Structural and functional studies of two enzymes from Arabidopsis thaliana in the ureide pathway: ureidoglycine aminohydrolase and ureidoglycolate amidohydrolase

Abstract

In plants, the ureide pathway is a metabolic route that converts the ring nitrogen atoms of purine to ammonia via sequential enzymatic reactions, playing an important role in nitrogen recovery. By using x-ray crystallography, crystal structures of two enzymes in this pathway were solved in this study: (S)-ureidoglycine aminohydrolase (UGlyAH) and the (S)-ureidoglycolate amidohydroalse (UAH), both from Arabidopsis thaliana. UGlyAH enzyme converts (S)-ureidoglycine into (S)-ureidoglycolate and ammonia, providing the final substrate to the pathway. Here, a structural and functional analysis of this enzyme from Arabidopsis thaliana (AtUGlyAH) is reported. The crystal structure of AtUGlyAH in the ligand-free form shows a monomer structure in the bi-cupin fold of the β-barrel and an octameric functional unit, as well as an Mn2+ ion binding site. The structure of AtUGlyAH in complex with (S)-ureidoglycine revealed that the Mn2+ ion acts as a molecular anchor to bind (S)-ureidoglycine and its binding mode dictates the enantioselectivity of the reaction. Further kinetic analysis characterized the functional roles of the active site residues, including the Mn2+ ion binding site and residues in the vicinity of (S)-ureidoglycine. These analyses provide molecular insights into the structure of the enzyme and its possible catalytic mechanism. In the final step of the pathway, UAH catalyzes the conversion of (S)-ureidoglycolate into glyoxylate and releases two molecules of ammonia as by-products. UAH is homologous in structure and sequence with allantoate amidohydrolase (AAH), an upstream enzyme in the pathway with a similar function as that of an amidase but with a different substrate. Both enzymes exhibit strict substrate specificity and catalyze reactions in a concerted manner, resulting in purine degradation. Here, three crystal structures of Arabidopsis thaliana UAH: bound with substrate, reaction intermediate, and product, and a structure of Escherichia coli AAH complexed with allantoate are reported. Structural analyses of UAH revealed a distinct binding mode for each ligand in a bimetal reaction center with the active site in a closed conformation. The ligand directly participates in the coordination shell of two metal ions and is stabilized by the surrounding residues. In contrast, AAH, which exhibits a substrate-binding site similar to that of UAH, requires a larger active site due to the additional ureido group in allantoate. Structural analyses and mutagenesis revealed that both enzymes undergo an open-to-close conformational transition in response to ligand binding, and that the active site size as well as the interaction environments in each UAH and AAH are determinants for substrate specificity between two structurally homologous enzymes.