Identification of the nucleophile catalytic residue of GH51 α-l-arabinofuranosidase from Pleurotus ostreatus

Amore, Antonella; Iadonisi, Alfonso; Vincent, Florence; Faraco, Vincenza

doi:10.1186/s13568-015-0164-x

Title: Identification of the nucleophile catalytic residue of GH51 α-l-arabinofuranosidase from Pleurotus ostreatus

Journal Article · Mon Dec 21 00:00:00 EST 2015 · AMB Express

DOI:https://doi.org/10.1186/s13568-015-0164-x· OSTI ID:1236038

Amore, Antonella ^[1]; Iadonisi, Alfonso ^[2]; Vincent, Florence ^[3]; Faraco, Vincenza ^[2]

Univ. of Naples Federico II (Italy); National Renewable Energy Lab. (NREL), Golden, CO (United States)
Univ. of Naples Federico II (Italy)
Aix-Marseille Univ. (France)

In this paper, the recombinant α-l-arabinofuranosidase from the fungus Pleurotus ostreatus (rPoAbf) was subjected to site-directed mutagenesis in order to identify the catalytic nucleophile residue. Based on bioinformatics and homology modelling analyses, E449 was revealed to be the potential nucleophilic residue. Thus, the mutant E449G of PoAbf was recombinantly expressed in Pichia pastoris and its recombinant expression level and reactivity were investigated in comparison to the wild-type. The design of a suitable set of hydrolysis experiments in the presence or absence of alcoholic arabinosyl acceptors and/or formate salts allowed to unambiguously identify the residue E449 as the nucleophile residue involved in the retaining mechanism of this GH51 arabinofuranosidase. ¹H NMR analysis was applied for the identification of the products and the assignement of their anomeric configuration.

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Research Organization:: National Renewable Energy Laboratory (NREL), Golden, CO (United States)

Sponsoring Organization:: Ministero dell'Universita e della Ricerca Scientifica-Industrial Research Project

Grant/Contract Number:: AC36-08GO28308

OSTI ID:: 1236038

Report Number(s):: NREL/JA-2700-65692

Journal Information:: AMB Express, Vol. 5, Issue 1; Related Information: AMB Express; ISSN 2191-0855

Country of Publication:: United States

Language:: English

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Cited by: 1 work

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37 INORGANIC, ORGANIC, PHYSICAL, AND ANAYLYTICAL CHEMISTRY
arabinofuranosidase
lignocellulose
site-directed mutagenesis

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