Biochemical and structural characterization of a novel 4‐O‐α‐l‐rhamnosyl‐β‐d‐glucuronidase from Fusarium oxysporum

Abstract

In this study, we have isolated the novel enzyme 4‐O‐α‐l‐rhamnosyl‐β‐d‐glucuronidase (FoBGlcA), which releases α‐l‐rhamnosyl (1→4) glucuronic acid from gum arabic (GA), from Fusarium oxysporum 12S culture supernatant, and for the first time report an enzyme with such catalytic activity. The gene encoding FoBGlcA was cloned and expressed in Pichia pastoris. When GA was subjected to the recombinant enzyme, > 95% of the l‐rhamnose (Rha) and d‐glucuronic acid in the substrate were released, which indicates that almost all Rha binds to the glucuronic acid at the end of the GA side chains. The crystal structure of FoBGlcA was determined using a single‐wavelength anomalous dispersion at 1.51 Å resolution. FoBGlcA consisted of an N‐terminal (β/α)8‐barrel domain and a C‐terminal antiparallel β‐sheet domain. This configuration is characteristic of glycoside hydrolase (GH) family 79 proteins. A structural similarity search showed that FoBGlcA mostly resembled GH79 β‐d‐glucuronidase (AcGlcA79A) of Acidobacterium capsulatum; however, the root‐mean‐square deviation value was 3.2 Å, indicating that FoBGlcA has a high structural divergence. FoBGlcA had a low sequence identity with AcGlcA79A (19%) and differed from other GH79 β‐glucuronidases. The structures of FoBGlcA and AcGlcA79A also differed in terms of the loop structure location near subsite –2 of their catalytic sites, which may account for the unique substrate specificity of FoBGlcA. The amino acid residues involved in the catalytic activity of this enzyme were determined by evaluating the activity levels of various mutant enzymes based on the crystal structure analysis of the FoBGlcA reaction product complex. Database Atomic coordinates and structure factors (codes https://doi.org/10.2210/pdb7DFQ/pdb and https://doi.org/10.2210/pdb7DFS/pdb) have been deposited in the Protein Data Bank (http://wwpdb.org/).

Structural and functional analysis of gum arabic l-rhamnose-α-1,4-d-glucuronate lyase establishes a novel polysaccharide lyase family

“…Since GA has an Rha–GlcA structure at the end of the side chains ( 10 , 18 ), FoRham1 seemed to act specifically on Rha residues bound to GlcA by α-1,4-linkages. GH145 α- l -rhamnosidase BT3686 also shows activity on GA and Rha–GlcA, but not on p NP-α- l -Rha, RG-I, or RG-II ( 12 ).…”

“…When GA was thoroughly digested with FoRham1, approximately 95% of Rha in GA was released, indicating that most Rha residues were present at the nonreducing ends of the side chains, and were bound to GlcA by α-1,4 linkages in GA. We evaluated whether FoRham1 prefers a polymer or an oligomer. In addition to natural GA, GA degraded by GH79 4- O -α- l -rhamnosyl-β- d -glucuronidase (FoBGlcA; GenBank accession number: LC534636) ( 18 ), which we termed GA-RG, was used as the substrate. FoBGlcA hydrolyzes the β-1,6-glucuronic linkage of GA and releases Rha–GlcA ( Fig.…”

“…This enzyme has not yet been isolated from F. oxysporum . GH79 4- O -α- l -rhamnosyl-β- d -glucuronidase (FoBGlcA; ( 18 )) degrades GSO4 to Rha–GlcA and β-1,6-galactobiose ( Fig. 5 E ).…”

“…p NP-α- l -Rha and GA (Lot number: 120M0035V) were purchased from Sigma–Aldrich Co. LWAG and rutin were purchased from Tokyo Chemical Industry Co, Ltd. RG-I oligosaccharides were prepared as described previously ( 37 ). GSOs (GSO4−7) were prepared from GA using the Sphingomonas exo-β-1,3-galactanase (Exo-1,3-Gal; ( 38 )) as described previously ( 18 ). Rha–GlcA was prepared by incubating 0.1 mg of 4- O -α- l -rhamnosyl-β- d -glucuronidase (FoBGlcA; ( 18 )) with 5% GA (500 ml) in 20 mM sodium acetate buffer (pH 4.0) at 37 °C for 48 h. The reaction product was recovered by ultrafiltration using a 10-kDa cut-off membrane (10K Minimate TFF Capsule with Omega membrane; Pall Life Sciences), concentrated by an evaporator, and purified by activated carbon resin (0–50% linear gradient of ethanol).…”

“…Previously, we isolated, cloned, and characterized various GA-degrading enzymes from Fusarium oxysporum 12S, a phytopathogenic fungus that can grow using GA as the sole carbon source ( 14 , 15 , 16 , 17 , 18 ). In addition to these enzymes, we recently observed that Rha was released from GA by the culture supernatant of this fungus.…”