Hydrolysis of Glycosyl Thioimidates by Glycoside Hydrolase Requires Remote Activation for Efficient Activity

Chemoenzymatic synthesis of glycosides relies on efficient glycosyl donor substrates able to react rapidly and efficiently, yet with increased stability towards chemical or enzymatic hydrolysis. In this context, glycosyl thioimidates have previously been used as efficient donors, in the case of hydrolysis or thioglycoligation. In both cases, the release of the thioimidoyl aglycone was remotely activated through a protonation driven by a carboxylic residue in the active site of the corresponding enzymes. A recombinant glucosidase (DtGly) from Dictyoglomus themophilum, previously used in biocatalysis, was also able to use such glycosyl thioimidates as substrates. Yet, enzymatic kinetic values analysis, coupled to mutagenesis and in silico modelling of DtGly/substrate complexes demonstrated that the release of the thioimidoyl moiety during catalysis is only driven by its leaving group ability, without the activation of a remote protonation. In the search of efficient glycosyl donors, glycosyl thioimidates are attractive and efficient. Their utility, however, is limited to enzymes able to promote leaving group release by remote activation.

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Regioselective enzymatic hydrolysis of acetylated pyranoses and pyranosides using immobilised lipases. An easy chemoenzymatic synthesis of α- and β-d-glucopyranose acetates bearing a free secondary C-4 hydroxyl group

Carbohydrate Research ◽

10.1016/s0008-6215(02)00113-1 ◽

2002 ◽

Vol 337 (18) ◽

pp. 1615-1621 ◽

Cited By ~ 32

Author(s):

Marco Terreni ◽

Raul Salvetti ◽

Laura Linati ◽

Roberto Fernandez-Lafuente ◽

Gloria Fernández-Lorente ◽

...

Keyword(s):

Enzymatic Hydrolysis ◽

Chemoenzymatic Synthesis ◽

Hydroxyl Group ◽

Hydrolysis Of

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Leaving Group Ability Observably Affects Transition State Structure in a Single Enzyme Active Site

Journal of the American Chemical Society ◽

10.1021/jacs.6b03156 ◽

2016 ◽

Vol 138 (23) ◽

pp. 7386-7394 ◽

Cited By ~ 29

Author(s):

Daniel Roston ◽

Darren Demapan ◽

Qiang Cui

Keyword(s):

Transition State ◽

Active Site ◽

State Structure ◽

Transition State Structure ◽

Enzyme Active Site ◽

Leaving Group ◽

Leaving Group Ability ◽

Single Enzyme

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Transition State Interactions in a Promiscuous Enzyme: Sulfate and Phosphate Monoester Hydrolysis byPseudomonas aeruginosaArylsulfatase

10.1101/327643 ◽

2018 ◽

Cited By ~ 1

Author(s):

Bert van Loo ◽

Ryan Berry ◽

Usa Boonyuen ◽

Mark F. Mohamed ◽

Marko Golicnik ◽

...

Keyword(s):

Active Site ◽

Isotope Effects ◽

Charge Compensation ◽

Linear Free Energy ◽

Uncatalyzed Reaction ◽

Leaving Group ◽

Energy Relationships ◽

Increased Sensitivity ◽

Leaving Group Ability ◽

Active Site Mutants

ABSTRACTPseudomonas aeruginosaarylsulfatase (PAS) hydrolyses sulfate and, promiscuously, phosphate monoesters. Enzyme-catalyzed sulfate transfer is crucial to a wide variety of biological processes, but detailed studies of the mechanistic contributions to its catalysis are lacking. We present an investigation based on linear free energy relationships (LFERs) and kinetic isotope effects (KIEs) of PAS and active site mutants that suggest a key role for leaving group (LG) stabilization. In LFERs wild type PAS has a much less negative Br0nsted coefficient (βleaving groupobs-Enz= −0.33) than the uncatalyzed reaction (βleavingroupobs= −1.81). This situation is diminished when cationic active site groups are exchanged for alanine. The considerable degree of bond breaking during the TS is evidenced by an18ObridgeKIE of 1.0088. LFER and KIE data for several active site mutants point to leaving group stabilization by active-site lysine K375, in cooperation with histidine H211.15N KIEs combined with an increased sensitivity to leaving group ability of the sulfatase activity in neat D2O (Δβleaving groupH-D= +0.06) suggest that the mechanism for S-Obridgebond fission shifts, with decreasing leaving group ability, from charge compensation via Lewis acid interactions towards direct proton donation.18OnonbridgeKIEs indicate that the TS for PAS-catalyzed sulfate monoester hydrolysis has a significantly more associative character compared to the uncatalyzed reaction, while PAS-catalyzed phosphate monoester hydrolysis does not show this shift. This difference in enzyme-catalyzed TSs appears to be the major factor favoring specificity toward sulfate over phosphate in this promiscuous hydrolase, since other features are either too similar (uncatalyzed TS) or inherently favor phosphate (charge).

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Crystal structures of the GH6 Orpinomyces sp. Y102 CelC7 enzyme with exo and endo activity and its complex with cellobiose

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798319013597 ◽

2019 ◽

Vol 75 (12) ◽

pp. 1138-1147

Author(s):

Hsiao-Chuan Huang ◽

Liu-Hong Qi ◽

Yo-Chia Chen ◽

Li-Chu Tsai

Keyword(s):

Enzymatic Hydrolysis ◽

Crystal Structures ◽

Active Site ◽

Binding Sites ◽

Glycoside Hydrolase ◽

Catalytic Domain ◽

Glycoside Hydrolase Family ◽

Substrate Binding Sites ◽

Key Residues ◽

Hydrolase Family

The catalytic domain (residues 128–449) of the Orpinomyces sp. Y102 CelC7 enzyme (Orp CelC7) exhibits cellobiohydrolase and cellotriohydrolase activities. Crystal structures of Orp CelC7 and its cellobiose-bound complex have been solved at resolutions of 1.80 and 2.78 Å, respectively. Cellobiose occupies subsites +1 and +2 within the active site of Orp CelC7 and forms hydrogen bonds to two key residues: Asp248 and Asp409. Furthermore, its substrate-binding sites have both tunnel-like and open-cleft conformations, suggesting that the glycoside hydrolase family 6 (GH6) Orp CelC7 enzyme may perform enzymatic hydrolysis in the same way as endoglucanases and cellobiohydrolases. LC-MS/MS analysis revealed cellobiose (major) and cellotriose (minor) to be the respective products of endo and exo activity of the GH6 Orp CelC7.

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Jack bean urease (EC 3.5.1.5). V. On the mechanism of action of urease on urea, formamide, acetamide, N-methylurea, and related compounds

Canadian Journal of Biochemistry ◽

10.1139/o80-181 ◽

1980 ◽

Vol 58 (12) ◽

pp. 1335-1344 ◽

Cited By ~ 134

Author(s):

Nicholas E. Dixon ◽

Peter W. Riddles ◽

Carlo Gazzola ◽

Robert L. Blakeley ◽

Burt Zerner

Keyword(s):

Enzymatic Hydrolysis ◽

Mechanism Of Action ◽

Active Site ◽

Ph Dependence ◽

Jack Bean Urease ◽

Nickel Ion ◽

Jack Bean ◽

First Time ◽

Hydrolysis Of ◽

Hydrolysis Of Urea

Acetamide and N-methylurea have been shown for the first time to be substrates for jack bean urease. In the enzymatic hydrolysis of urea, formamide, acetamide, and N-methylurea at pH 7.0 and 38 °C, kcat has the values 5870, 85, 0.55, and 0.075 s−1, respectively. The urease-catalyzed hydrolysis of all these substrates involves the active-site nickel ion(s). Enzymatic hydrolysis of the following compounds could not be detected: phenyl formate, p-nitroformanilide, trifluoroacetamide, p-nitrophenyl carbamate, thiourea, and O-methylisouronium ion. In the enzymatic hydrolysis of urea, the pH dependence of kcat between pH 3.4 and 7.8 indicates that at least two prototropic forms are active. Enzymatic hydrolysis of urea in the presence of methanol gave no detectable methyl carbamate. A mechanism of action for urease is proposed which involves initially an O-bonded complex between urea and an active-site Ni2+ ion and subsequently an O-bonded carbamato–enzyme intermediate.

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Enzymatic Hydrolysis of Lignocellulosic Biomass Using Engineered Microorganisms and In Silico Approaches for Enhanced Enzyme Production

Microbial Fermentation and Enzyme Technology ◽

10.1201/9780429061257-19 ◽

2020 ◽

pp. 299-317

Author(s):

Manish Paul ◽

Sonali Mohapatra ◽

Hrudayanath Thatoi

Keyword(s):

Enzymatic Hydrolysis ◽

Lignocellulosic Biomass ◽

In Silico ◽

Enzyme Production ◽

Hydrolysis Of

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Contribution of a family 1 carbohydrate-binding module in thermostable glycoside hydrolase 10 xylanase from Talaromyces cellulolyticus toward synergistic enzymatic hydrolysis of lignocellulose

Biotechnology for Biofuels ◽

10.1186/s13068-015-0259-2 ◽

2015 ◽

Vol 8 (1) ◽

Cited By ~ 19

Author(s):

Hiroyuki Inoue ◽

Seiichiro Kishishita ◽

Akio Kumagai ◽

Misumi Kataoka ◽

Tatsuya Fujii ◽

...

Keyword(s):

Enzymatic Hydrolysis ◽

Glycoside Hydrolase ◽

Carbohydrate Binding ◽

Carbohydrate Binding Module ◽

Talaromyces Cellulolyticus ◽

Hydrolysis Of

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Bacillus licheniformistrehalose-6-phosphate hydrolase structures suggest keys to substrate specificity

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798315020756 ◽

2016 ◽

Vol 72 (1) ◽

pp. 59-70 ◽

Cited By ~ 3

Author(s):

Min-Guan Lin ◽

Meng-Chun Chi ◽

Vankadari Naveen ◽

Yi-Ching Li ◽

Long-Liu Lin ◽

...

Keyword(s):

Enzymatic Activity ◽

Bacillus Licheniformis ◽

Active Site ◽

Glycoside Hydrolase ◽

Positive Charge ◽

Glycoside Hydrolase Family ◽

Erwinia Rhapontici ◽

Hydrolysis Of ◽

Hydrolase Family ◽

Glycoside Hydrolase Family 13

Trehalose-6-phosphate hydrolase (TreA) belongs to glycoside hydrolase family 13 (GH13) and catalyzes the hydrolysis of trehalose 6-phosphate (T6P) to yield glucose and glucose 6-phosphate. The products of this reaction can be further metabolized by the energy-generating glycolytic pathway. Here, crystal structures ofBacillus licheniformisTreA (BlTreA) and its R201Q mutant complexed withp-nitrophenyl-α-D-glucopyranoside (R201Q–pPNG) are presented at 2.0 and 2.05 Å resolution, respectively. The overall structure ofBlTreA is similar to those of other GH13 family enzymes. However, detailed structural comparisons revealed that the catalytic site ofBlTreA contains a long loop that adopts a different conformation from those of other GH13 family members. Unlike the homologous regions ofBacillus cereusoligo-1,6-glucosidase (BcOgl) andErwinia rhaponticiisomaltulose synthase (NX-5), the surface potential of theBlTreA active site exhibits a largely positive charge contributed by the four basic residues His281, His282, Lys284 and Lys292. Mutation of these residues resulted in significant decreases in the enzymatic activity ofBlTreA. Strikingly, the281HHLK284motif and Lys292 play critical roles in substrate discrimination byBlTreA.

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