Elucidating the Nature of the<i>Streptomyces plicatus</i>β-Hexosaminidase-Bound Intermediate Using ab initio Molecular Dynamics Simulations

Greig, Ian R.; Zahariev, Federico; Withers, Stephen G.

doi:10.1021/ja805640c

Cited by 37 publications

(48 citation statements)

References 63 publications

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“…This asparagine takes the place of an enzymic carboxylate that typically forms a hydrogen bond with the amide nitrogen of the acetamido group within enzymes from GH families 18, 20, 56, and 84 (19 -23). Studies have variously proposed this carboxylate acts within these enzymes using substrate-assisted catalysis to stabilize an oxazolinium ion intermediate (23,27) or, alternatively, act as a general base to enhance the nucleophilicity of the acetamido group (15). Within GH85, however, previous studies into the functional consequences of deleting the side chain of this active site asparagine through site-directed mutagenesis have indicated that this conserved residue is absolutely critical; in the closely sequence-related Endo-M or Endo-A deletion results in apparently complete loss of activity toward a biantennary complex-type sialyl glycopeptide (29).…”

Section: Inhibition Of Spgh85 By Nag-thiazoline-nag-thiazolinementioning

confidence: 99%

“…A Conserved Proton Shuttle-As the cyclization step of the reaction occurs, the pK a of the amide proton of the substrate acetamido group goes from ϳ18 to ϳ5 for a putative oxazolinium ion intermediate (26,27). It therefore seems quite possible that in SpGH85, Asn-335 in its imidic acid form acts as a general base to facilitate catalysis because the pK a of this group will be higher than that of the putative oxazolinium ion intermediate with which it forms a hydrogen bond.…”

Section: Inhibition Of Spgh85 By Nag-thiazoline-nag-thiazolinementioning

confidence: 99%

“…The acetamido group is oriented, and its nucleophilicity enhanced, through donation of a hydrogen bond to a second suitably positioned carboxylate residue. This residue has been postulated to act as a general base to aid formation of an oxazoline intermediate (26) or, alternatively, to stabilize an oxazolinium ion intermediate (23,27). The resulting intermediate is broken down through a process that is the near microscopic reverse of the first step; the general acid/base residue now facilitates the attack of water, and the second residue facilitates departure of the 2-acetamido group to form the sugar hemiacetal product with overall retention of stereochemistry.…”

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Streptococcus pneumoniae Endohexosaminidase D, Structural and Mechanistic Insight into Substrate-assisted Catalysis in Family 85 Glycoside Hydrolases

Abbott

Macauley

Vocadlo

et al. 2009

Journal of Biological Chemistry

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Endo-␤-D-glucosaminidases from family 85 of glycoside hydrolases (GH85 endohexosaminidases) act to cleave the glycosidic linkage between the two N-acetylglucosamine units that make up the chitobiose core of N-glycans. Endohexosaminidase D (Endo-D), produced by Streptococcus pneumoniae, is believed to contribute to the virulence of this organism by playing a role in the deglycosylation of IgG antibodies. Endohexosaminidases have received significant attention for this reason and, moreover, because they are powerful tools for chemoenzymatic synthesis of proteins having defined glycoforms. Here we describe mechanistic and structural studies of the catalytic domain (SpGH85) of Endo-D that provide compelling support for GH85 enzymes using a catalytic mechanism involving substrate-assisted catalysis. Furthermore, the structure of SpGH85 in complex with the mechanism-based competitive inhibitor NAG-thiazoline (K d ‫؍‬ 28 M) provides a coherent rationale for previous mutagenesis studies of Endo-D and other related GH85 enzymes. We also find GH85, GH56, and GH18 enzymes have a similar configuration of catalytic residues. Notably, GH85 enzymes have an asparagine in place of the aspartate residue found in these other families of glycosidases. We propose that this residue, as the imidic acid tautomer, acts analogously to the key catalytic aspartate of GH56 and GH18 enzymes. This topographically conserved arrangement of the asparagine residue and a conserved glutamic acid, coupled with previous kinetic studies, suggests these enzymes may use an unusual proton shuttle to coordinate effective general acid and base catalysis to aid cleavage of the glycosidic bond. These results collectively provide a blueprint that may be used to facilitate protein engineering of these enzymes to improve their function as biocatalysts for synthesizing glycoproteins having defined glycoforms and also may serve as a guide for generating inhibitors of GH85 enzymes.

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Section: Inhibition Of Spgh85 By Nag-thiazoline-nag-thiazolinementioning

confidence: 99%

Section: Inhibition Of Spgh85 By Nag-thiazoline-nag-thiazolinementioning

confidence: 99%

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confidence: 99%

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Streptococcus pneumoniae Endohexosaminidase D, Structural and Mechanistic Insight into Substrate-assisted Catalysis in Family 85 Glycoside Hydrolases

Abbott

Macauley

Vocadlo

et al. 2009

Journal of Biological Chemistry

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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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“…In addition, the QM/MM methods based on the all-atom ab initio molecular dynamics, is performed on the β-hexosmaminidase of glycoside hydrolase family 20, which is similar to family 18 chitinases. Grei et al [84] have successfully performed the combined technique to elucidate the catalytic role of Asp313 and the nature of the enzymebound intermediate, therefore, QM/MM technique also can be applied to study the catalytic mechanism more clearly.…”

Section: Future Persepectivementioning

confidence: 99%

Investigation of Family 18 Chitinases and Inhibitors by Computer-Aided Approaches

Chu

Wang²,

Shen³

et al. 2012

CDT

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Chitinases belong to family 18 glycosyl hydrolases that can hydrolyze chitin by cleaving β-1,4-glycosidic bond, and are at key points in the life cycles of organism. The inhibitors of chitinases not only have chemotherapeutic potential against fungi, insects, but also hold anti-inflammatory efficacy against asthma and allergic disease in human. This review summarizes the structural characters of chitinases, the proposed catalytic mechanism, furthermore, also gives descriptions of currently existing inhibitors. In addition, computational studies of the interaction modes of chitinases with different inhibitors and substrates, as well as the inhibitor design of chitinases, are summarized so as to obtain an overall understanding for chitinases.Keywords: Chitinases, chitinase inhibitors, computer-aided approach. CHITIN AND CHITINASESChitin (C 8 H 13 O 5 N) n (shown in Fig. 1a), a linear insoluble homopolymer of N-acetyl-D-glucosamine (GlcNAc), is the second most abundant polysaccharide on the earth next to the cellulose [1, 2] and has been found in a wide range of organism, including bacteria, fungi, insects, and viruses [3][4][5].Accumulation of chitin in an organism is modulated by chitin synthase-mediated biosynthesis and chitinases-mediated hydrolytic degradation [6]. Since chitin synthases have become an ideal target for the development of pesticides and fugicides in agriculture, the design of chitin synthase's inhibitors has attracted much more attention [7,8]. Recently, some chitin synthase inhibitors have been successfully designed and synthesized in the group of Dr. Qing Yang [6,9], and these synthesized inhibitors were claimed to be novel potential agents due to their bioactivities. Chitinases (EC 3.2.1.14) play a key role in hydrolyzing β-1,4-glycosidic bond of the chitin into low molecular weight, soluble oligomers of GlcNAc [10,11]. Chitinases are currently classified into two different families of glycosyl hydrolases, namely family 18 and family 19 [12][13][14], and this review mainly focuses on family 18 chitinases studies, information available from their three-dimensional structures and the biochemistry of reaction. Chitinases are also widespread, and perform different functions in the different organism [15][16][17][18][19][20]. Although chitin does not exist in mammals, human chitinase family members, such as acidic mammalian chitinase (AMCase), have also been described in previous Therefore, the design of chitinases inhibitors is of great interest. The well-known inhibitor allosamidin (shown in Fig. 1b) is the most potent broad-spectrum inhibitor of chitinases. It inhibits chitinases by mimicing the reaction intermediate [24], but unfortunately, the synthesis of allosamidin and its derivatives are very difficult and expensive, which makes it a less suitable candidate targating a common chemotherapeutical therapy [25]. Izumida et al. found another effective chitinases inhibitor, the cyclic dipeptide [cyclo-(L-Arg-D-pro)] CI-4 (shown in Fig. 1c) [26]. CI-4 shows relatively weak inhibition ...

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Elucidating the Nature of theStreptomyces plicatusβ-Hexosaminidase-Bound Intermediate Using ab initio Molecular Dynamics Simulations

Cited by 37 publications

References 63 publications

Streptococcus pneumoniae Endohexosaminidase D, Structural and Mechanistic Insight into Substrate-assisted Catalysis in Family 85 Glycoside Hydrolases

Streptococcus pneumoniae Endohexosaminidase D, Structural and Mechanistic Insight into Substrate-assisted Catalysis in Family 85 Glycoside Hydrolases

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

Investigation of Family 18 Chitinases and Inhibitors by Computer-Aided Approaches

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