title: Characterization of a new α-l-fucosidase isolated from Fusarium proliferatum LE1 that is regioselective to α-(1 → 4)-l-fucosidic linkage in the hydrolysis of α-l-fucobiosides.
abstract: Here, we report the biochemical characterization of a novel α-l-fucosidase with broad substrate specificity (FpFucA) isolated from the mycelial fungus Fusarium proliferatum LE1. Highly purified α-l-fucosidase was obtained from several chromatographic steps after growth in the presence of l-fucose. The purified α-l-fucosidase appeared to be a monomeric protein of 67 ± 1 kDa that was able to hydrolyze the synthetic substrate p-nitrophenyl α-l-fucopyranoside (pNPFuc), with Km= 1.1 ± 0.1 mM and kcat= 39.8 ± 1.8 s-1. l-fucose, 1-deoxyfuconojirimycin and tris(hydroxymethyl)aminomethane inhibited pNPFuc hydrolysis, with inhibition constants of 0.2 ± 0.05 mM, 7.1 ± 0.05 nM, and 12.2 ± 0.1 mM, respectively. We assumed that the enzyme belongs to subfamily A of the GH29 family (CAZy database) based on its ability to hydrolyze practically all fucose-containing oligosaccharides used in the study and the phylogenetic analysis. We found that this enzyme was a unique α-l-fucosidase that preferentially hydrolyzes the α-(1 → 4)-L-fucosidic linkage present in α-L-fucobiosides with different types of linkages. As a retaining glycosidase, FpFucA is capable of catalyzing the transglycosylation reaction with alcohols (methanol, ethanol, and 1-propanol) and pNP-containing monosaccharides as acceptors. These features make the enzyme an important tool that can be used in the various modifications of valuable fucose-containing compounds.
authors.affiliation: National Research Center «Kurchatov Institute», B.P. Konstantinov Petersburg Nuclear Physics Institute, 188300, Gatchina, Orlova Roscha, Russia; Department of Medical Physics, Peter the Great St. Petersburg Polytechnic University, 195251, St. Petersburg, Chlopina Str. 5, Russia.
authors.forename: Svetlana V
authors.initials: SV
authors.lastname: Shvetsova
authors.affiliation: National Research Center «Kurchatov Institute», B.P. Konstantinov Petersburg Nuclear Physics Institute, 188300, Gatchina, Orlova Roscha, Russia; Peter the Great St. Petersburg Polytechnic University, 195251, St. Petersburg, Polytechnicheskaya Str., 29, Russia.
authors.forename: Konstantin A
authors.initials: KA
authors.lastname: Shabalin
authors.affiliation: National Research Center «Kurchatov Institute», B.P. Konstantinov Petersburg Nuclear Physics Institute, 188300, Gatchina, Orlova Roscha, Russia.
authors.forename: Kirill S
authors.initials: KS
authors.lastname: Bobrov
authors.affiliation: National Research Center «Kurchatov Institute», B.P. Konstantinov Petersburg Nuclear Physics Institute, 188300, Gatchina, Orlova Roscha, Russia.
authors.forename: Dina R
authors.initials: DR
authors.lastname: Ivanen
authors.affiliation: N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 47, Leninsky Pr., Moscow, 119991, Russia.
authors.forename: Nadezhda E
authors.initials: NE
authors.lastname: Ustyuzhanina
authors.affiliation: N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 47, Leninsky Pr., Moscow, 119991, Russia.
authors.forename: Vadim B
authors.initials: VB
authors.lastname: Krylov
authors.affiliation: N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 47, Leninsky Pr., Moscow, 119991, Russia.
authors.forename: Nikolay E
authors.initials: NE
authors.lastname: Nifantiev
authors.affiliation: Institute of Biomedical Chemistry, Pogodinskaya 10, Moscow, 119121, Russia; National Research Center «Kurchatov Institute», B.P. Konstantinov Petersburg Nuclear Physics Institute, 188300, Gatchina, Orlova Roscha, Russia.
authors.forename: Stanislav N
authors.initials: SN
authors.lastname: Naryzhny
authors.affiliation: Institute of Biomedical Chemistry, Pogodinskaya 10, Moscow, 119121, Russia.
authors.forename: Victor G
authors.initials: VG
authors.lastname: Zgoda
authors.affiliation: National Research Center «Kurchatov Institute», B.P. Konstantinov Petersburg Nuclear Physics Institute, 188300, Gatchina, Orlova Roscha, Russia.
authors.forename: Elena V
authors.initials: EV
authors.lastname: Eneyskaya
authors.affiliation: National Research Center «Kurchatov Institute», B.P. Konstantinov Petersburg Nuclear Physics Institute, 188300, Gatchina, Orlova Roscha, Russia; Department of Medical Physics, Peter the Great St. Petersburg Polytechnic University, 195251, St. Petersburg, Chlopina Str. 5, Russia. Electronic address: kulm@omrb.pnpi.spb.ru.
authors.forename: Anna A
authors.initials: AA
authors.lastname: Kulminskaya
mesh_terms: D000438:Alcohols / Q000378:metabolism; D000595:Amino Acid Sequence; D004187:Disaccharides / Q000378:metabolism; D005643:Fucose / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005670:Fusarium / Q000201:enzymology* / Q000235:genetics; D006027:Glycosides / Q000378:metabolism; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D013058:Mass Spectrometry; D009844:Oligosaccharides / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature; D005644:alpha-L-Fucosidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D000438:Alcohols; D004187:Disaccharides; D005656:Fungal Proteins; D006027:Glycosides; D009844:Oligosaccharides; D011134:Polysaccharides; C498070:fucopyranosyl-1-3-fucopyranose; D005643:Fucose; C007789:fucoidan; D005644:alpha-L-Fucosidase
keywords: Fusarium proliferatum; Translycosylation; α-(1 → 4)-l-fucosidic linkage; α-l-fucobiosides; α-l-fucosidase