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Crystal structure and pH-dependent allosteric regulation of human β-ureidopropionase, an enzyme involved in anticancer drug metabolism

DOI: 10.1042/BCJ20180222 DOI Help

Authors: Dirk Maurer (Uppsala University) , Bernhard Lohkamp (Karolinska Institutet) , Michael Krumpel (Karolinska Institutet) , Mikael Widersten (Uppsala University) , Doreen Dobritzsch (Uppsala University)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Biochemical Journal

State: Published (Approved)
Published: July 2018
Diamond Proposal Number(s): 15868

Abstract: β-Ureidopropionase catalyzes the third step of the reductive pyrimidine catabolic pathway responsible for breakdown of uracil, thymine and pyrimidine-based antimetabolites such as 5-fluorouracil. Nitrilase-like β-ureidopropionases use a tetrad of conserved residues (Cys233, Lys196, Glu119 and Glu207) for catalysis and occur in a variety of oligomeric states. Positive cooperativity towards the substrate N-carbamoyl-β-alanine and an oligomerization-dependent mechanism of substrate activation and product inhibition have been reported for the enzymes from some species but not others. Here, the activity of recombinant human β-ureidopropionase is shown to be similarly regulated by substrate and product, but in a pH-dependent manner. Existing as homodimer at pH 9, the enzyme increasingly associates to octamers and larger oligomers with decreasing pH. Only at physiological pH it is responsive to effector binding, with N-carbamoyl-β-alanine causing association to more active higher molecular mass species, and β-alanine dissociation to inactive dimers. The parallel between the pH and ligand-induced effects suggests protonation state changes to play a crucial role in the allosteric regulation mechanism. Disruption of dimer-dimer interfaces by site-directed mutagenesis generated dimeric, inactive enzyme variants. The crystal structure of the T299C variant refined to 2.08 Å resolution revealed high structural conservation between human and fruit fly β-ureidopropionase, and supports the hypothesis that enzyme activation by oligomer assembly involves ordering of loop regions forming the entrance to the active site at the dimer-dimer interface, effectively positioning the catalytically important Glu207 in the active site.

Journal Keywords: pyrimidine degradation; 5-fluorouracil metabolism; amidohydrolase; allosteric regulation; crystal structure

Subject Areas: Biology and Bio-materials, Medicine


Instruments: I03-Macromolecular Crystallography