Trehalases: A neglected carbon metabolism regulator?

Abstract

Trehalases are enzymes that carry out the degradation of the non-reducing disaccharide trehalose. Trehalase phylogeny unveiled three major branches comprising those from bacteria; plant and animals; and those from fungal origin. Comparative analysis between several deduced trehalase structures and the crystallographic structure of bacterial trehalase indicated that these enzyme’s structures are highly conserved in spite of the marked differences found at the sequence level. These results suggest a bacterial origin for the trehalases in contrast to an eukaryotic origin, as previously proposed. Trehalases structural analysis showed that they contain six discrete motifs which are characteristic of each phylogenetic group, suggesting a positive evolutionary selection pressure for the structural conservation. Interestingly, trehalases are involved in multiple regulatory functions: In the response against pathogens (plant-pathogen interactions); the regulation of bacterial viability in symbiotic interactions (legume-Rhizobium); carbon partitioning in plants; regulating chitin biosynthesis, as well as energy supply in the hemolymph for flight, in insects. In summary, trehalases seem to have a prokaryotic origin and play an active role in carbon metabolism and other diverse regulatory effects on cell physiology.

Trehalases (EC 3.2.1.28) are the enzymes that specifically carry out the degradation for the non-reducing disaccharide trehalose (α-D-glucopyranosyl-1,1-α-D-glucopyranoside), being this the only trehalose degradation pathway reported up to today. Interestingly, trehalases are not as widely distributed as the trehalose biosynthetic pathway, since trehalose-6-phosphate synthase/phosphatases (TPS/TPP) are found in monera, protist, fungi, plant and the animal kingdoms (including vertebrates).^1-3

Trehalase sequence analysis (ref. 33; Table 1) unveiled the three major branches (Fig. 1). These branches comprise those trehalases from bacteria, plants and animals, and a third group from fungi (neutral trehalases and acid trehalases). This distribution suggests that eukaryotic trehalases diversified from those from bacteria.^1,3 Interestingly, eukaryotic trehalases show a dispersed distribution (Fig. 1), contrasting the previous idea of an eukaryotic origin, as previously proposed.³ In addition, the distribution⁴ of six consensus discrete motifs, besides the catalytic domain, were found.^5-9 These motifs are: a transmembrane span (LFFFFFFFLCFSFTTSML); a cAMP-dependent phosphorylation site (RRXS); an EF-like Ca²⁺-binding motif (DTXGDXQITIXD); two trehalase signature motifs 1 and 2, (PGGRFXEXYXWDXY) and (QWDXPX[G/A]W[P/A/S]P), respectively; and the glycosylphosphatidylinositol (GPI) membrane anchor motif (CRTNYGYSAA). The catalytic residues are distributed from the trehalase signature 1 to the C-terminal end. Bacterial trehalases have a transmembrane span at the N-terminal region, trehalase signature 1 and trehalase signature 2 and GPI anchor in the C-terminal region, where the catalytic residues are found. The catalytic residues are highly conserved, except in acid trehalases from fungi. Animal and plant trehalases have trehalase signatures 1 and 2 and the membrane GPI anchor motif at the C-terminal region. Interestingly, the transmembrane span was either present (plant trehalases) or absent (animal and plant trehalases). Fungi trehalases are clearly divided in neutral and acid trehalases. Fungi neutral trehalases have a phosphorylation site cAMP-dependent, an EF-like Ca²⁺-binding motif, trehalase signatures 1 and 2 and the conserved catalytic residues. Interestingly, these enzymes lack the transmembrane span domain, but instead have a GPI membrane-anchor motif at the C-terminal region. Contrary, fungi acid trehalases have a transmembrane span at the N-terminal region, albeit, lack signatures 1 and 2 and the GPI anchor in the N-terminal region and the catalytic residues are only slightly conserved. It has been reported that mamalian trehalases which lack the transmembrane spanning domain, are bound to the plasma membrane by the GPI anchor motif, suggesting that this motif is directly involved in binding to the plasma membrane.⁸

Table 1. Trehalase sequences source and corresponding accesion numbers.

Specie	Accesion Number
Xanthomonas campestris	YP_362391.1
Enterobacter sp	YP_001177075.1
Magnaporthe grisea (NTH)	AAB88889.1
Drosophila melanogaster	NP_524821.1
Erwinia amylovora	CBX82150.1
Spodoptera frugiperda	ACF94698.1
Homo sapiens	BAA24381.1
Ralstonia solanacearum	YP_003749251.1
Escherichia coli	EFW73826.1
Danio rerio	XP_001336187.3
Nicotiana tabacum	BAI63261.1
Apis mellifera	BAF81545.1
Medicago truncatula	ABJ98545.1
Physcomitrella patens	ABO61746.1
Ceanorhabditis elegans	CAD54512.1
Neurospora crassa (NTH)	CAD36994.1
Laccaria bicolor (NTH)	XP_001880715.1
Schyzosaccaromyces pombe (NTH)	CAA11904.1
Neurospora tetrasperma (NTH)	EGZ75693.1
Emericella nidulans (NTH)	AAB99831.1
Metarhizum acridum (NTH)	AAS67889.1
Saccharomyces cerevisiae (NTH)	EDN64617.1
Candida albicans (NTH)	CAA64476.1
Glycine max	NP_001238042.1
Magnaporthe oryzae (NTH)	XP_364626.1
Arabidopsis thaliana	AEE84844.1
Laccaria bicolor (ATH)	EDR13253.1
Metarhizium anisopliae (ATH)	ABO93464.1
Spodoptera exigua	ABY86218.1
Rattus norvegicus	AAC25985.1
Mus musculus	AAF61430.1
Saccharomyces cerevisiae (ATH)	CAA58961.1
Phaseolus vulgaris	Phvul.002G301500.1

                    NTH: Neutral trehalase; ATH: Acid trehalase

Figure 1. Trehalases: phylogenetic analysis, motifs and catalytic residues distribution are shown. The scale represents the estimated branch lengths (neighbor-joining method, 1,000 bootstrap replicates). Three phylogenetic groups are highlighted and the corresponding motifs in each group are described in the text.

In addition, using the trehalase crystallographic structure of (PDB code: 2WYN) we modeled the tridimensional structure from full sequences of all trehalases (33) reported.^5,10 The trehalase deduced structures from bacterial, animal, plant and fungi (neutral and acid) origin yielded a RMSD average of 0.062 Å, 0.079 Å, 0.078 Å, 0.080 Å and 0.076 Å, respectively (Fig. 2A). Moreover, we superimposed all the deduced structures to determine that the structural differences among trehalases are not significant (Fig. 2B).⁵ Additionally, we performed the maximum likelihood analysis of natural selection, on a codon-by-codon basis, for all trehalase sequences, to determine a marked trend for synonymous substitutions for the catalytic residues and trehalase signatures 1 and 2 (Fig. 2C).¹¹ These results indicate that trehalase structures are highly conserved in spite of their low amino acid identity. This suggests an evolutionary selection pressure for the structural conservation to maintain the trehalose degradation function. Interestingly, recently additional key regulatory roles of trehalases have been described, such as: in pathogen defense; in control of sucrose levels and homeostasis; in chitin synthesis, in cellular differentiation, and in stress tolerance, among others.^12-19 In addition, trehalase contrastingly different sub-cellular localizations have been reported, including the cytoplasm, the vacuole, the apoplast, or being bound to the plasma membrane and oriented to the apoplast.^16,20-23 In fungi, trehalase activity and localization have been directly involved in regulating the trehalose content and its mobilization into the cytoplasm and during the symbiotic interaction of the ectomycorrhizal fungi several genes for trehalose metabolism are strongly upregulated.^23,24 In insects the regulation of trehalose degradation by trehalase is essential to supply the energetic requirements for the flight and the direct regulation for chitin biosynthesis during the insect development.^{6,16,18,25,26} In higher plants, trehalose content is regulated by trehalase activity, which impacts directly on sucrose levels by negatively correlating sucrose synthase 1 expression levels and starch content.^14,15 Apparently, since trehalose accumulation is regulated by trehalase, this enzyme could be directly involved in regulating carbon partitioning, as well.^14,27-29 Recently, it has been reported that in Arabidopsis drought stress tolerance was achieved when the trehalase gene (AtTRE1) was overexpressed. In particular, lower trehalose levels induced stomatal closure through the ABA signaling pathway in these gain-of-function treahalase plants. It seems that ABA directly participates in the upregulation of AtTRE and AtTRE1 is involved in the regulation of ABA signaling in plants.³⁰ In addition, during the pathogenic interaction between Plasmodiophora brassicae and Arabidopsis thaliana a marked AtTRE1 upregulation was also reported.¹² In contrast, downregulation of PvTRE1 in Phaseolus vulgaris root nodules had a direct role into carbon partitioning, increased nodule biomass and bacteroid viability.¹⁵ Altogether, we propose that trehalase, by being a key modulator of trehalose levels, is a major carbon metabolism regulator. Studies are in progress to explore this possibility in the Phaseolus vulgaris-Rhizobium symbiosis. The remarkable and varied roles, in addition to the contrasting sub-cellular localizations where trehalases have been located, suggest that trehalase function should be considered central in carbon partitioning and energy homeostasis regulation, in eukaryotic organisms.

Figure 2. Homology model comparison with the EcTRE template structure (PDB code: 2WYN) and the trehalase deduced structures from diverse origins. (A) RMSD average values obtained from the comparison between the crystallographic structure (bacteria) and the deduced structures of trehalases from different origins. (B) Superimposition of all the trehalases (crystallographic and deduced structures). (C) Motifs and residues with a strong trend for synonymous mutations are depicted.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.