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Glycoside Hydrolase Family 37

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Glycoside Hydrolase Family GH37
Clan GH-G
Mechanism Inverting
Active site residues Known
CAZy DB link

Substrate specificities

To date, GH37 glycoside hydrolases have been shown to hydrolyze the α-1,1 bound trehalose (α-D-glucopyranosyl-(1→1)-α-D-glucopyranoside) into two molecules of D-glucose (EC GH37 enzymes are further classified by their optimal pH; neutral or acidic, and also by their cellular localization; soluble or membrane bound [1]. There is some evidence that organisms possessing multiple GH37 trehalases will utilize them for different purposes. This tends towards periplasmic trehalases being metabolically relevant while cytoplasmic trehalases participate in osmoregulation [2].

Kinetics and Mechanism

GH37 trehalases follow an inverting mechanism. This was first demonstrated through incubation of GH37 trehalases obtained from S. barbata, the flesh fly, with 18O-labelled water and observing its incorporation primarily into the beta-epimer [3]. This was further supported by the solved structure of E. coli Tre37A which demonstrated that the proposed catalytic residues were in a position consistent with an inverting mechanism [4].

Several fungal neutral trehalases; S. cerevisiae, A. nidulans, N. crassa, and C. albicans, show evidence of activation by calcium ion binding and cAMP-dependent phosphorylation [1, 5, 6].

Catalytic Residues

The catalytic residues were first predicted through structural determination of E. coli Tre37A in complex with inhibitors 1-thiatrehazolin (PDB ID 2JG0) and validoxylamine A (PDB ID 2JF4) [4]. These structures implicate an aspartate residue (Asp312 in E. coli) as the catalytic general acid, and a glutamate residue (Glu496 in E. coli) as the catalytic general base. A crystal structure of S. cerevisiae Nth1 with bound trehalose identified an aspartate residue (Asp478 in S. cerevisiae) as the catalytic general acid, and a glutamate residue (Glu674 in S. cerevisiae) as the general base. Superimposition of these structures indicates that the proposed catalytic residues align in both the E. coli Tre37A inhibitor bound and S. cerevisiae Nth1 trehalose bound structures.

Kinetic evidence for the catalytic residues was provided by site-directed mutagenesis of an S. frugiperda trehalase [7]. Mutation of the proposed catalytic acid and base residues; Asp322 and Glu520 respectively, resulted in dramatically reduced Kcat values compared to that of the Wild-type protein, and in the loss of ionization reflective of their predicted pKa values. The reduction in Kcat combined with loss of ionization strongly indicates that these function as catalytic residues [7].

Three-dimensional structures

The first three-dimensional structure of a GH37 trehalase was obtained from E. coli Tre37A in complex with the inhibitors 1-thiatrehazolin (PDB ID 2JG0) and validoxylamine A (PDB ID 2JF4) by x-ray crystallography [4]. The structure revealed a monomeric enzyme consisting of an (α/α)6 barrel fold, similar to other α-toroidal glycosidases. The structure revealed extensive hydrogen bonding and a distinct lack of hydrophobic stacking within the +1 subsite. The bound structure also revealed that the +1 and -1 subsites were buried within the enzyme structure and significant conformation changes would be required for substrate recognition.

The first eukaryotic GH37 structure was determined from an S. cerevisiae Nth1:Bmh1 complex, and provided the first structure in the presence of trehalose (PDB ID 5M4A) [5]. The catalytic domain consists of an (α/α)6 barrel formed by the interaction of one Bmh1 C-terminus with Nth1. Similar to the E. coli Tre37A structure, the substrate was found in a deep pocket. This structure provided the first evidence of a flexible “lid loop” structure, which would undergo significant conformational changes and complete the active site of Nth1. A similar, but shorter, structure was revealed in E. coli Tre37A once the solved structures were superimposed [5].

Further evidence of the “lid loop”, also referred to as a “hood-like domain”, in bacterial trehalases was observed in E. cloacae Tre [8]. Comparison of the solved structures for unbound (PDB ID 5Z6H) and validoxylamine A bound trehalase (PDB ID 5Z66) revealed both “side loop” and “lid loop” residues which undergo significant conformational changes upon ligand binding. Structural comparisons to E. coli Tre37A with validoxylamine A ligand highlighted identically positioned loop structures [4, 8]. Three conserved residues identified within the lid loop structure were observed to contact validoxylamine A, one of which was also observed to form a salt bridge upon lid loop closure. Mutation of this residue; Glu 511, resulted in significant decrease of enzyme activity likely resulting from incomplete loop closure.

GH37 enzymes belong to the clan GH-G.

Family Firsts

First sterochemistry determination
The inversion of stereochemistry for a trehalase from the flesh fly Sarcophaga barbata was first demonstrated by Clifford in 1980 [3].
First general acid identification
First predicted in E. coli Tre37A from structure determination with inhibitors [4], experimentally observed in S. frugiperda through site-directed mutagenesis and kinetic determination [7].
First general base identification
First predicted in E. coli Tre37A from structure determination with inhibitors [4], experimentally observed in S. frugiperda through site-directed mutagenesis and kinetic determination [7].
First 3-D structure
The GH37 trehalase from Escherichia coli was solved by X-ray crystallography [4].


  1. d'Enfert C, Bonini BM, Zapella PD, Fontaine T, da Silva AM, and Terenzi HF. (1999). Neutral trehalases catalyse intracellular trehalose breakdown in the filamentous fungi Aspergillus nidulans and Neurospora crassa. Mol Microbiol. 1999;32(3):471-83. DOI:10.1046/j.1365-2958.1999.01327.x | PubMed ID:10320571 [DEnfert1999]
  2. Argüelles JC (2000). Physiological roles of trehalose in bacteria and yeasts: a comparative analysis. Arch Microbiol. 2000;174(4):217-24. DOI:10.1007/s002030000192 | PubMed ID:11081789 [Arguelles2000]
  3. Clifford KH (1980). Stereochemistry of the hydrolysis of trehalose by the enzyme trehalase prepared from the flesh fly Sarcophaga barbata. Eur J Biochem. 1980;106(1):337-40. DOI:10.1111/j.1432-1033.1980.tb06028.x | PubMed ID:7341233 [Clifford1980]
  4. Gibson RP, Gloster TM, Roberts S, Warren RA, Storch de Gracia I, García A, Chiara JL, and Davies GJ. (2007). Molecular basis for trehalase inhibition revealed by the structure of trehalase in complex with potent inhibitors. Angew Chem Int Ed Engl. 2007;46(22):4115-9. DOI:10.1002/anie.200604825 | PubMed ID:17455176 [Gibson2007]
  5. Alblova M, Smidova A, Docekal V, Vesely J, Herman P, Obsilova V, and Obsil T. (2017). Molecular basis of the 14-3-3 protein-dependent activation of yeast neutral trehalase Nth1. Proc Natl Acad Sci U S A. 2017;114(46):E9811-E9820. DOI:10.1073/pnas.1714491114 | PubMed ID:29087344 [Alblova2017]
  6. Alblova M, Smidova A, Kalabova D, Lentini Santo D, Obsil T, and Obsilova V. (2019). Allosteric activation of yeast enzyme neutral trehalase by calcium and 14-3-3 protein. Physiol Res. 2019;68(2):147-160. DOI:10.33549/physiolres.933950 | PubMed ID:30628830 [Alblova2019]
  7. Silva MC, Terra WR, and Ferreira C. (2010). The catalytic and other residues essential for the activity of the midgut trehalase from Spodoptera frugiperda. Insect Biochem Mol Biol. 2010;40(10):733-41. DOI:10.1016/j.ibmb.2010.07.006 | PubMed ID:20691783 [Silva2010]
  8. Adhav A, Harne S, Bhide A, Giri A, Gayathri P, and Joshi R. (2019). Mechanistic insights into enzymatic catalysis by trehalase from the insect gut endosymbiont Enterobacter cloacae. FEBS J. 2019;286(9):1700-1716. DOI:10.1111/febs.14760 | PubMed ID:30657252 [Adhav2019]

All Medline abstracts: PubMed