The Reaction Coordinate of a Bacterial GH47 α‐Mannosidase: A Combined Quantum Mechanical and Structural Approach

Thompson, Andrew J.; Dabin, Jérôme; Iglésias-Fernández, Javier; Ardèvol, Albert; Dinev, Zoran; Williams, Spencer J.; Bande, Omprakash; Siriwardena, Aloysius; Moreland, Carl; Hu, Ting‐Chou; Smith, David K.; Gilbert, Harry J.; Rovira, Carme; Davies, G.J.

doi:10.1002/anie.201205338

Cited by 58 publications

(84 citation statements)

References 33 publications

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“…This model is one of the most complete protein models in the PDB examined so far (Table I), and has the smallest R-factor gap with R Ratio of 1.22 (see Methods). [4][5][6][7][8][9][10][11][12][13][14][15] Evidence will be provided below that its corresponding residual ED map is the closest to true featurelessness with all the residual peaks contributed mainly by random noise present in the intensity data.…”

Section: Resultsmentioning

confidence: 99%

Systematic analysis of residual density suggests that a major limitation in well‐refined X‐ray structures of proteins is the omission of ordered solvent

Wang

2017

Protein Science

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In the residual electron density map of a fully refined X-ray protein model, there should be no peaks arising from modeling errors or missing atoms. Any residual peaks that do occur should be contributed by random residual intensity differences between the model and the data. If the model is incomplete (i.e., some atoms are missing), there will be more positive peaks than negative ones. On the other hand, if the model includes inappropriately located atoms, there will be an excess of negative peaks. In this study, random residual peaks are quantified using the probability density function P(x), which is defined as the probability for a peak having peak height between x and x + dx. It is found that P(x) is single-exponential and symmetric for both positive and negative peaks. Thus, P(x) can be used to discriminate residual peaks contributed by random noise in complete models from residual peaks being attributable to modeling errors in incomplete models. For a number of representative structures in the PDB it is found that P(x) has far more large (greater than 5 sigma) positive peaks than large negative peaks. This excess of large positive peaks suggests that the main defect in these refined structures is the omission of ordered water molecules.

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Section: Resultsmentioning

confidence: 99%

Systematic analysis of residual density suggests that a major limitation in well‐refined X‐ray structures of proteins is the omission of ordered solvent

Wang

2017

Protein Science

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“…Such a conformational itinerary has never been reported for a β-mannosidase or β-mannanase; however, a reversed Southern hemisphere 3 S 1 → 3 H 4 ‡ → 1 C 4 conformational itinerary has been assigned to inverting α-mannosidases from family GH47. 34−36 …”

Section: Resultsmentioning

confidence: 99%

“…It is expected that once the product exits the active site it relaxes to its most stable 4 C 1 conformation in solution. 36 The computed mechanism can be considered an electrophilic migration of the anomeric carbon from the departing sugar residue to the nucleophilic water, assisted by E45 as general acid and D57 as general base (Figure 5). …”

Section: Resultsmentioning

confidence: 99%

A β-Mannanase with a Lysozyme-like Fold and a Novel Molecular Catalytic Mechanism

Jin

Petricevic²,

John

et al. 2016

ACS Cent. Sci.

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The enzymatic cleavage of β-1,4-mannans is achieved by endo-β-1,4-mannanases, enzymes involved in germination of seeds and microbial hemicellulose degradation, and which have increasing industrial and consumer product applications. β-Mannanases occur in a range of families of the CAZy sequence-based glycoside hydrolase (GH) classification scheme including families 5, 26, and 113. In this work we reveal that β-mannanases of the newly described GH family 134 differ from other mannanase families in both their mechanism and tertiary structure. A representative GH family 134 endo-β-1,4-mannanase from a Streptomyces sp. displays a fold closely related to that of hen egg white lysozyme but acts with inversion of stereochemistry. A Michaelis complex with mannopentaose, and a product complex with mannotriose, reveal ligands with pyranose rings distorted in an unusual inverted chair conformation. Ab initio quantum mechanics/molecular mechanics metadynamics quantified the energetically accessible ring conformations and provided evidence in support of a 1C4 → 3H4‡ → 3S1 conformational itinerary along the reaction coordinate. This work, in concert with that on GH family 124 cellulases, reveals how the lysozyme fold can be co-opted to catalyze the hydrolysis of different polysaccharides in a mechanistically distinct manner.

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“…Thisc onformation is unusualf or an iminosugar. Relative to the proposed conformational pathway 3 16,17] for this family of enzymes, it pro-vides conformational mimicryo ft he product. Of relevance to this observation, ac omplex of noeuromycin 2 bound to ab acterial a-mannosidase of family GH47, Caulobacter sp.…”

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confidence: 99%

“…1 C 4 conformational itinerary that this family of enzymes is believed to follow. [2b, 16,17] We highlight that this analysiss uggests that potency is achieved in the absence of transition state mimicry.I ti ss urprising that this conformational restraint is achieved by introducing an oxalamide bridge without interfering with binding in the active site, as ap reviousa ttemptw ith as imilar goal to synthetically introduce ab ridge into an iminosugar failed owing to steric clashes in the actives ite. [5] The preference of kifunensinef or a 1 C 4 conformation likely arises not merely from the fusion of the bridge to the ring, but also from the sp 2 hybridisation of the endocyclic nitrogen as part of an amide.…”

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Conformational Analysis of the Mannosidase Inhibitor Kifunensine: A Quantum Mechanical and Structural Approach

et al. 2017

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The varied yet family-specific conformational pathways used by individual glycoside hydrolases (GHs) offer at antalising prospectf or the design of tightly binding and specific enzyme inhibitors. Ac ardinal example of aG H-family-specific inhibitor, and one that finds widespread practical use, is the natural product kifunensine, which is al ow-nanomolar inhibitor that is selectivef or GH family 47 inverting a-mannosidases. Here we show,t hrough quantum-mechanical approaches, that kifunensine is restrained to a" ring-flipped" 1 C 4 conformation with another accessible, but higher-energy,r egion aroundt he 1,4 B conformation.T he conformationso fk ifunensine in complex with ar ange of GH47 enzymes-including an atomic-levelr esolution (1 )s tructure of kifunensinew ith Caulobacter sp. CkGH47 reportedh erein and with GH family 38 and 92 a-mannosidases-werem apped ontot he kifunensine free-energy landscape. These studies revealed that kifunensine has the ability to mimic the product state of GH47 enzymesb ut cannot mimic any conformational states relevant to the reactionc oordinate of mannosidases from other families.There is compelling evidence that the enzymatic hydrolysis of glycosides, catalysed by glycoside hydrolases (GHs) or glycosidases,o ccurs via transition states with significant oxocarbenium ion character.F or pyranoside-active enzymes,S innott was the first to argue that the allowed canonical conformationso f the transition state sugar ring were two half-chairs ( 4 H 3 and 3

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The Reaction Coordinate of a Bacterial GH47 α‐Mannosidase: A Combined Quantum Mechanical and Structural Approach

Cited by 58 publications

References 33 publications

Systematic analysis of residual density suggests that a major limitation in well‐refined X‐ray structures of proteins is the omission of ordered solvent

Systematic analysis of residual density suggests that a major limitation in well‐refined X‐ray structures of proteins is the omission of ordered solvent

A β-Mannanase with a Lysozyme-like Fold and a Novel Molecular Catalytic Mechanism

Conformational Analysis of the Mannosidase Inhibitor Kifunensine: A Quantum Mechanical and Structural Approach

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