02370nas a2200265   4500008004100000022001400041245011700055210006900172260000800241300001400249490000800263520148700271653002501758653002401783653002401807653001401831653002101845100001701866700002001883700001601903700001601919700002401935700001801959856012701977       2019                        eng d  a1742-464X00aMechanistic insights into enzymatic catalysis by trehalase from the insect gut endosymbiont Enterobacter cloacae0 aMechanistic insights into enzymatic catalysis by trehalase from   cMAY  a1700-17160 v2863 a<p>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 K-M of 1.47 (+/- 0.05) mm, k(cat) of 6254.72 min(-1) and V-max 0.2 (+/- 0.002) mm center dot min(-1) at 55 degrees 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.</p>
10aEnterobacter cloacae10aPlutella xylostella10astructural analysis10atrehalase10avalidoxylamine A1 aAdhav, Anmol1 aHarne, Shrikant1 aBhide, Amey1 aGiri, Ashok1 aGayathri, Pananghat1 aJoshi, Rakesh  uhttps://library.ncl.res.in/content/mechanistic-insights-enzymatic-catalysis-trehalase-insect-gut-endosymbiont-enterobacter