Family - HR6

F6IEX3
Q76LC4

Literature Information

Title	Structural and functional insights into RHA-P, a bacterial GH106 α-L-rhamnosidase from Novosphingobium sp. PP1Y
Author	Francesca Mensitieri , Federica De Lise, Andrea Strazzulli, Marco Moracci, Eugenio Notomista , Valeria Cafaro, Emiliano Bedini, Matthew Howard Sazinsky, Marco Trifuoggi, Alberto Di Donato, Viviana Izzo
DOI	10.1016/j.abb.2018.04.013
Abstract	α-L-Rhamnosidases (α-RHAs, EC 3.2.1.40) are glycosyl hydrolases (GHs) hydrolyzing terminal α-l-rhamnose residues from different substrates such as heteropolysaccharides, glycosylated proteins and natural flavonoids. Although the possibility to hydrolyze rhamnose from natural flavonoids has boosted the use of these enzymes in several biotechnological applications over the past decades, to date only few bacterial rhamnosidases have been fully characterized and only one crystal structure of a rhamnosidase of the GH106 family has been described. In our previous work, an α-l-rhamnosidase belonging to this family, named RHA-P, was isolated from the marine microorganism Novosphingobium sp. PP1Y. The initial biochemical characterization highlighted the biotechnological potential of RHA-P for bioconversion applications. In this work, further functional and structural characterization of the enzyme is provided. The recombinant protein was obtained fused to a C-terminal His-tag and, starting from the periplasmic fractions of induced recombinant cells of E. coli strain BL21(DE3), was purified through a single step purification protocol. Homology modeling of RHA-P in combination with a site directed mutagenesis analysis confirmed the function of residues D503, E506, E644, likely located at the catalytic site of RHA-P. In addition, a kinetic characterization of the enzyme on natural flavonoids such as naringin, rutin, hesperidin and quercitrin was performed. RHA-P showed activity on all flavonoids tested, with a catalytic efficiency comparable or even higher than other bacterial α-RHAs described in literature. The results confirm that RHA-P is able to hydrolyze both α-1,2 and α-1,6 glycosidic linkages, and suggest that the enzyme may locate different polyphenolic aromatic moities in the active site.

Experimental results

Enzyme

Uniprot ID: F6IEX3

Protein: Glycoside hydrolase family protein

Organism: Novosphingobium sp. PP1Y

Length: 1,146 AA

Taxonomic identifier: 702113 [NCBI]

Pfam

Source	Domain	Start	End	E-value (Domain)	Coverage
Pfam-A	Glyco_hydro_106	47	945	0	0.999

Program: hmmscan

Version: 3.1b2 (February 2015)

Method: hmmscan --domtblout hmmscan.tbl --noali -E 1e-5 pfam query.fa

Date: Mon Jul 20 14:32:16 2020

Description:

Glyco_hydro_106

Pfam

No Pfam abstract.

InterPro

No InterPro results.

Information is taken from Pfam and InterPro web site.

Reaction

naringin + H₂O ⇒ naringenin + L-rhamnose

References

Literature Information

Title	Structural and functional insights into RHA-P, a bacterial GH106 α-L-rhamnosidase from Novosphingobium sp. PP1Y
Author	Francesca Mensitieri , Federica De Lise, Andrea Strazzulli, Marco Moracci, Eugenio Notomista , Valeria Cafaro, Emiliano Bedini, Matthew Howard Sazinsky, Marco Trifuoggi, Alberto Di Donato, Viviana Izzo
DOI	10.1016/j.abb.2018.04.013
Abstract	α-L-Rhamnosidases (α-RHAs, EC 3.2.1.40) are glycosyl hydrolases (GHs) hydrolyzing terminal α-l-rhamnose residues from different substrates such as heteropolysaccharides, glycosylated proteins and natural flavonoids. Although the possibility to hydrolyze rhamnose from natural flavonoids has boosted the use of these enzymes in several biotechnological applications over the past decades, to date only few bacterial rhamnosidases have been fully characterized and only one crystal structure of a rhamnosidase of the GH106 family has been described. In our previous work, an α-l-rhamnosidase belonging to this family, named RHA-P, was isolated from the marine microorganism Novosphingobium sp. PP1Y. The initial biochemical characterization highlighted the biotechnological potential of RHA-P for bioconversion applications. In this work, further functional and structural characterization of the enzyme is provided. The recombinant protein was obtained fused to a C-terminal His-tag and, starting from the periplasmic fractions of induced recombinant cells of E. coli strain BL21(DE3), was purified through a single step purification protocol. Homology modeling of RHA-P in combination with a site directed mutagenesis analysis confirmed the function of residues D503, E506, E644, likely located at the catalytic site of RHA-P. In addition, a kinetic characterization of the enzyme on natural flavonoids such as naringin, rutin, hesperidin and quercitrin was performed. RHA-P showed activity on all flavonoids tested, with a catalytic efficiency comparable or even higher than other bacterial α-RHAs described in literature. The results confirm that RHA-P is able to hydrolyze both α-1,2 and α-1,6 glycosidic linkages, and suggest that the enzyme may locate different polyphenolic aromatic moities in the active site.