Human UDP-glucose dehydrogenase in complex with Uridine-5'-diphosphoglucose

Target ID:

UGDHA

Aliases:

GDHUDP-GlcDHUDPGDHUGDUGDH

Deposition Date:

Saturday 31st October 2009

Families:

Oxidoreductases

Related Diseases:

MetabolismSignallingNeurobiologyDrug metabolism and toxicology

Structure type:

protein-ligand

Download Coordinates:

3KHU

Authors:

Chaikuad, A., Egger, S., Yue, W.W., Sethi, R., Filippakopoulos, P., Muniz, J.R.C., von Delft, F., Bountra, C., Arrowsmith, C. H., Weigelt, J., Edwards, A.M., Kavanagh, K. L., Nidetzky, B., Oppermann, U.

Site:

Oxford

3KHU

tabs

Structure Details

UDP glucuronic acid is an essential metabolite required for the synthesis of extracellular matrix components (such as glycosaminoglycans involving hyaluronan, chondroitin sulfate, and heparan sulfate). A further important role is its role in the esterification of lipophilic endogenous (e.g. steroids) or xenobiotic compounds, thus forming excretable products.

The enzyme UDP-glucose dehydrogenase (UGDH) produces in a NAD + dependent reaction UDP glucuronic acid from UDP-glucose, which then serves as substrate for UDP-glucuronyltransferases, resulting in the multiple metabolites described above.

The enzyme is expressed in many gastrointestinal tissues with highest levels observed in the liver; and expression of the enzyme is regulated by several proinflammatory cytokines, growth factors such as transforming growth factor beta or IL-beta, by androgens and xenobiotics like eugenol and rifampicin.

UGDH is a potential therapeutic target for the treatment of cancer as inhibition of hyaluronan synthesis has been shown to reduce tumor angiogenesis and thereby restrict in vivo growth of human prostate tumors. Furthermore UGDH also plays an important role in the control of sex steroid inactivation via glucuronidation in breast cancer cells.

We previously determined structures of UGDH in complex with NADH and UDP-glucose (substrate-bound structure) and in complex with NAD + and UDP-glucuronic acid (product-bound structure). The enzyme utilizes a multi-step mechanism that consumes 2 equivalents of NAD + per UDP-glucuronic acid produced, but the reaction mechanism has never been well characterized. The first part of the reaction is similar to an alcohol dehydrogenase reaction whereby a hydroxyl group is converted to an aldehyde. The second part is equivalent to an aldehyde dehydrogenase reaction, which proceeds via a covalent intermediate. In the last step an activated water molecule hydrolyzes the intermediate, releasing UDP-glucuronic acid.

In this structure we have captured a covalent thio-hemiacetal intermediate formed between Cys276 and the aldehyde produced by the first NAD +-dependent step. This tetrahedral intermediate will subsequently be reduced by a second NAD + to a planar thioester before hydrolysis occurs. Major questions still remaining include identifying the rate limiting step as well as functional assignment of some catalytic residues. A better understanding of the UGDH mechanism will aid in designing structure-based inhibitors.

Materials and Methods

Materials and Method
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Entry Clone Source: Site-directed mutagenesis

Entry Clone Accession: n/a

SGC Construct ID: UGDHA-c603

GenBank GI number: gi|4507813

Vector: pBEN1-SGC

Final protein sequence (tag sequence in lowercase):
mdpeeasvtsteetltpaqeaartraank
arkeaelaaataeqtsdekttgwrgghvv
eglageleqlrarlehhpqgqrepsggck
lglgtenlyfq*sMFEIKKICCIGAGYVG
GPTCSVIAHMCPEIRVTVVDVNESRINAW
NSPTLPIYEPGLKEVVESCRGKNLFFSTN
IDDAIKEADLVFISVNTPTKTYGMGKGRA
ADLKYIEACARRIVQNSNGYKIVTEKSTV
PVRAAESIRRIFDANTKPNLNLQVLSNPQ
FLAEGTAIKDLKNPDRVLIGGDETPEGQR
AVQALCAVYEHWVPREKILTTNTWSSELS
KLAANAFLAQRISSINSISALCEATGADV
EEVATAIGMDQRIGNKFLKASVGFGGSCF
QKDVLNLVYLCEALNLPEVARYWQQVIDM
NDYQRRRFASRIIDSLFNTVTDKKIAILG
FAFKKDTGDTRESSSIYISKYLMDEGAHL
HIYDPKVPREQIVVDLSHPGVSEDDQVSR
LVTISKDPYEACDGAHAVVICTEWDMFKE
LDYERIHKKMLKPAFIFDGRRVLDGLHNE
LQTIGFQIETIGKKV

Tag sequence: N-terminal SET1 and SBP tags, followed by a TEV protease cleavage site:
mdpeeasvtsteetltpaqeaartraank
arkeaelaaataeqtsdekttgwrgghvv
eglageleqlrarlehhpqgqrepsggck
lglgtenlyfq*s
(* - TEV cleavage site)

Tag removed: yes

Host: BL21(DE3)-R3-pRARE2

Expression protocol: A glycerol stock of host strain BL21(DE3)-R3-pRARE2 harbouring the expression plasmid pBEN-SGC1 encoding UGDH E161Q construct was used to inoculate 50 ml of TB (terrific broth) supplemented with 50 µg/ml kanamycin and 34 µg/ml chloramphenicol. This starter culture was grown overnight at 37°C and used to inoculate 6x 1 liter culture in the same media to a starting OD₆₀₀ of 0.01. The culture was grown at 37°C until the OD₆₀₀ reached ~ 0.8. Afterwards the temperature was lowered to 18°C and protein production was induced with 0.2 mM IPTG. Recombinant UGDH was expressed at that temperature overnight. Cells were harvested by centrifugation at 5000 rpm for 20 minutes and the pellet was resuspended in 150 ml Strepavidin binding buffer supplemented with Complete Protease Inhibitors (1 tablet/50 ml) and stored at -20°C.

Cell extraction: Frozen cell pellets were thawed and lysed by passing the crude cell extract 5 times through a high pressure homogenizer. The lysate was clarified by centrifuging for 60 minutes at 21,000 rpm at 4C. Before applying to the column the lysate was passed through a 1.2µm syringe filter. Strepavidin binding buffer: 20 mM Tris, pH 8.0, 150 mM NaCl.

Column 1: Streptavidin sepharose

Buffers:
Binding buffer: 20 mM Tris, pH 8.0, 150 mM NaCl
Elution buffer: 20 mM Tris, pH 8.0, 150 mM NaCl, 2 mM Biotin.

Procedure: A final bed volume of 10 ml Streptavidin sepharose was equilibrated with 100 ml of binding buffer. The clarified cell lysate from the 6L culture was applied to the column and the resin was washed with 60 ml of binding buffer. The protein was eluted with 20 ml elution buffer containing 2 mM Biotin. The eluted protein was concentrated (Vivaspin centricon MWCO 30kDa) and exchanged into gel filtration buffer using a PD-10 desalting column.

Enzymatic treatment: His-tagged TEV protease was added to the protein using a 1:20 TEV to protein ratio (mg/mg). It was incubated for a total of 48h at 4°C; the cleavage was monitored to ensure completion.

Column 2: Gel filtration, Hiload 16/60 Superdex S200 prep grade, 120 ml bed volume (GE Healthcare)

Gel Filtration Buffer: 20 mM Tris pH 7.5, 300 mM NaCl

Procedure: The protein sample was loaded onto a S200 column at a flow rate of 1 ml/min using an AKTA Purifier system (GE Healthcare) at 4°C. After SDS-Page analysis of the fractions, pure protein was pooled and concentrated using a Vivaspin centricon with a MWCO of 30kDa.

Mass spectrometry characterization: ESI-MS confirmed the correct mass of the protein of 52019 Da (expected mass: 52019 Da).

Crystallization: Crystals of the UGDH thiohemiacetal intermediate complex was obtained after incubating 16mg/ml protein solution with 2mM NAD⁺ and 5mM UDP-glucose. Crystals were grown by vapour diffusion in sitting drops at 4°C using 18% PEG smears, 0.1M HEPES pH 7.5 and 5% ethylene glycol as precipitant at a protein to precipitant ratio of 2:1. A crystal was cryo-protected using well solution supplemented with 20% (v/v) ethylene glycol and flash-cooled in liquid nitrogen.

Data Collection: Resolution (scaled): 2.3 Å; Diffraction data were collected at Diamond beamline I03.