Identification of a small protein domain present in all plant lineages that confers high prephenate dehydratase activity

Abstract

SUMMARYL-Phenylalanine serves as a building block for the biosynthesis of proteins, but also as a precursor for a wide range of plant-derived compounds essential for plants and animals. Plants can synthesize Phe within the plastids using arogenate as a precursor; however, an alternative pathway using phenylpyruvate as an intermediate, described for most microorganisms, has recently been proposed. The functionality of this pathway requires the existence of enzymes with prephenate dehydratase (PDT) activity (EC 4.2.1.51) in plants. Using phylogenetic studies, functional complementation assays in yeast and biochemical analysis, we have identified the enzymes displaying PDT activity in Pinus pinaster. Through sequence alignment comparisons and site-directed mutagenesis we have identified a 22-amino acid region conferring PDT activity (PAC domain) and a single Ala314 residue critical to trigger this activity. Our results demonstrate that all plant clades include PAC domain-containing ADTs, suggesting that the PDT activity, and thus the ability to synthesize Phe using phenylpyruvate as an intermediate, has been preserved throughout the evolution of plants. Moreover, this pathway together with the arogenate pathway gives plants a broad and versatile capacity to synthesize Phe and its derived compounds. PAC domain-containing enzymes are also present in green and red algae, and glaucophytes, the three emerging clades following the primary endosymbiont event resulting in the acquisition of plastids in eukaryotes. The evolutionary prokaryotic origin of this domain is discussed.

When an enzyme isn’t just an enzyme anymore

“…The new study provides an in-depth analysis of the localization of the six Arabidopsis ADT isoforms, all of which are targeted, by virtue of a transit peptide, to the stroma and stromules of chloroplasts. This extends previous reports on the presence of these six proteins in chloroplasts, as also reported for four ADTs from Pinus pinaster (Rippert et al , 2009; El-Azaz et al , 2016). Bross et al now show that two of the isoforms have additional localizations that point to non-catalytic functions.…”

“…Comprehensive phylogenetic analyses previously classified the plant ADTs into four subgroups that are distinct from the groups encompassing microbial ADTs, including those in the Bacterioidetes/Chlorobi, which represent the closest bacterial homologs (Dornfeld et al , 2014; El-Azaz et al , 2016). This analysis further suggests that plant ADTs diversified following the split of plants and chlorophytes (green algae), with present-day ADTs having evolved from the subfamily I-type enzymes to which ADT2 belongs.…”

Harnessing evolutionary diversification of primary metabolism for plant synthetic biology

“…3). Although model microbes, such as E. coli and yeast, only have prephenate dehydratase (PDT) and dehydrogenase (TyrA p ), some bacteria have ADT and TyrA a enzymes, which likely evolved through enzyme neofunctionalization of PDT and TyrA p , respectively, and switch in their substrate specificity from prephenate to arogenate (136,138,(141)(142)(143)(144)(145)(146). Interestingly, all known plant ADTs are most closely related to those of Chlorobi/Bacteroidetes (137), suggesting that both PPA-AT and ADT enzymes required for the arogenate Phe pathway were transferred from Chlorobi/Bacteroidetes to the common ancestor of green algae and land plants.…”

“…The cytosolic CM orthologs are present in all angiosperms, but appear to be absent in gymnosperms, ferns, mosses, and Amborella trichopoda, an early diverged flowering plant (148,150,152). Because plastidic ADT isoforms having PDT activity were found in Pinus pinaster (144), a part of the alternative phenylpyruvate Phe pathway may take place also in the plastids in nonflowering plants (153). Thus, some variations exist in the phenylpyruvate Phe pathway at least for its enzyme subcellular localization among different plant groups.…”

Moonlighting proteins: putting the spotlight on enzymes

“…The development of additional protein functions might be easier than originally anticipated, and can probably occur over a relatively short evolutionary time, as it has been shown that only a few amino acid changes can be sufficient to implement a new function. [36][37][38][39] In fact, even a single amino acid change can be sufficient to alter protein function. For example, a single substitution is enough to alter the substrate specificity of a β-glucosidase, 40 or to introduce new substrate recognition to a cyclic nucleotide phosphodiesterase.…”