Three-dimensional Structure of the Barley β-d-Glucan Glucohydrolase in Complex with a Transition State Mimic

Abstract

Glucophenylimidazole (PheGlcIm), a tetrahydroimidazopyridine-type inhibitor and 4 H 3 conformer mimic of a glucoside, binds very tightly to a barley ␤-D-glucan glucohydrolase, with a K i constant of 2 ؋ 10 ؊9 M and a ⌬G of 51 kJ mol ؊1 . PheGlcIm binds to the barley ␤-D-glucan glucohydrolase ϳ2 ؋ 10 5 times tighter than laminarin, which is the best non-synthetic ground-state substrate found so far for this enzyme, 10 6 times tighter than 4-nitrophenyl ␤-D-glucopyranoside, and 2 ؋ 10 7 tighter than glucose. The three-dimensional structure of the ␤-D-glucan glucohydrolase with bound PheGlcIm indicates that the complex resembles a hypothetical transition state during the hydrolytic cycle, that the enzyme derives substrate binding energy from the "aglycone" portion of the ligand, and that it also reveals an anti-protonation trajectory for hydrolysis. Continuous electron densities at the 1.6 level form between the three active site residues Asp 95 , His 207 , and Asp 285 , and the C6OH, C7OH, C8OH, and C9OH groups of PheGlcIm. These electron densities correspond to the most favorable interactions in the three-dimensional structure of the ␤-D-glucan glucohydrolase-PheGlcIm complex and indicate atomic distances equal to or less than 2.55 Å. The crystallographic data were corroborated with ab initio molecular orbital calculations. The data indicate that the 4 E conformation of the glucose part of PheGlcIm is critical for tight binding and provide the first evidence for probable substrate distortion during catalysis by this enzyme.

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.…”

Functional Diversity of Four Glycoside Hydrolase Family 3 Enzymes from the Rumen Bacterium Prevotella bryantii B 1 4

“…The catalytic acid/ base that serves to protonate the leaving-group oxygen and subsequently activate an attacking water has been identified for several GH3 ␤-D-glucosidases (9,35,40). In addition to these two critical residues, GH3 enzymes possess three additional highly conserved amino acid residues (Arg, Lys, and His) that are predicted to be involved in catalysis (27,29). The residues that contribute to substrate specificity for this family of enzymes, however, have not been thoroughly investigated.…”

Identification of a Catalytic Residue ofClostridium paraputrificumN-Acetyl-β-D-glucosaminidase Nag3A by Site-Directed Mutagenesis

“…In this enzyme, Asp285 in the N-terminal domain and Glu491 in the C-terminal domain are considered to be involved in catalysis as nucleophile and proton donor, respectively. [12][13][14] Based on this finding, a putative active site of Nags in this family was also deduced to be Asp242 of Vibrio furnissii ExoII 10) and Asp303 of C. paraputrificum Nag3A, 6) corresponding to Asp285 of ExoI. 12) However, another active site, a putative proton donor, was not expected in single-domain enzymes, including C. paraputrificum Nag3A, from the information about barley ExoI, since Nag3A and other single-domain enzymes of family 3 did not contain a second domain.…”

“…Barley -glucan glucanohydrolase ExoI in family 3 is composed of 605 amino acid residues and is the only enzyme of which crystalline structure has been reported. 12,13) Structural analysis of ExoI indicated that this enzyme is a globular protein y To whom correspondence should be addressed. Tel: +81-59-231-9621; Fax: +81-59-231-9684; E-mail: sakka@bio.mie-u.ac.jp Abbreviations: Nag, -N-acetylglucosaminidase; GlcNAc, N-acetylglucosamine; 4-MU-(GlcNAc), 4-methylumbelliferyl -D-N-acetylglucosamine; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; Tris, Tris(hydroxymethyl)aminomethane composed of two domains: the first 357 residues constitute an ð=Þ 8 TIM-barrel domain, and the second domain is arranged in a six-stranded sandwich with three helices on either side of the sheet.…”

“…Furthermore, Asp285 in the first domain and Glu491 in the second domain were identified as the catalytic nucleophile and proton donor respectively. [12][13][14] On the other hand, C. paraputrificum Nag3A consists of 413 amino acid Amino acids conserved in at least 9 of 11 sequences are highlighted; -gap left to improve alignment. Asterisks indicate the amino acids selected for site-directed mutagenesis.…”