Mechanistic evidence for a front-side, SNi-type reaction in a retaining glycosyltransferase

Lee, Seung Seo; Hong, Sung You; Errey, James C.; Izumi, Atsushi; Davies, G.J.; Davis, Benjamin G.

doi:10.1038/nchembio.628

Cited by 144 publications

(209 citation statements)

References 55 publications

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“…The structure showed that hydrogen bonding existed between the leaving group oxygen of UDP and the nucleophile mimic of the sugar moiety, suggestive of an S N i-like reaction mechanism. Kinetic isotope effects and linear free energy relationship experiments (40), along with computational studies (41) on the inhibitor complex, confirmed that VA6P and UDP acted together as synergistic transition state mimics, supporting front-face nucleophilic attack involving hydrogen bonding between leaving group and nucleophile. Structural comparison with our TarM ternary-like complex reveals a very similar active site composition with good structural agreement between UDP and donor sugar glycoside products (Fig.…”

Section: Discussionmentioning

confidence: 78%

Structure and mechanism ofStaphylococcus aureusTarM, the wall teichoic acid α-glycosyltransferase

Sobhanifar

Worrall

Gruninger

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Unique to Gram-positive bacteria, wall teichoic acids are anionic glycopolymers cross-stitched to a thick layer of peptidoglycan. The polyol phosphate subunits of these glycopolymers are decorated with GlcNAc sugars that are involved in phage binding, genetic exchange, host antibody response, resistance, and virulence. The search for the enzymes responsible for GlcNAcylation in Staphylococcus aureus has recently identified TarM and TarS with respective α-and β-(1-4) glycosyltransferase activities. The stereochemistry of the GlcNAc attachment is important in balancing biological processes, such that the interplay of TarM and TarS is likely important for bacterial pathogenicity and survival. Here we present the crystal structure of TarM in an unusual ternary-like complex consisting of a polymeric acceptor substrate analog, UDP from a hydrolyzed donor, and an α-glyceryl-GlcNAc product formed in situ. These structures support an internal nucleophilic substitution-like mechanism, lend new mechanistic insight into the glycosylation of glycopolymers, and reveal a trimerization domain with a likely role in acceptor substrate scaffolding.glycosyltransferase | wall teichoic acid | cell wall | crystal structure | bacterial pathogenicity

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Section: Discussionmentioning

confidence: 78%

Structure and mechanism ofStaphylococcus aureusTarM, the wall teichoic acid α-glycosyltransferase

Sobhanifar

Worrall

Gruninger

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Recently, it was reported that relatively high values for -SD(T)KIEs were observed in the synthesis reaction of ,-trehalose 6-phosphate by ,-trehalose 6-phosphat synthase (UDP forming) as a retaining glycosyltransferase (EC 2.4.1.15): the values forand -SDKIEs are 1.20 and 1.16, respectively, and the value for -STKIEs, 1.16. 38,39) This implies that, in the enzyme classified into the category of transferase (EC 2 groups) too, the splitting reaction of the C 1 -O bond of UDPglucose occurs through resonance between oxocarbenium ion and the carbocation intermediate.…”

Section: )mentioning

confidence: 99%

A Historical Perspective for the Catalytic Reaction Mechanism of Glycosidase; So As to Bring about Breakthrough in Confusing Situation

Chiba

2012

Bioscience, Biotechnology, and Biochemistry

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Various catalytic reaction models have been proposed as the reaction mechanisms of glycosidases, but a reasonable and unitary model capable of interpreting both ''inverting'' and ''retaining'' glycosidase reactions remains to be established. As for the models proposed to date, the nucleophilic displacement mechanism and the oxocarbenium ion intermediate mechanism are widely known, but recently the former is widely accepted, and so the general tendency of world opinion appears to favor it. This reaction model, however, is considered to comprise some inconsistencies that cannot be neglected from the viewpoint of reactivity in organic chemistry. While the nucleophilic displacement mechanism is often applied to reactions of glycosidases, it appears unlikely that such reactions actually occur. This review argues that the oxocarbenium ion intermediate reaction mechanism is more rational than the nucleophilic displacement reaction mechanism, as the action mode of glycosidases and related enzymes.

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“…Recently, in a detailed exhaustive study (15), the reaction catalyzed by OtsA has been shown to proceed through a highly dissociative oxocarbenium ion-like transition state, with a substantial participation of the incoming nucleophile through hydrogen bonding donation to the leaving group. This finding and the demonstration that the OtsA/UDP/validoxamine A 6 0 -Ophosphate represents a transition state mimics have been interpreted as a mechanistic evidence of a front-side S N i-like reaction mechanism (Path A, Fig.…”

Section: Discussionmentioning

confidence: 99%

“…4), as long as the hydrogen bonding interaction between the donor and acceptor molecules is preserved. In particular, the KIE (KIE 5 1.164) observed when the hydrogen atom attached to the C2 of the glucosyl moiety of the donor was substituted by a deuterium was interpreted as a large b-secondary effect, which highlighted the hyperconjugation of the C2À ÀH r-bond and the electron-deficient C1 carbon of the oxocarbenium (15). The difference between extensive hyperconjugation and full donation of the C2À ÀH r-bond electrons is subtle, and the formation of a C1-C2 double bond could be viewed as an extreme case of hyperconjugation.…”

Section: Discussionmentioning

confidence: 99%

“…Quantum mechanics/molecular mechanics metadynamics simulations of the reaction catalyzed by treahalose-6-phosphate synthase (OtsA), a retaining GT classed in family 20, indicated that a stepwise S N i-like mechanism could provide a reaction pathway with a low activation energy (20), consistent with the experimental data. Furthermore, the measurement of several kinetic isotope effects (KIEs) in the OtsA-catalyzed reaction supported the existence of a highly dissociative oxocarbenium ion-like transition state, in which the bond of the anomeric carbon to the leaving group was almost broken and the acceptor was partially deprotonated at an early stage (15).…”

Section: Introductionmentioning

confidence: 91%

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Lyase activity of glycogen synthase: Is an elimination/addition mechanism a possible reaction pathway for retaining glycosyltransferases?

et al. 2012

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Despite the biological relevance of glycosyltrasferases (GTs) and the many efforts devoted to this subject, the catalytic mechanism through which a subclass of this large family of enzymes, namely those that operate with net retention of the anomeric configuration, has not been fully established. Here, we show that in the absence of an acceptor, an archetypal retaining GT such as Pyrococcus abyssi glycogen synthase (PaGS) reacts with its glucosyl donor substrate, uridine 5'‐diphosphoglucose (UDP‐Glc), to produce the scission of the covalent bond between the terminal phosphate oxygen of UDP and the sugar ring. X‐ray diffraction analysis of the PaGS/UDP‐Glc complex shows no electronic density attributable to the UDP moiety, but establishes the presence in the active site of the enzyme of a glucose‐like derivative that lacks the exocyclic oxygen attached to the anomeric carbon. Chemical derivatization followed by gas chromatography/mass spectrometry of the isolated glucose‐like species allowed us to identify the molecule found in the catalytic site of PaGS as 1,5‐anhydro‐D‐arabino‐hex‐1‐enitol (AA) or its tautomeric form, 1,5‐anhydro‐D‐fructose. These findings are consistent with a stepwise SNi‐like mechanism as the modus operandi of retaining GTs, a mechanism that involves the discrete existence of an oxocarbenium intermediate. Even in the absence of a glucosyl acceptor, glycogen synthase (GS) promotes the formation of the cationic intermediate, which, by eliminating the proton of the adjacent C2 carbon atom, yields AA. Alternatively, these observations could be interpreted assuming that AA is a true intermediate in the reaction pathway of GS and that this enzyme operates through an elimination/addition mechanism. © 2012 IUBMB Life, 64(7): 649–658, 2012.

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Mechanistic evidence for a front-side, SNi-type reaction in a retaining glycosyltransferase

Abstract: 3

Cited by 144 publications

References 55 publications

Structure and mechanism ofStaphylococcus aureusTarM, the wall teichoic acid α-glycosyltransferase

Structure and mechanism ofStaphylococcus aureusTarM, the wall teichoic acid α-glycosyltransferase

A Historical Perspective for the Catalytic Reaction Mechanism of Glycosidase; So As to Bring about Breakthrough in Confusing Situation

Lyase activity of glycogen synthase: Is an elimination/addition mechanism a possible reaction pathway for retaining glycosyltransferases?

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