Biochemical characteristics and crystallographic evidence for substrate-assisted catalysis of a ß-N-acetylhexosaminidase in Akkermansia muciniphila.
Biochem Biophys Res Commun
; 517(1): 29-35, 2019 09 10.
Article
en En
| MEDLINE
| ID: mdl-31345574
In this paper, we characterized Am2136 as a ß-N-acetylhexosaminidase from Akkermansia muciniphila to perform the biochemical characteristics and the crystal structure of selenomethionine-labeled Am2136 with GlcNAc complex. Crystallographic evidence suggests that an oxazolinium ion was formed intermediately by the 2-acetamido group during the substrate-assisted catalytic procedure. Structural and kinetic analysis of native Am2136 and D412A, E413A mutants were investigated and the results revealed substantial difference. The Kcat/Km value of D412A was decreased 4297-fold compared to native Am2136 revealed that mutation of Asp-412 results in preventing the 2-acetamido substituent from providing anchimeric assistance and thus reducing the catalytic efficiency. Moreover, Am2136 has a wide dependence on pH and temperature, while sensitive to divalent metal ions such as Ca2+ and Mn2+. These biochemical and crystallographic results provide evidences that Asp-412 residue assists to orient the 2-acetamido group for catalysis. Based on crystallographic evidence and sequence alignment with other GH family 20 enzymes, Asp-412 residue is possibly fundamental for Am2136 during substrate-assisted catalysis.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Proteínas Bacterianas
/
Beta-N-Acetilhexosaminidasas
/
Verrucomicrobia
Tipo de estudio:
Prognostic_studies
Idioma:
En
Revista:
Biochem Biophys Res Commun
Año:
2019
Tipo del documento:
Article
País de afiliación:
China
Pais de publicación:
Estados Unidos