Configurationally selective transition state analogue inhibitors of glycosidases. A study with nojiritetrazoles, a new class of glycosidase inhibitors

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The label was cleaved from the peptide by treatment with ammonia, and the resultant unlabeled peptide was purified and sequenced by Edman degradation. The peptide identified contained only one candidate for the catalytic nucleophile, an aspartic acid. This residue was contained within the sequence Gly-Trp-GlnIle-Asp-Pro-Phe-Gly-His-Ser, which showed excellent sequence similarity with regions in mammalian lysosomal and Golgi ␣-mannosidase sequences. These mammalian ␣-mannosidases belong to family 38 (or class II ␣-mannosidases) in which the Asp in the above sequence is totally conserved. This finding therefore assigns jack bean ␣-mannosidase to family 38, validating it as a model for other pharmaceutically interesting enzymes and thereby identifying the catalytic nucleophile within this family.

Section: Resultsmentioning

confidence: 99%

Identification of the Active Site Nucleophile in Jack Bean α-Mannosidase Using 5-Fluoro-β-l-Gulosyl Fluoride

Howard

Withers

1998

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“…Mutated cDNAs were constructed by the PCR-mediated overlap extension technique using a specific pair of complementary primers (ED fwd , 5Ј-GACCTTTAATGAC-CCGGAG-3Ј; and ED rev , 5Ј-CTCCGGGTCATTAAAGGTC-3Ј) and a pair of vector-specific primers (T7 prom , 5Ј-TAATACGACTCACTAT-AGGG-3Ј; and T7 term , 5Ј-ATGCTAGTTATTGCTCAGCGGT-3Ј) as described (24). Because of a codon usage bias for E. coli expression with two consecutive rare codons (AGG) in the cDNA of SbDhr1, the cDNA was mutated using the complementary primer pair SbRR fwd (5Ј-CCT-GGGAAATCCCTCGCCGTGACTGG-3Ј) and SbRR rev (5Ј-CCAGT-CACGGCGAGGGATTTCCCAGG-3Ј) to have the best E. coli codon usage for Arg 21 and Arg 22 . The DNA template used in this study was pET28a-SbDhr1, which was previously used for expression in E. coli and subsequent purification in our laboratory (17).…”

Section: Methodsmentioning

confidence: 99%

“…To address these questions, we have produced and purified SbDhr1 and an inactive mutant of SbDhr1 (referred to as SbDhr1-E189D hereafter) obtained by site-directed mutagenesis for three-dimensional analysis by x-ray crystallography of the native enzyme and mutant enzyme⅐substrate complexes. To complete the picture of enzyme-substrate or enzyme-inhibitor interactions, this study also describes the crystal structure of inactive ZmGlu1-E191D in complex with a cyclic glucotetrazole inhibitor that mimics a partially planar reaction transition state (21). In this work, we report the identification of the crucial amino acids within the active site of SbDhr1, a ␤-glucosidase known for its strict specificity for its natural substrate, and compare its mode of substrate binding with that of the homologous maize ␤-glucosidase (22), an enzyme with broad substrate specificity.…”

mentioning

confidence: 99%

Structural Determinants of Substrate Specificity in Family 1 β-Glucosidases

Verdoucq¹,

Moriniere

Bevan

et al. 2004

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126

Plant ␤-glucosidases play a crucial role in defense against pests. They cleave, with variable specificity, ␤-glucosides to release toxic aglycone moieties. The Sorghum bicolor ␤-glucosidase isoenzyme Dhr1 has a strict specificity for its natural substrate dhurrin (p-hydroxy-(S)-mandelonitrile-␤-D-glucoside), whereas its close homolog, the maize ␤-glucosidase isoenzyme Glu1, which shares 72% sequence identity, hydrolyzes a broad spectrum of substrates in addition to its natural substrate 2-O-␤-Dglucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxaxin-3-one. Structural data from enzyme⅐substrate complexes of Dhr1 show that the mode of aglycone binding differs from that previously observed in the homologous maize enzyme. Specifically, the data suggest that Asn Carbohydrates and their glycoconjugates are one of the most diverse groups of organic molecules in the biosphere. The selective cleavage of glycosidic bonds is crucial in a variety of fundamental biological processes for all living organisms. The large (and growing) number of glycoside hydrolase families reflects this diversity of substrates and the need for selective cleavage of the glycosidic bond. Ninety-one glycoside hydrolase families are currently available on the continuously updated CAZY web server (1).1 ␤-Glucosidases constitute a major group in glycoside hydrolase families 1 and 3 and hydrolyze either O-linked ␤-glycosidic bonds (␤-D-glucoside glucohydrolase, EC 3.2.1.21) or S-linked ␤-glycosidic bonds (myrosinase or ␤-Dthioglucoside glucohydrolase, EC 3.2.3.1). More precisely, enzymes of glycoside hydrolase family 1 hydrolyze substrates of the type G-O/S-X, where G indicates the glycosyl residue and X can be either another glycosyl residue or a non-glycosyl aglycone group. In higher plants, the major functions of ␤-glucosidases are defense against pests with the release of bitter or toxic aglycones and their breakdown products (2, 3), phytohormone activation (4, 5), lignification (6), and cell wall catabolism (7). The nature of the aglycone moiety of substrates is believed to be critical for the specificity and physiological functions of these enzymes.Because plants possess a large number of ␤-glucosidases (most of the 48 genes identified as putative ␤-glucosidase genes in Arabidopsis thaliana do not have a known function), the understanding of the mechanism of substrate specificity is important for accurately predicting the diverse physiological functions of these proteins. The roles of the 2 catalytic glutamates (8, 9) included in the TFNEP and Y(I/V)TENG peptide motifs of ␤-glucosidases as the general acid/base catalyst and the nucleophile, respectively (10), are now well understood. These residues are 5.5-6 Å apart within the active-site pocket on opposite sides of the glycosidic bond (8,11) and are required in the two steps of the substrate hydrolysis that results in retention of the anomeric conformation of C-1 at the point of cleavage. The conformational change in the glucose moiety prior to nucleophilic attack and the determinants of binding and d...

“…Although the K i constant for PheImGlc is in the low nanomolar ranges, the potential to design even more powerful mimics of the transition states exists. It has been suggested that these analogues should bind 10 10 -10 15 times tighter (6,14,56) or even 10 19 -10 23 times tighter (3, 57) than the ground-state substrates.…”

Section: ␤-D-glucan Glucohydrolase Transition State Complexmentioning

confidence: 99%

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

Hrmová

Gori

Smith

et al. 2004

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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.