Triphosphate Tunnel Metalloenzyme Function in Senescence Highlights a Biological Diversification of This Protein Superfamily

Abstract

ORCID IDs: 0000-0003-3889-6183 (W.M.); 0000-0002-3797-4277 (K.Y.).The triphosphate tunnel metalloenzyme (TTM) superfamily comprises a group of enzymes that hydrolyze organophosphate substrates. They exist in all domains of life, yet the biological role of most family members is unclear. Arabidopsis (Arabidopsis thaliana) encodes three TTM genes. We have previously reported that AtTTM2 displays pyrophosphatase activity and is involved in pathogen resistance. Here, we report the biochemical activity and biological function of AtTTM1 and diversification of the biological roles between AtTTM1 and 2. Biochemical analyses revealed that AtTTM1 displays pyrophosphatase activity similar to AtTTM2, making them the only TTMs characterized so far to act on a diphosphate substrate. However, knockout mutant analysis showed that AtTTM1 is not involved in pathogen resistance but rather in leaf senescence. AtTTM1 is transcriptionally up-regulated during leaf senescence, and knockout mutants of AtTTM1 exhibit delayed dark-induced and natural senescence. The double mutant of AtTTM1 and AtTTM2 did not show synergistic effects, further indicating the diversification of their biological function. However, promoter swap analyses revealed that they functionally can complement each other, and confocal microscopy revealed that both proteins are tail-anchored proteins that localize to the mitochondrial outer membrane. Additionally, transient overexpression of either gene in Nicotiana benthamiana induced senescence-like cell death upon dark treatment. Taken together, we show that two TTMs display the same biochemical properties but distinct biological functions that are governed by their transcriptional regulation. Moreover, this work reveals a possible connection of immunity-related programmed cell death and senescence through novel mitochondrial tail-anchored proteins.

Identity and functions of inorganic and inositol polyphosphates in plants

“…While the physiological roles of AtTTM3 remain unclear, it is of note that functional connections between polyPs and cell cycle regulation are well established in other prokaryotes and eukaryotes (Bru et al, 2016;Racki et al, 2017). There are at least two additional TTM proteins in plants (AtTTM1 and AtTTM2), where the TTM domain is located at the C-terminus of a uridine kinase domain (Ung et al, 2014(Ung et al, , 2017. Both enzymes have been reported to have pyrophosphatase activity, but it is not known which domain harbors this activity (Ung et al, 2014(Ung et al, , 2017.…”

A genetically validated approach to detect inorganic polyphosphates in plants

“…PolyP in Chlamydomonas is synthesized by the vacuolar transporter chaperone VTC complex (Aksoy et al, 2014), whose catalytic subunit Vtc4 vacuole/acidocalcisome using a membrane pore (Gerasimaitė et al, 2014). While the VTC complex is apparently absent in higher plants, proteins sharing structural homology with the Vtc4 catalytic domain exist in Arabidopsis (Moeder et al, 2013;Martinez et al, 2015;Ung et al, 2017). We have previously characterized AtTTM3 as a specific short-chain inorganic polyphosphatase (Martinez et al, 2015), which is transcribed in a bicistronic transcript also encoding the cell cycle regulator AtCDC26 (Lorenzo-Orts, Witthoeft, et al, 2019).…”

Brachypodium distachyon triphosphate tunnel metalloenzyme 3 is both a triphosphatase and an adenylyl cyclase upregulated by mechanical wounding

“…As an example, we can site three known plant TTMs from A. thaliana that differ in their affinity for the substrate. AtTTM3 had a strong preference for tripolyphoshpate (PPPi), whereas AtTTM1 and AtTTM2 had a high affinity for PPi [9,10]. Differences in the substrate preferences of individual TTM family members from E. coli, mice, yeast, and some fungi have also been studied and published previously [5, 32,33].…”

“…AtTTM1-AtTTM3 from Arabidopsis thaliana and mammalian ThTPases [5, [8][9][10][11] did not show adenylate cyclase activity.…”

“…TTMs in many bacteria, for example, Clostridium thermocellum (CthTTM), N. europaea (NeuTTM), or Escherichia coli (Ygif), are known to be PPPases with various affinities for different substrates [5, [15][16][17][18]. A similar situation for organophosphate substrate specificity applies to three plant TTM proteins identified in A. thaliana [8][9][10]. Biochemical analyses revealed that AtTTM1 and AtTTM2 have the highest affinity for pyrophosphate (PPi), whereas AtTTM3 has a strong PPPase activity similar to that of TTMs from C. thermocellum and N. europaea.…”