Structural basis of cooperativity in human UDP-glucose dehydrogenase

Abstract

Background: UDP-glucose dehydrogenase (UGDH) is the sole enzyme that catalyzes the conversion of UDP-glucose to UDP-glucuronic acid. The product is used in xenobiotic glucuronidation in hepatocytes and in the production of proteoglycans that are involved in promoting normal cellular growth and migration. Overproduction of proteoglycans has been implicated in the progression of certain epithelial cancers, while inhibition of UGDH diminished tumor angiogenesis in vivo. A better understanding of the conformational changes occurring during the UGDH reaction cycle will pave the way for inhibitor design and potential cancer therapeutics.

Methodology: Previously, the substrate-bound of UGDH was determined to be a symmetrical hexamer and this regular symmetry is disrupted on binding the inhibitor, UDP-α-D-xylose. Here, we have solved an alternate crystal structure of human UGDH (hUGDH) in complex with UDP-glucose at 2.8 Å resolution. Surprisingly, the quaternary structure of this substrate-bound protein complex consists of the open homohexamer that was previously observed for inhibitor-bound hUGDH, indicating that this conformation is relevant for deciphering elements of the normal reaction cycle.

Conclusion: In all subunits of the present open structure, Thr131 has translocated into the active site occupying the volume vacated by the absent active water and partially disordered NAD+ molecule. This conformation suggests a mechanism by which the enzyme may exchange NADH for NAD+ and repolarize the catalytic water bound to Asp280 while protecting the reaction intermediates. The structure also indicates how the subunits may communicate with each other through two reaction state sensors in this highly cooperative enzyme.

Figure 1. Mechanism of hUGDH catalysis.

(A) In the first phase of the mechanism established from the structure of bacterial UGDH (Easley et al., 2007), a water molecule, activated by Asp280, is predicted to be the general acid/base catalyst that abstracts a proton from the C6′ hydroxyl to initiate oxidation. Mutation of Asp280 to Asn is detrimental to catalysis. An aldehyde intermediate is transiently formed at the active site (Ia). This aldehyde has not been detected experimentally , however, added aldehyde functions as a substrate for the second oxidation . The intermediate is then proposed to rapidly convert to the thiohemiacetal adduct (IIa), via Cys276, without being released and the second NAD⁺ to replace the first reduced cofactor, NADH. (B) In an updated mechanism (Ib and IIb), the reaction proceeds directly to the thiohemiacetal adduct without proceeding through the aldehyde intermediate . (C) In the second phase of both mechanisms, the thiohemiacetal is then oxidized to the thioester via transfer of hydrogen to NAD⁺ (III). Finally the acid product is released through spontaneous hydrolysis , . Here the water bound to Asp280 is highlighted as the probable active water molecule. Evidence for a covalent thiohemiacetal (II) lies in the observation that the second deprotonation step (III) is reversible while the overall conversion to acid is irreversible (V) . Mutation of Cys276 to serine led to the build up of covalently attached adduct, however, the C276A mutation, while not able to proceed to completion from UDP-glucose, was able to catalyze the oxidation of the aldehyde intermediate . Lys220 provides charge stabilization to the anionic transition state during the second oxidation step (III), and for the course of the hydrolysis of the thioester (IV and V). Mutation of Lys220 significantly, but not completely, reduced the enzyme function suggesting that it does not form a Schiff's base , . Features in blue and red indicate that Thr131 and Asp280 coordinate the movements of NAD⁺/NADH and the active water molecule.

Figure 2. Structure of hUGDH.

(A) Schematic representation of a hUGDH protomer taken from the dodecamer. N-terminal, central and C-terminal domains are colored blue, green and red, respectively. UDP glucose is shown in purple, NAD⁺ in yellow. (B) The closely associated dimeric unit taken from the dodecamer. The second copy of the central domain is tinted orange. (C) The dodecameric hUGDH arrangement that is present in the crystal asymmetric unit comprised of two open hexamers. Upper panel shows the top layer of the dodecamer. Central and lower panels depict the full dodecamer in space filled representation. The twisted conformation is evident from the lower panel. (D) For comparison, a typical symmetric hexamer of hUGDH taken from PDB code 2Q3E. Movies S1 and S2 show morphs between these two structures. Protein representations were generated here, and in the figures that follow, using PYMOL (http://pymol.sourceforge.net/).

Figure 3. Secondary structure and topology of the hUGDH protomer.

(A) Secondary structure elements shown above the hUGDH amino acid sequence in domain colors (N-terminal, blue; central, green; C-terminal, red). Important active site residues are boxed. (B) Topology of the protomer. The figures were generated in ALSCRIPT and TOPDRAW .

Figure 4. Cooperativity within the hUGDH dimeric unit.

(A) Residues 265–280 wrap around and orient UDP-glucose in one subunit while Arg260 forms two hydrogen bonds to the glucose moiety of UDP-glucose in the second protomer. Hence, residues 260–280, which include active site residues Cys276 and Asp280, may act as a sensor by transmitting the state of one UDP-glucose binding site to the other protomer at the dimer interface. (B) Close up of the UDP-glucose binding site.

Figure 5. Cooperativity within the hUGDH trimeric unit.

Here, movement of the protomer A Thr131 loop into the active water-binding site is associated with an adjustment to the α6-helix, which mediates contact to the next protomer E in the trimeric unit. (A) Portions of protomers A (green), B (brown) and E (gray surface) from the open hexamer. (B) Close up of Thr131 in the active water-binding site. (C) Portions of protomers A (teal), B (orange) and E (pink surface) from the closed hexamer. (D) Close up of the active water-binding site. Movie S3 shows a morph between these two structures.

Figure 6. The UDP-glucose binding site.

(A, B) The active site conformations of the open and closed hexamers, respectively. (C, D) The position and hydrogen-bonding pattern of Asp280 in the open and closed hexamers, respectively. The active water molecule is shown as a black sphere. Movie S4 shows a morph between these two structures.