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

Piniello B. ; Lira-Navarrete E. (Universidad de Zaragoza) ; Takeuchi H. ; Takeuchi M. ; Haltiwanger R.S. ; Hurtado-Guerrero R. ; Rovira C.

Resumen: 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.

Idioma: Inglés

DOI: 10.1021/acscatal.1c01785

Año: 2021

Publicado en: ACS CATALYSIS 11, 15 (2021), 9926-9932
ISSN: 2155-5435

Factor impacto JCR: 13.7 (2021)

Categ. JCR: CHEMISTRY, PHYSICAL rank: 19 / 165 = 0.115 (2021) - Q1 - T1

Factor impacto CITESCORE: 20.8 - Chemical Engineering (Q1)

Factor impacto SCIMAGO: 4.202 - Chemistry (miscellaneous) (Q1) - Catalysis (Q1)

Financiación: info:eu-repo/grantAgreement/ES/DGA-FEDER/E35-20R
Financiación: info:eu-repo/grantAgreement/ES/DGA/LMP58-18
Financiación: info:eu-repo/grantAgreement/ES/MICINN/IJCI-2017-32874
Financiación: info:eu-repo/grantAgreement/ES/MICINN/PID2019-105451GB-I00

Tipo y forma: Article (Published version)

Área (Departamento): Área Bioquímica y Biolog.Mole. (Dpto. Bioq.Biolog.Mol. Celular)