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  Retaining Glycosyltransferase Mechanism Studied by QM/MM Methods: Lipopolysaccharyl-α-1,4-galactosyltransferase C Transfers α-Galactose via an Oxocarbenium Ion-like Transition State

Gomez, H., Polyak, I., Thiel, W., Lluch, J. M., & Masgrau, L. (2012). Retaining Glycosyltransferase Mechanism Studied by QM/MM Methods: Lipopolysaccharyl-α-1,4-galactosyltransferase C Transfers α-Galactose via an Oxocarbenium Ion-like Transition State. Journal of the American Chemical Society, 134(10), 4743-4752. doi:10.1021/ja210490f.

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 Creators:
Gomez, Hansel1, Author           
Polyak, Iakov1, Author           
Thiel, W.1, Author           
Lluch, Jose M.2, Author
Masgrau, Laura2, Author
Affiliations:
1Research Department Thiel, Max-Planck-Institut für Kohlenforschung, Max Planck Society, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr, DE, ou_1445590              
2Univ Autonoma Barcelona, Inst Biotecnol & Biomed, E-08193 Barcelona, Spain, ou_persistent22              

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Free keywords: APPROXIMATE COULOMB POTENTIALS; ENZYMATIC GLYCOSYL TRANSFER; AUXILIARY BASIS-SETS; GAUSSIAN-BASIS SETS; NEISSERIA-MENINGITIDIS; GALACTOSYLTRANSFERASE LGTC; MOLECULAR-DYNAMICS; CORRELATION-ENERGY; COMPLEX MATERIALS; ZEOLITE STRUCTURE
 Abstract: Glycosyltransferases (GTs) catalyze the highly specific biosynthesis of glycosidic bonds and, as such, are important both as drug targets and for biotechnological purposes. Despite their broad interest, fundamental questions about their reaction mechanism remain to be answered, especially for those GTs that transfer the sugar with net retention of the configuration at the anomeric carbon (retaining glycosyltransferases, ret-GTs). In the present work, we focus on the reaction catalyzed by lipopolysaccharyl-alpha-1,4-galactosyltransferase C (LgtC) from Neisseria meningitides. We study and compare the different proposed mechanisms (S(N)i, S(N)i-like, and double displacement mechanism via a covalent glycosyl-enzyme intermediate, CGE) by using density functional theory (DFT) and quantum mechanics/molecular mechanics (QM/MM) calculations on the full enzyme. We characterize a dissociative single-displacement (S(N)i) mechanism consistent with the experimental data, in which the acceptor substrate attacks on the side of the UDP leaving group that acts as a catalytic base. We identify several key interactions that help this front-side attack by stabilizing the transition state. Among them, Gln189, the putative nucleophile in a double displacement mechanism, is shown to favor the charge development at the anomeric center by about 2 kcal/mol, compatible with experimental mutagenesis data. We predict that using 3-deoxylactose as acceptor would result in a reduction of k(cat) to 0.6-3% of that for the unmodified substrates. The reactions of the Q189A and Q189E mutants have also been investigated. For Q189E, there is a change in mechanism since a CGE can be formed which, however, is not able to evolve to products. The current findings are discussed in the light of the available experimental data and compared with those for other ret-GTs.

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Language(s): eng - English
 Dates: 2012-02-21
 Publication Status: Issued
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1021/ja210490f
 Degree: -

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Title: Journal of the American Chemical Society
  Other : J. Am. Chem. Soc.
Source Genre: Journal
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Publ. Info: American Chemical Society
Pages: - Volume / Issue: 134 (10) Sequence Number: - Start / End Page: 4743 - 4752 Identifier: ISSN: 0002-7863
CoNE: https://pure.mpg.de/cone/journals/resource/954925376870