Computational analyses of the conformational itinerary along the reaction pathway of GH94 cellobiose phosphorylase

Fushinobu, Shinya; Mertz, Blake; Hill, Arthur V.; Hidaka, Masafumi; Kitaoka, Motomitsu; Reilly, Peter J.

doi:10.1016/j.carres.2008.02.026

Cited by 23 publications

(26 citation statements)

References 35 publications

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“…One of the four oxygen atoms in P i is located near the anomeric C1 carbon of glucose at subsite À1 (dis-tance = 3.2 A). This observation indicates that the phosphorolysis reaction of GH65 begins with direct nucleophilic attack by phosphate, as has been shown in other inverting GH-type phosphorylases [22,23]. All of the hydroxyl groups of the b-glucose at subsite À1 form hydrogen bonds with Trp343, Asp344, Lys596, and Gln597.…”

Section: Active Sitesupporting

confidence: 67%

Structural and mutational analysis of substrate recognition in kojibiose phosphorylase

et al. 2013

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Glycoside hydrolase (GH) family 65 contains phosphorylases acting on maltose (Glc-a1,4-Glc), kojibiose (Glc-a1,2-Glc), trehalose (Glc-a1,a1,-Glc), and nigerose (Glc-a1,3-Glc). These phosphorylases can efficiently catalyze the reverse reactions with high specificities, and thus can be applied to the practical synthesis of a-glucosyl oligosaccharides. Here, we determined the crystal structures of kojibiose phosphorylase from Caldicellulosiruptor saccharolyticus in complex with glucose and phosphate and in complex with kojibiose and sulfate, providing the first structural insights into the substrate recognition of a glycoside hydrolase family 65 enzyme. The loop 3 region comprising the active site of kojibiose phosphorylase is significantly longer than the active sites of other enzymes, and three residues around this loop, Trp391, Glu392, and Thr417, recognize kojibiose. Various mutants mimicking the residue conservation patterns of other phosphorylases were constructed by mutation at these three residues. Activity measurements of the mutants against four substrates indicated that Trp391 and Glu392, especially the latter, are required for the kojibiose activity.

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Section: Active Sitesupporting

confidence: 67%

Structural and mutational analysis of substrate recognition in kojibiose phosphorylase

et al. 2013

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“…These structural features suggest that the inverting phosphorolytic reaction begins with a direct nucleophilic attack by phosphate on the anomeric carbon, but the evidence for the mechanism has yet to be obtained. This type of mechanism has also been proposed for cellobiose phosphorylase, the other inverting sugar phosphorylase in GH94 (53)(54)(55). In the crystal structures of cellobiose phosphorylase, subsite ϩ1 is occupied by glucose, and the adjacent subsite Ϫ1 and phosphate binding site are occupied by glycerol and SO 4 2Ϫ or phosphate, respectively.…”

Section: Discussionmentioning

confidence: 73%

“…Efficient catalysis on the hydrolytic or phosphorolytic cleavage of ␤-glucosidic bonds is considered to require distortion of the glycon sugar ring (55)(56)(57)(58). In this study, we tried to dock LNB with several distorted conformers at its glycon (e.g.…”

Section: Discussionmentioning

confidence: 99%

The Crystal Structure of Galacto-N-biose/Lacto-N-biose I Phosphorylase

Hidaka

Nishimoto

Kitaoka

et al. 2009

Journal of Biological Chemistry

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. GLNBP homologues are often found in human pathogenic and commensal bacteria, and their substrate specificities potentially define the nutritional acquisition ability of these microbes in their habitat. We report the crystal structures of GLNBP in five different ligand-binding forms. This is the first three-dimensional structure of glycoside hydrolase (GH) family 112. The GlcNAc-and GalNAc-bound forms provide structural insights into distinct substrate preferences of GLNBP and its homologues from pathogens. The catalytic domain consists of a partially broken TIM barrel fold that is structurally similar to a thermophilic ␤-galactosidase, strongly supporting the current classification of GLNBP homologues as one of the GH families. Anion binding induces a large conformational change by rotating a half-unit of the barrel. This is an unusual example of molecular adaptation of a TIM barrel scaffold to substrates.

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“…5,6 The conformational interconversions that occur in the active sites of specific GHs have been the subject of many recent studies. [7][8][9][10][11][12][13][14] Nevertheless, direct evidence of conformational itineraries is still missing for many GH families. Furthermore, detailed structural features of the enzymatic transition state (TS) in GHs are still fairly unknown.…”

Section: Introductionmentioning

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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Computational analyses of the conformational itinerary along the reaction pathway of GH94 cellobiose phosphorylase

Cited by 23 publications

References 35 publications

Structural and mutational analysis of substrate recognition in kojibiose phosphorylase

Structural and mutational analysis of substrate recognition in kojibiose phosphorylase

The Crystal Structure of Galacto-N-biose/Lacto-N-biose I Phosphorylase

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

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