8I4D

X-ray structure of a L-rhamnose-alpha-1,4-D-glucuronate lyase from Fusarium oxysporum 12S, L-Rha complex at 100K

Summary for 8I4D

• Entry DOI: 10.2210/pdb8i4d/pdb
• Related: 7YQS
• Descriptor: L-Rhamnose-alpha-1,4-D-glucuronate lyase, alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]alpha-D-mannopyranose-(1-6)-[alpha-D-mannopyranose-(1-3)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose, alpha-L-rhamnopyranose, ... (8 entities in total)
• Functional Keywords: polysaccharide lyase, gum arabic, carbohydrate-active enzyme, lyase
• Biological source: Fusarium oxysporum
• Total number of polymer chains: 1
• Total formula weight: 51728.72

Authors

Yano, N.,Kondo, T.,Kusaka, K.,Yamada, T.,Arakawa, T.,Sakamoto, T.,Fushinobu, S. (deposition date: 2023-01-19, release date: 2024-01-24, Last modification date: 2024-10-16)

Primary citation

Yano, N.,Kondo, T.,Kusaka, K.,Arakawa, T.,Sakamoto, T.,Fushinobu, S.
Charge neutralization and beta-elimination cleavage mechanism of family 42 L-rhamnose-alpha-1,4-D-glucuronate lyase revealed using neutron crystallography.
J.Biol.Chem., 300:105774-105774, 2024

PubMed Abstract: Gum arabic (GA) is widely used as an emulsion stabilizer and edible coating and consists of a complex carbohydrate moiety with a rhamnosyl-glucuronate group capping the non-reducing ends. Enzymes that can specifically cleave the glycosidic chains of GA and modify their properties are valuable for structural analysis and industrial application. Cryogenic X-ray crystal structure of GA-specific L-rhamnose-α-1,4-D-glucuronate lyase from Fusarium oxysporum (FoRham1), belonging to the polysaccharide lyase (PL) family 42, has been previously reported. To determine the specific reaction mechanism based on its hydrogen-containing enzyme structure, we performed joint X-ray/neutron crystallography of FoRham1. Large crystals were grown in the presence of L-rhamnose (a reaction product), and neutron and X-ray diffraction datasets were collected at room temperature at 1.80 and 1.25 Å resolutions, respectively. The active site contained L-rhamnose and acetate, the latter being a partial analog of glucuronate. Incomplete H/D exchange between Arg166 and acetate suggested that a strong salt-bridge interaction was maintained. Doubly deuterated His105 and deuterated Tyr150 supported the interaction between Arg166 and the acetate. The unique hydrogen-rich environment functions as a charge neutralizer for glucuronate and stabilizes the oxyanion intermediate. The NE2 atom of His85 was deprotonated and formed a hydrogen bond with the deuterated O1 hydroxy of L-rhamnose, indicating the function of His85 as the base/acid catalyst for bond cleavage via β-elimination. Asp83 functions as a pivot between the two catalytic histidine residues by bridging them. This His-His-Asp structural motif is conserved in the PL 24, 25, and 42 families.

PubMed: 38382672
DOI: 10.1016/j.jbc.2024.105774

Experimental method

X-RAY DIFFRACTION (1.06 Å)