<i>Trypanosoma c</i><i>ruzi</i> Trans-sialidase Operates through a Covalent Sialyl−Enzyme Intermediate:  Tyrosine Is the Catalytic Nucleophile

Watts, Andrew G.; Damager, Iben; Amaya, M.F.; Buschiazzo, Alejandro; Alzari, Pedro M.; Frasch, Alberto C.C.; Withers, Stephen G.

doi:10.1021/ja0344967

Cited by 203 publications

(164 citation statements)

References 13 publications

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“…The observed decrease in turnover rate, therefore, indicates either that this residue is not the catalytic nucleophile or that an uncharged nucleophile is required for optimal activity on a charged substrate. The recent finding that an active site tyrosine acts as the nucleophile in the double displacement mechanism of a trans-sialidase, which uses charged sialic acid-containing substrates, supports this latter interpretation (32). Alternatively, this finding is consistent with the notion that this enzyme uses a mechanism that differs from that involving the discrete nucleophilic catalysis of a double displacement mechanism.…”

Section: Resultsmentioning

confidence: 74%

Intermediate Trapping on a Mutant Retaining α-Galactosyltransferase Identifies an Unexpected Aspartate Residue

Lairson

Chiu

et al. 2004

Journal of Biological Chemistry

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Lipopolysaccharyl-␣-1,4-galactosyltransferase C (LgtC), a glycosyltransferase family 8 ␣-1,4-galactosyltransferase from Neisseria meningitidis, catalyzes the transfer of galactose from UDP galactose to terminal lactose-containing acceptor sugars with net retention of anomeric configuration. To investigate the potential role of discrete nucleophilic catalysis suggested by the double displacement mechanism generally proposed for retaining glycosyltransferases, the side chain amide of Gln-189, which is suitably positioned to act as the catalytic nucleophile of LgtC, was substituted with the more nucleophilic carboxylate-containing side chain of glutamate in the hope of accumulating a glycosyl-enzyme intermediate. The resulting mutant was subjected to kinetic, mass spectrometric, and x-ray crystallographic analysis. Although the K m for UDP-galactose is not significantly altered, the k cat was reduced to 3% that of the wild type enzyme. Electrospray mass spectrometric analysis revealed that a steady state population of the Q189E variant contains a covalently bound galactosyl moiety. Liquid chromatographic/mass spectrometric analysis of fragmented proteolytic digests identified the site of labeling not as Glu-189 but, surprisingly, as the sequentially adjacent Asp-190. However, the side chain carboxylate of Asp-190 is located 8.9 Å away from the donor substrate in the available crystal structure. Kinetic analysis of a D190N mutant at this position revealed a k cat value 3000-fold lower than that of the wild type enzyme. A 2.6-Å crystal structure of the Q189E mutant with bound uridine 5 -diphospho-2-deoxy-2-fluoro-␣-D-galactopyranose revealed no significant perturbation of the mode of donor sugar binding nor of active site configuration. This is the first trapping of an intermediate in the active site of a retaining glycosyltransferase and, although not conclusive, implicates Asp-190 as an alternative candidate catalytic nucleophile, thereby rekindling a longstanding mechanistic debate.Oligosaccharides on glycoproteins and glycolipids distributed on cell surfaces and within extracellular matrices are known to play key roles in normal cell functions including cell growth and differentiation, recognition by the immune system, and cell-cell interactions (1-3). Changes in the composition of these glycoconjugates are often associated with disease states, including the metastasis of cancerous cells and autoimmune responses (4 -7). They are also known to modulate interactions with viral and bacterial pathogens leading to infection and are involved in mechanisms to evade host immune responses (8 -10). Glycosyltransferases, the anabolic enzymes responsible for the highly specific construction of these carbohydrate structures, therefore, not only represent an attractive class of therapeutic targets but also are important tools for the enzymatic synthesis of this synthetically challenging class of therapeutic agents. Of central importance to both of these applications is a detailed understanding of the mechanisms by which this c...

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Section: Resultsmentioning

confidence: 74%

Intermediate Trapping on a Mutant Retaining α-Galactosyltransferase Identifies an Unexpected Aspartate Residue

Lairson

Chiu

et al. 2004

Journal of Biological Chemistry

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“…The mutation of this residue to phenylalanine leads to an almost complete loss of activity in GH8 members. Even though tyrosine can act as nucleophile in some retaining GHs, 64 the role of Tyr215 in GH8 is apparently to fix the water molecule in a correct position for catalysis. 26 To confirm the identity of the proton acceptor in GH8 C. thermocellum EG, we calculated where the proton released from the nucleophilic water molecule after hydrolysis was likely to go by performing a series of QM/MM geometric optimizations for three different situations: (1) after Asp278 takes the proton; (2) after Tyr215, which forms a second hydrogen bond with the nucleophilic water molecule, takes the proton leading to the tyrosine hydroxyl group becoming positively charged; (3) after the solvent becomes the proton acceptor, forming a H 3 O + cation.…”

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confidence: 99%

Mechanism of Cellulose Hydrolysis by Inverting GH8 Endoglucanases: A QM/MM Metadynamics Study

et al. 2009

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A detailed understanding of the catalytic strategy of cellulases is key to finding alternative ways to hydrolyze cellulose to mono-, di-, and oligosaccharides. Endoglucanases from glycoside hydrolase family 8 (GH8) catalyze the hydrolysis of β-1,4-glycosidic bonds in cellulose by an inverting mechanism believed to involve a oxacarbenium ion-like transition state (TS) with a boat-type conformation of the glucosyl unit in subsite −1. In this work, hydrolysis by Clostridium thermocellum endo-1,4-glucanase A was computationally simulated with quantum mechanics/molecular mechanics metadynamics based on density functional theory. Our calculations show that the glucosyl residue in subsite −1 in the Michaelis complex is in a distorted 2 S O / 2,5 B ring conformation, agreeing well with its crystal structure. In addition, our simulations capture the cationic oxacarbenium ion-like character of the TS with a partially formed double bond between the ring oxygen and C5′ carbon atoms. They also provide previously unknown structural information of important states along the reaction pathway. The simulations clearly show for the first time in GH8 members that the TS features a boattype conformation of the glucosyl unit in subsite −1. The overall catalytic mechanism follows a D N *A N -like mechanism and a β-2 S O → 2,5 B [TS] → α-5 S 1 conformational itinerary along the reaction coordinate, consistent with the anti-periplanar lone pair hypothesis. Because of the structural similarities and sequence homology among all GH8 members, our results can be extended to all GH8 cellulases, xylanases, and other endoglucanases. In addition, we provide evidence supporting the role of Asp278 as the catalytic proton acceptor (general base) for GH8a subfamily members. Disciplines Biochemical and Biomolecular Engineering | Biological Engineering | Chemical Engineering CommentsPosted with permission from The Journal of Physical Chemistry B, 113, no. 20 (2009) ReceiVed: December 29, 2008; ReVised Manuscript ReceiVed: March 18, 2009 A detailed understanding of the catalytic strategy of cellulases is key to finding alternative ways to hydrolyze cellulose to mono-, di-, and oligosaccharides. Endoglucanases from glycoside hydrolase family 8 (GH8) catalyze the hydrolysis of -1,4-glycosidic bonds in cellulose by an inverting mechanism believed to involve a oxacarbenium ion-like transition state (TS) with a boat-type conformation of the glucosyl unit in subsite -1. In this work, hydrolysis by Clostridium thermocellum endo-1,4-glucanase A was computationally simulated with quantum mechanics/molecular mechanics metadynamics based on density functional theory. Our calculations show that the glucosyl residue in subsite -1 in the Michaelis complex is in a distortedB ring conformation, agreeing well with its crystal structure. In addition, our simulations capture the cationic oxacarbenium ion-like character of the TS with a partially formed double bond between the ring oxygen and C5′ carbon atoms. They also provide previously unknown structural inf...

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“…A tyrosine residue is a neutral nucleophile, but requires a general base to enhance its nucleophilicity. This mechanism was implied from X-ray structures, and was supported by experiments involving trapping of the intermediate with fluoro sugars followed by peptide mapping and then crystallography [6,7], and also through mechanism studies on mutants [8] (Figure 7). …”

Section: Alternative Nucleophilesmentioning

confidence: 74%

Extremophilic Carbohydrate Active Enzymes (CAZymes)

Lee¹,

Park²,

Park³

2017

JNHFE

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Carbohydrate active enzymes (CAZymes) are a large class of enzymes, which build and breakdown the complex carbohydrates of the cell. On the basis of their amino acid sequences they are classified in families that show conserved catalytic mechanism, structure, and active site residues, but may conflict each other in substrate specificity. The CAZymes provides a continuously updated list of the glycoside hydrolase families, GHs. This group of enzymes is classified based on functional similarity, but today they are classified into 108 GHs on the basis of amino acid sequence similarity. Despite their similarities to enzymes with known functions, their primary functions are still unclear. Based on these criteria, β-galactosidase activities are now divided into four different families: GH1, GH2, GH35 and GH42, among which the better studied GH2 includes β-galactosidase from Escherichia coli, Aspergillus, Bacillus megatherium, and Sulfolobus solfataricus, while those from thermophilic, psychrophilic and halophilic organisms belong to GH42. Lactase is often confused as an alternate name for β-galactosidase, but it is actually simply a subclass (small subunit) of β-galactosidase.(β-D-galactoside galactohydrolase, EC 3.2.1.23) that catalyses hydrolysis of the galactosyl moiety from non-reducing termini of oligosaccharides or from glycosides. Most genes encoding GH42 enzymes are from prokaryotes that are unable to grow on lactose as a sole carbon source and at least two GH42 β-galactosidase do not cleave lactose in vitro. The determination of growth on lactose can be complicated by the multiple β-galactosidases because not all of the β-galactosidase is acting as lactases in vitro. Β-Galactosidase hydrolyses the β-1, 4-D-galactosidic linkage of lactose, as well as those of related chromogens, o-nitrophenyl-β-dgalactopyranoside (ONPG), p-nitrophenyl-β-d-galactopyranoside (PNPG) and 6-bromo-2-naphthyl-galactopyranoside (BNG). This enzyme has been purified and characterized from various sources, including plants, animals, and many microorganisms. In humans, lactase is present predominantly along the brush border membrane of the differentiated enterocytes lining the villi of the small intestine. Lactase is essential for digestive hydrolysis of lactose in milk. Deficiency of the enzyme causes lactose intolerance. Several 3D structures are available for the GH1 and GH2 β-galactosidase for which the catalytic residues have experimentally been determined, while three 3D structures of β-galactosidase from E. coli, Penicillium sp and Thermus thermophilus A4 are available for both the GH35 and GH42 families. Glycoside hydrolasesGlycoside hydrolases are enzymes that catalyze the hydrolysis of the glycosidic linkage of glycosides, leading to the formation of a sugar hemiacetal or hemiketal and the corresponding free aglycon. Glycoside hydrolases are also referred to as glycosidases, and sometimes also as glycosyl hydrolases. Glycoside hydrolases can catalyze the hydrolysis of O-, N-and S-linked glycosides ( Figure 1). Endo/Exo:Exo / ...

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Trypanosoma cruzi Trans-sialidase Operates through a Covalent Sialyl−Enzyme Intermediate: Tyrosine Is the Catalytic Nucleophile

Cited by 203 publications

References 13 publications

Intermediate Trapping on a Mutant Retaining α-Galactosyltransferase Identifies an Unexpected Aspartate Residue

Intermediate Trapping on a Mutant Retaining α-Galactosyltransferase Identifies an Unexpected Aspartate Residue

Mechanism of Cellulose Hydrolysis by Inverting GH8 Endoglucanases: A QM/MM Metadynamics Study

Extremophilic Carbohydrate Active Enzymes (CAZymes)

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