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

Abstract

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Charge neutralization and β-elimination cleavage mechanism of family 42 L-rhamnose-α-1,4-D-glucuronate lyase revealed using neutron crystallography

“…We observed GlcNAc2-Man5 heptasaccharide in the XN structure (Fig. S2), whereas only tetrasaccharide (GlcNAc2-Man2) was previously observed in the highresolution X-ray structure (1.05 Å; PDB ID:7ESK) (21). Two monoatomic cations (Na + and Ca 2+ ) were observed at the same positions as those in the previous X-ray structures (21).…”

“…S2), whereas only tetrasaccharide (GlcNAc 2 -Man 2 ) was previously observed in the high-resolution X-ray structure (1.05 Å; PDB ID:7ESK) (21). Two monoatomic cations (Na + and Ca 2+ ) were observed at the same positions as those in the previous X-ray structures (21). A Tris molecule, which was derived from the reservoir solution for growing huge crystals, was bound on the surface of the posterior side (Fig.…”

“…1B) based on the neutron scattering length density (NSLD) maps. Similar to the previous X-ray structure (21), the C-terminal polyhistidine and c-Myc tags were not modeled due to disorder, whereas a high-mannose N-glycan at Asn247 was modeled (Fig. 1C).…”

“…We have studied various GA-degrading enzymes from Fusarium oxysporum 12S, a phytopathogenic fungus that can grow using GA as the sole carbon source (16)(17)(18)(19)(20)(21). L-Rhamnose-α-1,4-D-glucuronate lyase from F. oxysporum 12S (FoRham1, EC 4.2.2.-) specifically cleaves the glycosidic bond in the Rha-GlcA moiety of GA and releases Rha (21). FoRham1 generates Δ4,5-unsaturated GlcA via β-elimination.…”

“…Therefore, FoRham1 and its homologs in the former glycoside hydrolase 145 family were reclassified into a novel PL42 family in the Carbohydrate-Active enZYmes database (http://www.cazy.org) (22). Based on structural and mutational analyses, we proposed a possible catalytic mechanism of FoRham1 that involves charge neutralization of uronate carboxyl by Arg166 and proton abstraction and donation by His85 in the lytic cleavage of the O4–C4 bond (21). His105 may support the base-acid dual function of His85.…”