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

Abstract

AbstractGum 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 tools 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 up to 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 deuteronated His105 and deuteronated Tyr150 supported this interaction. The unusually 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, and this His–His–Asp structural motif is conserved in the three PL families.Significance StatementAlthough hydrogen transfer plays an important role in enzymatic reactions, hydrogen atoms are generally invisible in macromolecular X-ray crystallography. In the reaction of polysaccharide lyases, substrate activation by negative charge stabilization of uronic acid and base/acid-catalyzed β-elimination reaction have been postulated. Here, we report the neutron crystallography of polysaccharide lyase. Joint X-ray/neutron crystallography of L-rhamnose-α-1,4-D-glucuronate lyase from Fusarium oxysporum (FoRham1) complexed with L-rhamnose was performed, and the hydrogen and deuterium atoms were visualized at a high resolution. FoRham1 catalyzes the specific cleavage of the cap structure of gum arabic, which is useful for various applications in the food, cosmetic, and pharmaceutical industries. A detailed catalytic mechanism for FoRham1 was proposed based on the key structural features of its active site.