Structural Rationale for Low-Nanomolar Binding of Transition State Mimics to a Family GH3 β-d-Glucan Glucohydrolase from Barley^,

Abstract

The interactions of a transition state mimic anilinomethyl glucoimidazole (AmGlcIm), with a K(i) constant of 0.6 x 10(-)(9) M and a Gibbs free energy value of -53.5 kJ/mol, with a family GH3 beta-d-glucan glucohydrolase from barley have been analyzed crystallographically and by ab initio quantum mechanical modeling. AmGlcIm binds 3 times more tightly to the beta-d-glucan glucohydrolase than a previously investigated phenyl glucoimidazole. In the enzyme-AmGlcIm complex, an additional residue, Tyr253, and a water molecule positioned between subsites -1 and +1 are recruited for binding. Analyses of the two binary complexes reveal the following. (i) An intricate network exists in which hydrogen bonds between the enzyme's catalytic pocket residues Lys206, His207, Tyr253, Asp285, and Glu491 and the glucoimidazoles are shorter by 0.15-0.53 A, compared with distances of hydrogen bonds in the Michaelis complex. (ii) The "glucose" moiety of the glucoimidazoles adopts a (4)E conformation that is vital for the low-nanomolar binding. (iii) The N1 atoms of the glucoimidazoles are positioned nearly optimally for in-line protonation by the Oepsilon1 atom of the catalytic acid/base Glu491. (iv) The enzyme derives binding energies from both glycone and aglycone components of the glucoimidazoles. (iv) The prevalent libration motion of the two domains of the enzyme could play a significant role during induced fit closure in the active site. (v) Modeling based on the structural data predicts that protons could be positioned on the N1 atoms of the glucoimidazoles, and the catalytic acid/base Glu491 could carry an overall negative charge. (vi) The enzyme-AmGlcIm complex reveals the likely structure of an early transition state during hydrolysis. Finally, the high-resolution structures enabled us to define minimal structures of oligosaccharides attached to Asn221, Asn498, and Asn600 N-glycosylation sites.

Biochemical Characterization and Crystal Structures of a Fungal Family 3 β-Glucosidase, Cel3A from Hypocrea jecorina

“…Clear density is observed for this glucose accommodated at the Ϫ1 subsite. The Ϫ1 subsite seems to have a highly conserved composition among the structures of HjCel3A, TnBgl3B, and HvExoI (17,18,38,66) , and Glu 441 of HjCel3A. Superposition of the structure of HjCel3A with those of TnBgl3B and HvExoI show that most of the loops forming the active site of TnBgl3B are present in HjCel3A and also that the ϩ1 subsite is situated in a similar position to the corresponding site in TnBgl3B and HvExoI.…”

“…In the case of HjCel3A, the search model was a polyalanine model that was generated using two different structures: (i) H. vulgare Exo1 (HvExo1; Protein Data Bank code 1X39 (38)) and (ii) T. neapolitana Bgl3B (TnBgl3B; Protein Data Bank code 2X41 (18)). The three structures were superimposed by the program Coot (39 -41), and the elements of the structures that were not superimposable were deleted.…”

Thermostable Enzymes as Biocatalysts in the Biofuel Industry

“…Both families hydrolyze their cognate substrate with net retention of configuration of the anomeric carbon. The crystal structures for a GH family 3 β- d -glucan exohydrolase in complex with a variety of transition-state analogs have been reported (Hrmova et al ., 2002, 2004, 2005; Varghese et al ., 1999). This enzyme exhibits two domains: an N-terminal (α/β) 8 TIM-barrel domain and a C-terminal six-stranded β-sandwich domain.…”

Structural and Functional Analyses of β-Glucosidase 3B from Thermotoga neapolitana: A Thermostable Three-Domain Representative of Glycoside Hydrolase 3

“…However, only the H. vulgare enzyme has been characterized in detail with regard to its structure, enzymatic mechanism, and substrate specificity. 17,[20][21][22] Distinct phylogenetic clusters have been identified within GH3, and six major branches have been identified. 23 TnBgl3B, analyzed in this work, is located in a cluster (no.…”