Rational engineering of AA5_2 copper radical oxidases to probe the molecular determinants governing their substrate selectivity.
AA5
alcohol oxidase
fungal CRO
galactose oxidase
site-directed mutagenesis
structure-activity relationships
Journal
The FEBS journal
ISSN: 1742-4658
Titre abrégé: FEBS J
Pays: England
ID NLM: 101229646
Informations de publication
Date de publication:
05 2023
05 2023
Historique:
revised:
20
12
2022
received:
10
11
2022
accepted:
23
12
2022
medline:
18
5
2023
pubmed:
21
1
2023
entrez:
20
1
2023
Statut:
ppublish
Résumé
Fungal copper radical oxidases (CROs) from the Auxiliary Activity family 5 (AA5) constitute a group of metalloenzymes that oxidize a wide panel of natural compounds, such as galactose-containing saccharides or primary alcohols, into product derivatives exhibiting promising biotechnological interests. Despite a well-conserved first copper-coordination sphere and overall fold, some members of the AA5_2 subfamily are incapable of oxidizing galactose and galactosides but conversely efficiently catalyse the oxidation of diverse aliphatic alcohols. The objective of this study was to understand which residues dictate the substrate preferences between alcohol oxidases and galactose oxidases within the AA5_2 subfamily. Based on structural differences and molecular modelling predictions between the alcohol oxidase from Colletotrichum graminicola (CgrAlcOx) and the archetypal galactose oxidase from Fusarium graminearum (FgrGalOx), a rational mutagenesis approach was developed to target regions or residues potentially driving the substrate specificity of these enzymes. A set of 21 single and multiple CgrAlcOx variants was produced and characterized leading to the identification of six residues (W39, F138, M173, F174, T246, L302), in the vicinity of the active site, crucial for substrate recognition. Two multiple CgrAlcOx variants, i.e. M4F (W39F, F138W, M173R and T246Q) and M6 (W39F, F138W, M173R, F174Y, T246Q and L302P), exhibited a similar affinity for carbohydrate substrates when compared to FgrGalOx. In conclusion, using a rational site-directed mutagenesis approach, we identified key residues involved in the substrate selectivity of AA5_2 enzymes towards galactose-containing saccharides.
Substances chimiques
Copper
789U1901C5
Galactose
X2RN3Q8DNE
Oxidoreductases
EC 1.-
Galactose Oxidase
EC 1.1.3.9
Ceruloplasmin
EC 1.16.3.1
Alcohols
0
Banques de données
RefSeq
['AAO95371', 'EFQ30446', 'EFQ30446.1']
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
2658-2672Informations de copyright
© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.
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