title: Human UDP-α-d-xylose synthase forms a catalytically important tetramer that has not been observed in crystal structures.
abstract: Human UDP-α-d-xylose synthase (hUXS) is a member of the extended short chain dehydrogenase/reductase (SDR) family of enzymes. Previous crystallographic studies have shown that hUXS conserves the same dimeric quaternary structure observed in other SDR enzymes. Here, we present evidence that hUXS also forms a tetramer in solution that is important for activity. Sedimentation velocity studies show that two hUXS dimers undergo a concentration-dependent association to form a tetramer with a Kd of 2.9 μM. The tetrameric complex is also observed in small-angle X-ray scattering (SAXS). The specific activity for the production of the reaction intermediate UDP-α-d-4-keto-xylose displays a hyperbolic dependence on protein concentration that is well modeled by an isotherm using the 2.9 μM Kd of the tetramer. Likewise, the rate of UDP-α-d-xylose production in the presence of increasing concentrations of the small molecule crowder trimethylamine N-oxide is consistent with the formation of a higher activity tetramer. We present several possible structural models of the hUXS tetramer based on (i) hUXS crystal packing, (ii) homology modeling, or (iii) ab initio simulated annealing of dimers. We analyze the models in terms of packing quality and agreement with SAXS data. The higher activity of the tetramer coupled with the relative instability of the complex suggests that an association-dissociation mechanism may regulate hUXS activity.
authors: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, United States.
authors: Samuel J
authors: SJ
authors: Polizzi
authors: Richard M
authors: RM
authors: Walsh
authors: Pierre
authors: P
authors: Le Magueres
authors: Angela R
authors: AR
authors: Criswell
authors: Zachary A
authors: ZA
authors: Wood
mesh_terms: D002262:Carboxy-Lyases / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D006801:Humans; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D053838:Scattering, Small Angle; D012996:Solutions; D014461:Ultracentrifugation
chemical_list: D012996:Solutions; D002262:Carboxy-Lyases; C023257:UDPglucuronate decarboxylase