7C24

Glycosidase F290Y at pH8.0

Summary for 7C24

• Entry DOI: 10.2210/pdb7c24/pdb
• Descriptor: Isomaltose glucohydrolase, GLYCEROL, AMMONIUM ION, ... (4 entities in total)
• Functional Keywords: glycoside hydrolase, hydrolase
• Biological source: Kribbella flavida (strain DSM 17836 / JCM 10339 / NBRC 14399)
• Total number of polymer chains: 1
• Total formula weight: 44538.57

Authors

Tagami, T.,Kikuchi, A.,Okuyama, M.,Kimura, A. (deposition date: 2020-05-07, release date: 2021-05-12, Last modification date: 2023-11-29)

Primary citation

Tagami, T.,Chen, M.,Furunaga, Y.,Kikuchi, A.,Sadahiro, J.,Lang, W.,Okuyama, M.,Tanaka, Y.,Iwasaki, T.,Yao, M.,Kimura, A.
Structural insights reveal the second base catalyst of isomaltose glucohydrolase.
Febs J., 289:1118-1134, 2022

PubMed Abstract: Glycoside hydrolase family 15 (GH15) inverting enzymes contain two glutamate residues functioning as a general acid catalyst and a general base catalyst, for isomaltose glucohydrolase (IGHase), Glu178 and Glu335, respectively. Generally, a two-catalytic residue-mediated reaction exhibits a typical bell-shaped pH-activity curve. However, IGHase is found to display atypical non-bell-shaped pH-k and pH-k /K profiles, theoretically better-fitted to a three-catalytic residue-associated pH-activity curve. We determined the crystal structure of IGHase by the single-wavelength anomalous dispersion method using sulfur atoms and the cocrystal structure of a catalytic base mutant E335A with isomaltose. Although the activity of E335A was undetectable, the electron density observed in its active site pocket did not correspond to an isomaltose but a glycerol and a β-glucose, cryoprotectant, and hydrolysis product. Our structural and biochemical analyses of several mutant enzymes suggest that Tyr48 acts as a second catalytic base catalyst. Y48F mutant displayed almost equivalent specific activity to a catalytic acid mutant E178A. Tyr48, highly conserved in all GH15 members, is fixed by another Tyr residue in many GH15 enzymes; the latter Tyr is replaced by Phe290 in IGHase. The pH profile of F290Y mutant changed to a bell-shaped curve, suggesting that Phe290 is a key residue distinguishing Tyr48 of IGHase from other GH15 members. Furthermore, F290Y is found to accelerate the condensation of isomaltose from glucose by modifying a hydrogen-bonding network between Tyr290-Tyr48-Glu335. The present study indicates that the atypical Phe290 makes Tyr48 of IGHase unique among GH15 enzymes.

PubMed: 34665923
DOI: 10.1111/febs.16237

Experimental method

X-RAY DIFFRACTION (1.71 Å)