4.8 Article

Asparagine Tautomerization in Glycosyltransferase Catalysis. The Molecular Mechanism of Protein O-Fucosyltransferase 1

期刊

ACS CATALYSIS
卷 11, 期 15, 页码 9926-9932

出版社

AMER CHEMICAL SOC
DOI: 10.1021/acscatal.1c01785

关键词

enzymes; O-glycosylation; carbohydrates; glycosyltransferases; quantum mechanics/molecular mechanics; metadynamics

资金

  1. Spanish Ministry of Science, Innovation and Universities (MICINN/AEI/FEDER, UE) [CTQ2017-85496-P, PID2019-105451GB-I00]
  2. Aragon Government [E35_R20, LMP58_18]
  3. FEDER (2014-2020) funds for Building Europe from Aragon
  4. Agency for Management of University and Research Grants of Generalitat de Catalunya (AGAUR) [2017SGR-1189]
  5. National Institute of General Medical Sciences [GM061126]
  6. Red Nacional de Supercomputacion (RES) [BCV2020-2-0009]
  7. MICINN [IJCI-201732874]
  8. AGAUR [2020 FI_B 00423]
  9. Spanish Structures of Excellence Maria de Maeztu [MDM-2017-0767]

向作者/读者索取更多资源

O-glycosylation is essential for life and involves enzymes like POFUT1, which fucosylates specific residues in EGF-LD. POFUT1 lacks a catalytic base in its active site, but uses a mechanism involving proton shuttling through a conserved asparagine residue. Mutations in this residue can abolish enzyme activity, providing insights for developing inhibitors for O-glycosylation disorders.
O-glycosylation is a post-translational protein modification essential to life. One of the enzymes involved in this process is protein O-fucosyltransferase 1 (POFUT1), which fucosylates threonine or serine residues within a specific sequence context of epidermal growth factor-like domains (EGF-LD). Unlike most inverting glycosyltransferases, POFUT1 lacks a basic residue in the active site that could act as a catalytic base to deprotonate the Thr/Ser residue of the EGF-LD acceptor during the chemical reaction. Using quantum mechanics/molecular mechanics (QM/MM) methods on recent crystal structures, as well as mutagenesis experiments, we uncover the enzyme catalytic mechanism, revealing that it involves proton shuttling through an active site asparagine, conserved among species, which undergoes tautomerization. This mechanism is consistent with experimental kinetic analysis of Caenorhabditis elegans POFUT1 Asn43 mutants, which ablate enzyme activity even if mutated to Asp, the canonical catalytic base in inverting glycosyltransferases. These results will aid inhibitor development for Notch-associated O-glycosylation disorders.

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