Mechanistic insights into enzymatic catalysis by trehalase from the insect gut endosymbiont Enterobacter cloacae

Abstract

Energy metabolism in the diamondback moth Plutella xylostella is facilitated by trehalase, an enzyme which assists in trehalose hydrolysis, from the predominant gut bacterium Enterobacter cloacae. We report the biochemical and structural characterization of recombinant trehalase from E. cloacae (Px_EclTre). Px_EclTre showed KM of 1.47 (±0.05) mm, kcat of 6254.72 min−1 and Vmax 0.2 (±0.002) mm·min−1 at 55 °C and acidic pH. Crystal structures of Px_EclTre were determined in the ligand‐free form and bound to the inhibitor Validoxylamine A. The crystal structure of the ligand‐free form, unavailable until now for any other bacterial trehalases, enabled us to delineate the conformational changes accompanying ligand binding in trehalases. Multiple salt bridges were identified that potentially facilitated closure of a hood over the substrate‐binding site. A cluster of five tryptophans lined the −1 substrate‐binding subsite, interacted with crucial active site residues and contributed to both trehalase activity and stability. The importance of these residues in enzyme activity was further validated by mutagenesis studies. Many of these identified residues form part of signature motifs and other conserved sequences in trehalases. The structure analysis thus led to the assignment of the functional role to these conserved residues. This information can be further explored for the design of effective inhibitors against trehalases.

The Promoting Effect of Gut Microbiota on Growth and Development of Red Palm Weevil, Rhynchophorus ferrugineus (Olivier) (Coleoptera: Dryophthoridae) by Modulating Its Nutritional Metabolism

“…Furthermore, the concentration of some important nutritional indices, containing hemolymph protein, glucose, triglyceride, and trehalose which can indicate the nutrition and metabolic status of animal host well (Ridley et al, 2012; Newell and Douglas, 2014), were measured to reveal the physiological consequences of gut microbiota on their host phenotype. It has been verified that Enterobacter cloacae can produce various glycolytic enzymes, containing cellulases, trehalases and other glucosidases, which participate in food digestion (Xia et al, 2017; Adhav et al, 2019). Moreover, Lactococcus lactis is a lactic acid bacterium which is involved in digesting and fermenting some plant polymers to improve insect nutrition acquisition (Passerini et al, 2013; Zhou H. et al, 2017).…”

Synthesis, trehalase hydrolytic resistance and inhibition properties of 4- and 6-substituted trehalose derivatives

“…All manipulations were performed with the molecular modelling program YASARA and the YASARA/WHATIF twinset and figures were created with PyMOL 2.0. The ligand free and inhibitor bound crystal structures of trehalase from Enterobacter cloacae (GH37 family) (PDB code 5Z6H and 5Z66, respectively) 33 were used as template for docking. Trehalose derivative structures were created with YASARA Structure and subsequently minimised with the AMBER03 force field.…”

“…Unfortunately, no structural elucidation was possible for the enzyme from M. smegmatis as no crystal structure of a trehalase from family GH15 has yet been determined. However, several crystal structures of trehalases from family GH37 are described in literature, namely from Escherichia coli, Saccharomyces cerevisiae and Enterobacter cloacae 33,[35][36][37] . Interestingly, the latter has been crystallised in two different forms, i.e.…”

“…with and without the inhibitor validoxylamine A 36, which resulted in structures that can be described as 'closed' and 'open', respectively. Indeed, the so-called lid loop (G 506 -G 519 ) and side loop (Y 147 -Y 159 ) were found to undergo a significant conformational change upon ligand binding (Figure 2) 33 . Docking simulations were performed with these structures as representative scenario for family GH37 trehalases, such as the one from porcine kidney.…”

Duplication and diversification of trehalase confers evolutionary advantages on lepidopteran insects

“…In insects, it is a major haemolymph sugar, serving as a prime source of energy for flight and tolerance to stress (Becker, Schlöder, Steele, & Wegener, ). The elucidation of trehalose metabolism has been one of the most exciting developments in our understanding of insect metabolism in recent years (Adhav et al, ; Cornette et al, ). A number of studies have demonstrated a correlation between intracellular trehalose levels and the ability of insects to survive various environmental stresses, such as starvation, osmotic and oxidative stresses, and extremes in temperature (Benoit, Lopez‐Martinez, Elnitsky, Lee, & Denlinger, ; Cornette et al, ; Juliano, O'Meara, Morrill, & Cutwa, ).…”