent study (Graindorge et al, 2014) revealed that aminotransferases having PPA-AT activity are present not only in plants but also in a broader range of microbes. Plant PPA-ATs belong to the AspAT Ib class (Graindorge et al, 2010;Dal Cin et al, 2011;Maeda et al, 2011), which falls within subfamily Ig (Mehta et al, 1993;Jensen and Gu, 1996) and is distantly related to class-Ia AspATs (subfamily Ia) commonly present in all organisms (Alfano and Kahn, 1993;Okamoto et al, 1996;Nobe et al, 1998).…”

“…The Synechocystis enzyme also displayed strong Asp-AT activity (K m of 486 mM, k cat of 54.2 s 21 ; Table 1) similar to the C. tepidum and Arabidopsis enzymes. Although the same Synechocystis enzyme was reported to be devoid of PPA-AT activity in a previous study based on a coupled spectrophotometric assay (Graindorge et al, 2014), we detected some PPA-AT activity through direct detection of the arogenate product by HPLC; however, its K m (2.5 mM) and k cat (1 s 21 ) with prephenate were much higher and lower, respectively, than those of C. tepidum and Arabidopsis enzymes (Table 1). Taken together, these results show that, while all three enzymes exhibit comparable Asp-AT activity, the PPA-AT activity of the C. tepidum enzyme is more similar to that of Arabidopsis PPA-AT than that of the Synechocystis enzyme.…”

“…a-proteobacteria (Rhizobium meliloti and Rhodobacter sphaeroides; Graindorge et al, 2014). The most extensively studied AspAT Ib enzyme from T. thermophilus (Nobe et al, 1998;Nakai et al, 1999;Ura et al, 2001) also showed a comparable level of PPA-AT activity to plant and C. tepidum enzymes (Figure 4; Supplemental Table 3).…”

“…PPA-AT activities have been detected in plants and some microbes (Stenmark et al, 1974;Fazel and Jensen, 1979;Bonner and Jensen, 1985;Siehl et al, 1986;Abou-Zeid et al, 1995). Three types of aminotransferases, including class-Ib aspartate aminotransferases (AspAT Ibs), branched-chain aminotransferases (BCATs), and N-succinyl-diaminopimelate aminotransferases (S-DAPAT), were recently shown to have PPA-AT activity in different microbes (Graindorge et al, 2014). Several lines of evidence support that AspAT Ib-type PPA-AT is the major enzyme responsible for PPA-AT activity in plants.…”

“…In addition, detailed biochemical characterizations of C. tepidum (Chlorobi) and Synechocystis (cyanobacterium) PPA-AT homologs showed that the C. tepidum homolog exhibits biochemical properties more similar to plant PPA-ATs (e.g., strong PPA-AT activity; Graindorge et al, 2010;Maeda et al, 2011) than does the Synechocystis counterpart (Table 1). The weak to no PPA-AT activity detected in the Synechocystis AspAT Ib enzyme (Table 1; Graindorge et al, 2014) is consistent with the presence of BCAT-type enzymes having strong PPA-AT activity with broad substrate specificity in cyanobacteria (Stenmark et al, 1974;Jensen and Stenmark, 1975;Graindorge et al, 2014). Also, the ADT homolog of C. tepidum, previously reported as PDT (Tan et al, 2008), showed 10-fold higher catalytic efficiency for ADT than PDT activity (Table 2), whereas cyanobacteria such as Synechocystis process a bifunctional CM-PDT enzyme (Kaneko et al, 1996) and lack ADT activity (Stenmark et al, 1974;Hall et al, 1982).…”