α1,4-N-Acetylhexosaminyltransferase EXTL2: The Missing Link for Understanding Glycosidic Bond Biosynthesis with Retention of Configuration

Fernanda Mendoza, Hansel Gómez, José M. Lluch, Laura Masgrau

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Glycosyltransferases (GTs) are biocatalysts that synthesize a wide variety of glycans present in nature. The mechanism by which they form a new glycosidic bond, preserving the configuration at the anomeric carbon, has been strongly debated. In the last few years, new experimental and computational results have provided very valuable knowledge: the proposed front-side reaction seems to be the preferred path, whereas some systems (family GT6 members) can switch mechanism toward the double displacement. However, why would retaining GTs have evolved to catalyze sugar transfer by different mechanisms? The present QM(DFT)/MM work on α1,4-N-acetylhexosaminyltransferase (EXTL2) fills this gap in the understanding of retaining glycosyltransferases catalysis. We show that EXTL2, despite having a nucleophilic residue (Asp246) that is structurally analogous to the Glu nucleophile found in GT6, clearly follows a front-side mechanism. Our results show that Asp246 is used to facilitate UDP-GalNAc bond cleavage by stabilizing the oxocarbenium species and to compensate for the close presence of a positively charged residue (Arg293) that interacts electrostatically with the acceptor substrate carboxylate (and with Asp246). Altogether, a more complete picture of retaining GT catalysis can now be provided: the front-side mechanism is the preferred path, with the crucial contribution from substrate-assisted catalysis in facilitating UDP departure; in some cases though, an extra stabilization of the forming oxocarbenium species is needed, which is achieved by the presence of a nucleophilic residue (nucleophilically assisted catalysis). This can eventually shift (not switch) the mechanism toward a double displacement, a strategy that within this context may be regarded as an extreme case of oxocarbenium stabilization. Importantly, we also conclude that the identity of the residues found on the β-face of the sugar in retaining GTs is strongly linked to both the required binding orientation of the acceptor substrate (for regiospecificity) and to its chemical identity (both aspects having also an influence on UDP stabilization). These are aspects that may help in the engineering of enzymes for glycan synthesis.

Original languageEnglish
Pages (from-to)2577-2589
Number of pages13
JournalACS Catalysis
Volume6
Issue number4
DOIs
Publication statusPublished - 1 Apr 2016
Externally publishedYes

Fingerprint

N-Acetylhexosaminyltransferases
Glycosyltransferases
Biosynthesis
Catalysis
Uridine Diphosphate
Stabilization
Sugars
Substrates
Switches
Polysaccharides
Nucleophiles
Biocatalysts
Discrete Fourier transforms
Enzymes
Carbon

Keywords

  • acceptor binding
  • front-side nucleophilic substitution
  • glycosyltransferases
  • oxocarbenium
  • QM/MM
  • reaction mechanism

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)

Cite this

Mendoza, Fernanda ; Gómez, Hansel ; Lluch, José M. ; Masgrau, Laura. / α1,4-N-Acetylhexosaminyltransferase EXTL2 : The Missing Link for Understanding Glycosidic Bond Biosynthesis with Retention of Configuration. In: ACS Catalysis. 2016 ; Vol. 6, No. 4. pp. 2577-2589.
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α1,4-N-Acetylhexosaminyltransferase EXTL2 : The Missing Link for Understanding Glycosidic Bond Biosynthesis with Retention of Configuration. / Mendoza, Fernanda; Gómez, Hansel; Lluch, José M.; Masgrau, Laura.

In: ACS Catalysis, Vol. 6, No. 4, 01.04.2016, p. 2577-2589.

Research output: Contribution to journalArticle

TY - JOUR

T1 - α1,4-N-Acetylhexosaminyltransferase EXTL2

T2 - The Missing Link for Understanding Glycosidic Bond Biosynthesis with Retention of Configuration

AU - Mendoza, Fernanda

AU - Gómez, Hansel

AU - Lluch, José M.

AU - Masgrau, Laura

PY - 2016/4/1

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N2 - Glycosyltransferases (GTs) are biocatalysts that synthesize a wide variety of glycans present in nature. The mechanism by which they form a new glycosidic bond, preserving the configuration at the anomeric carbon, has been strongly debated. In the last few years, new experimental and computational results have provided very valuable knowledge: the proposed front-side reaction seems to be the preferred path, whereas some systems (family GT6 members) can switch mechanism toward the double displacement. However, why would retaining GTs have evolved to catalyze sugar transfer by different mechanisms? The present QM(DFT)/MM work on α1,4-N-acetylhexosaminyltransferase (EXTL2) fills this gap in the understanding of retaining glycosyltransferases catalysis. We show that EXTL2, despite having a nucleophilic residue (Asp246) that is structurally analogous to the Glu nucleophile found in GT6, clearly follows a front-side mechanism. Our results show that Asp246 is used to facilitate UDP-GalNAc bond cleavage by stabilizing the oxocarbenium species and to compensate for the close presence of a positively charged residue (Arg293) that interacts electrostatically with the acceptor substrate carboxylate (and with Asp246). Altogether, a more complete picture of retaining GT catalysis can now be provided: the front-side mechanism is the preferred path, with the crucial contribution from substrate-assisted catalysis in facilitating UDP departure; in some cases though, an extra stabilization of the forming oxocarbenium species is needed, which is achieved by the presence of a nucleophilic residue (nucleophilically assisted catalysis). This can eventually shift (not switch) the mechanism toward a double displacement, a strategy that within this context may be regarded as an extreme case of oxocarbenium stabilization. Importantly, we also conclude that the identity of the residues found on the β-face of the sugar in retaining GTs is strongly linked to both the required binding orientation of the acceptor substrate (for regiospecificity) and to its chemical identity (both aspects having also an influence on UDP stabilization). These are aspects that may help in the engineering of enzymes for glycan synthesis.

AB - Glycosyltransferases (GTs) are biocatalysts that synthesize a wide variety of glycans present in nature. The mechanism by which they form a new glycosidic bond, preserving the configuration at the anomeric carbon, has been strongly debated. In the last few years, new experimental and computational results have provided very valuable knowledge: the proposed front-side reaction seems to be the preferred path, whereas some systems (family GT6 members) can switch mechanism toward the double displacement. However, why would retaining GTs have evolved to catalyze sugar transfer by different mechanisms? The present QM(DFT)/MM work on α1,4-N-acetylhexosaminyltransferase (EXTL2) fills this gap in the understanding of retaining glycosyltransferases catalysis. We show that EXTL2, despite having a nucleophilic residue (Asp246) that is structurally analogous to the Glu nucleophile found in GT6, clearly follows a front-side mechanism. Our results show that Asp246 is used to facilitate UDP-GalNAc bond cleavage by stabilizing the oxocarbenium species and to compensate for the close presence of a positively charged residue (Arg293) that interacts electrostatically with the acceptor substrate carboxylate (and with Asp246). Altogether, a more complete picture of retaining GT catalysis can now be provided: the front-side mechanism is the preferred path, with the crucial contribution from substrate-assisted catalysis in facilitating UDP departure; in some cases though, an extra stabilization of the forming oxocarbenium species is needed, which is achieved by the presence of a nucleophilic residue (nucleophilically assisted catalysis). This can eventually shift (not switch) the mechanism toward a double displacement, a strategy that within this context may be regarded as an extreme case of oxocarbenium stabilization. Importantly, we also conclude that the identity of the residues found on the β-face of the sugar in retaining GTs is strongly linked to both the required binding orientation of the acceptor substrate (for regiospecificity) and to its chemical identity (both aspects having also an influence on UDP stabilization). These are aspects that may help in the engineering of enzymes for glycan synthesis.

KW - acceptor binding

KW - front-side nucleophilic substitution

KW - glycosyltransferases

KW - oxocarbenium

KW - QM/MM

KW - reaction mechanism

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