Functional characterization of Arabidopsis thaliana transthyretin-like protein

Abstract

BackgroundArabidopsis thaliana transthyretin-like (TTL) protein is a potential substrate in the brassinosteroid signalling cascade, having a role that moderates plant growth. Moreover, sequence homology revealed two sequence domains similar to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) decarboxylase (N-terminal domain) and 5-hydroxyisourate (5-HIU) hydrolase (C-terminal domain). TTL is a member of the transthyretin-related protein family (TRP), which comprises a number of proteins with sequence homology to transthyretin (TTR) and the characteristic C-terminal sequence motif Tyr-Arg-Gly-Ser. TRPs are single domain proteins that form tetrameric structures with 5-HIU hydrolase activity. Experimental evidence is fundamental for knowing if TTL is a tetrameric protein, formed by the association of the 5-HIU hydrolase domains and, in this case, if the structural arrangement allows for OHCU decarboxylase activity. This work reports about the biochemical and functional characterization of TTL.ResultsThe TTL gene was cloned and the protein expressed and purified for biochemical and functional characterization. The results show that TTL is composed of four subunits, with a moderately elongated shape. We also found evidence for 5-HIU hydrolase and OHCU decarboxylase activities in vitro, in the full-length protein.ConclusionsThe Arabidopsis thaliana transthyretin-like (TTL) protein is a tetrameric bifunctional enzyme, since it has 5-HIU hydrolase and OHCU decarboxylase activities, which were simultaneously observed in vitro.

Probing the Evolution of Hydroxyisourate Hydrolase into Transthyretin through Active-Site Redesign

“…1 In plants, HIUase and OHCU decarboxylase are fused in a bifunctional protein with (S)-allantoin synthase activity. 2,3 These observations are consistent with a general role for uraH genes in the purine catabolic pathway. An exception is found in enterobacteria, for which the primary function of the uraH gene remains to be clarified.…”

Nucleotide Metabolism in Plants

“…29) and is hydrolyzed and decarboxylated by allantoin synthase (ALNS; Fig. 5B, #30) to (S)-allantoin Pessoa et al, 2010). Mutation of UOX leads to strong accumulation of uric acid, which is deleterious for peroxisome maintenance in the embryo, leading to a severe suppression of germination and seedling establishment (Hauck et al, 2014)-surprisingly, accumulation of similar amounts of xanthine in Arabidopsis plants lacking XDH does not lead to strong phenotypic alterations under standard growth conditions (Hauck et al, 2014;Schroeder et al, 2018;Soltabayeva et al, 2018).…”

The Brassinosteroid Signaling Pathway—New Key Players and Interconnections with Other Signaling Networks Crucial for Plant Development and Stress Tolerance

“…TTL is a tetrameric, bifunctional protein with decarboxylase and hydrolase activity [83], which is phosphorylated by BRI1 and functions as a negative regulator of BR signaling [46,84]. The exact role of TTL in regulation of this process is not known [33,85], however it has been recently reported that TTL binds kinase-active BRI1 with higher affinity than kinase-inactive BRI1, indicating that TTL may inhibit BRI1 signaling after its activation [15].…”