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.

Man o’ War Mutation in UDP-α-d-Xylose Synthase Favors the Abortive Catalytic Cycle and Uncovers a Latent Potential for Hexamer Formation

“…The widths of the peak and the tailing at both low and high s values were previously shown to indicate the presence of a small amount of monomer and tetramer in rapid equilibrium with the dimer (Figure 2A). 15 At the same concentration of protein, hUXS R236H sediments as a broad peak at a lower value of 3.4 S, close to the theoretical value for the hUXS monomer (3.3 S) ( Figure 2A). This confirms our earlier prediction that the mow mutation would weaken the dimer.…”

“…6 We have also shown that hUXS dimers undergo a concentration-dependent association to form a tetramer, which represents the catalytically important oligomeric state of hUXS. 15 This result prompted us to examine the structural consequences of the mow mutation in detail. Here we show that the R236H substitution weakens the dimeric complex as expected ( Figure 2A).…”

“…This hypothesis is consistent with our earlier work showing that wild-type hUXS tetramer is six times more active than the dimer (Figure 4). 15 Thus, the persistence of the hexamer-building interface in hUXS may be due to the functional significance of a tetramer with higher activity. The conservation of this oligomeric interface between hUXS and deeply divergent SDR enzymes also suggests that the ancestral UXS enzyme may have been a hexamer ( Figure 5B).…”

“…15 The formation of the hUXS tetramer increases the rate of UDP-Xyl production 6-fold while the abortive release of the reaction intermediates increases by only 20%. 15 The preferential increase in UDP-Xyl production suggests that the tetramer is the catalytically important species ( Figure 1B). The mow mutation (R236H) is buried in the dimer interface of hUXS, located approximately 16 Å away from the active site ( Figure 1C,D).…”

Weak protein–ligand interactions studied by small‐angle X‐ray scattering

“…Typically, a systematic search of K d values is carried out to determine the optimal relative fractions of A, B and AB that minimize the goodness‐of‐the‐fit chi‐squared between the resulting theoretical scattering profile and the experimental one using Eqns . This approach enables the testing of alternative structural models for a protein–ligand complex .…”