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Originally published In Press as doi:10.1074/jbc.M404234200 on July 7, 2004

J. Biol. Chem., Vol. 279, Issue 36, 37491-37498, September 3, 2004
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Importance of Gly-13 for the Coenzyme Binding of Human UDP-glucose Dehydrogenase*

Jae-Wan Huh, Hye-Young Yoon{ddagger}, Hyun-Ju Lee, Won-Beom Choi, Seung-Ju Yang, and Sung-Woo Cho§

From the Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine, Seoul 138-736, Korea

Received for publication, April 16, 2004 , and in revised form, July 6, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
UDP-glucose dehydrogenase (UGDH) is the unique pathway enzyme furnishing in vertebrates UDP-glucuronate for numerous transferases. In this report, we have identified an NAD+-binding site within human UGDH by photoaffinity labeling with a specific probe, [32P]nicotinamide 2-azidoadenosine dinucleotide (2N3 NAD+), and cassette mutagenesis. For this work, we have chemically synthesized a 1509-base pair gene encoding human UGDH and expressed it in Escherichia coli as a soluble protein. Photolabel-containing peptides were generated by photolysis followed by tryptic digestion and isolated using the phosphopeptide isolation kit. Photolabeling of these peptides was effectively prevented by the presence of NAD+ during photolysis, demonstrating a selectivity of the photoprobe for the NAD+-binding site. Amino acid sequencing and compositional analysis identified the NAD+-binding site of UGDH as the region containing the sequence ICCIGAXYVGGPT, corresponding to Ile-7 through Thr-19 of the amino acid sequence of human UGDH. The unidentified residue, X, can be designated as a photolabeled Gly-13 because the sequences including the glycine residue in question have a complete identity with those of other UGDH species known. The importance of Gly-13 residue in the binding of NAD+ was further examined with a G13E mutant by cassette mutagenesis. The mutagenesis at Gly-13 had no effects on the expression or stability of the mutant. Enzyme activity of the G13E point mutant was not measurable under normal assay conditions, suggesting an important role for the Gly-13 residue. No incorporation of [32P]2N3NAD+ was observed for the G13E mutant. These results indicate that Gly-13 plays an important role for efficient binding of NAD+ to human UGDH.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
UDP-glucose dehydrogenase (UGDH,1 EC 1.1.1.22 [EC] ) is a member of a small group of NAD+-linked, 4-electron-transferring oxidoreductases (1) and converts UDP-glucose to UDP-glucuronate. Kinetic studies of UGDH from bovine (2, 3), Streptococcus (4, 5), and plant (6, 7) show that the reaction of UGDH involves two successive oxidations to convert the 6'-hydroxyl of UDP-glucose to a carboxylate, together with the reduction of 2 molecules of NAD+ to NADH. In animals, it constitutes the unique pathway for glucuronate formation (8). Because glucuronate is a component of glycosaminoglycans (GAGs), the mutation inactivation of UGDH (sugarless) abolishes GAG assembly and, consequently, abolishes GAG-dependent growth factor signaling (9–11). The primary structure of the mammalian enzyme was obtained from the protein sequence of bovine UGDH (12). The human gene was also recently cloned and assigned to chromosome 4p15.1 (13). It contains 12 exons, extends over 26 kb, and has one major transcription start site (14).

GAG chains of proteoglycans and hyaluronan are ubiquitous components of extracellular matrix and pericellular spaces. There is a growing body of information on the implication of GAGs in cell behavior, including signal transduction, cell proliferation, spreading, migration, and cancer growth and metastasis (15–17). GAG synthesis is influenced by cytokines and growth factors. Transforming growth factor-{beta} is the most potent stimulator of proteoglycan and GAG synthesis, including that of hyaluronan. Its action, however, depends on the cell type (18). The synthesis of GAGs is also modulated by oxygen cell status. Hypoxic endothelial cells and lung fibroblasts enhanced the heparan sulfate/chondroitin sulfate ratio, which led to an increase of basic fibroblast growth factor reactivity on the cell surface (19, 20). It was also shown that the level of intracellular UDP-glucuronate could influence GAG synthesis (8). Interestingly, the human UGDH was shown to be an early response gene after interleukin-1 treatment of ocular fibroblasts (21), as well as an early androgen response gene in breast cancer (22).

Although a clone of human UGDH has been reported (13, 15), specific catalytic residues are not yet available. Therefore, further characterization of the active sites of human UGDH is needed to elucidate the physiological nature of the UGDH. In the present study, we have identified an NAD+-binding site using photoaffinity labeling and cassette mutagenesis to gain a deeper insight into the structural basis of human UGDH. For this study, a 1509-base pair gene that encodes human UGDH has been chemically synthesized and expressed in Escherichia coli as a soluble protein. Identification of the nucleotide-binding sites of a variety of proteins has been advanced by the use of nucleotide photoaffinity analogues that selectively insert into a site upon photoactivation with ultraviolet light. For instance, [32P]2N3NAD+ was shown to be a valid active site probe for several proteins (23–26). GTP- and ADP-binding sites of the bovine glutamate dehydrogenase have been reported using [32P]8N3GTP and [32P]8N3ADP, respectively (27–30). The ATP-binding site of adenylate kinase and creatine kinase and the protein unique to cerebrospinal fluids of Alzheimer's patients successfully also has been identified using 2N3ATP and 8N3ATP (31, 32).

Our results indicate that Gly-13 plays an important role for efficient binding of NAD+ to human UGDH. To our knowledge, this is the first report identifying a reactive residue critically involved in the coenzyme binding of the human UGDH.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—UDP-glucose, NAD+, ampicillin, isopropyl thio-{beta}-D-galactoside, phenylmethanesulfonyl fluoride, and L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin were purchased from Sigma. 2N3NAD+ and [32P]2N3NAD+ were synthesized using NMN adenyltransferase according to the same method as described previously (26). Monoclonal antibody against human UGDH was kindly provided by Dr. S. Choi (Hallym University, Chunchon, Korea). Precast gels for SDS-polyacrylamide gel electrophoresis were purchased from NOVEX. All other chemicals and solvents were of reagent grade or better.

Bacterial Strains—E. coli DH5{alpha} (33) was used as the host strain for plasmid-mediated transformations during the assembly of the synthetic human UGDH gene (pHUGDH) and for cassette mutagenesis. E. coli BL21 (DE3) (34) was used for high level expression of the recombinant hUGDH.

Design and Assembly of the Synthetic hUGDH Gene—The design of the synthetic hUGDH gene was based on the amino acid sequence of hUGDH (15) and used the following strategy. First, a DNA sequence containing 37 restriction sites located approximately every 42 bp throughout the entire length of the coding region was selected from the large number of possibilities. Only those sites recognized by commercially available restriction enzymes and that are not located in pUC18 (except in the polylinker region) were included in the final sequence of the gene. The hUGDH gene is flanked by unique EcoRI and HindIII restriction sites that render the gene portable to various E. coli expression vectors. Second, the codon usage of the resulting hUGDH gene was modified to include those triplets that are utilized in highly expressed E. coli genes (35, 36) while retaining the largest possible number of unique restriction sites. In some cases, suboptimal codons were used either to allow the inclusion of unique restriction sites or to preclude redundant sites. Third, a ribosome-binding site AGGAGG (37) was added 10 bases upstream of the coding region to direct the initiation of translation in E. coli. The sequence adjacent to the ribosome-binding site included an A at position –3 relative to the ATG, and the spacer region (–1 to –9) was made A + T-rich to reduce potential mRNA secondary structure in the vicinity of the translation start site (37). Addition of a ribosome-binding site made the synthetic hUGDH gene portable to any of a number of commonly available plasmid vectors that carry inducible E. coli promoters. The hUGDH gene was assembled from seven gene segments that were initially cloned into pUC18. Each of the gene segments was constructed with 2–4 oligonucleotides. 6–7 isolates of each segment were examined by restriction analysis and DNA sequencing. The final synthetic hUGDH gene, designated as pHUGDH, was used for gene expression and mutagenesis studies.

Protein Purification and Characterization—Fresh overnight cultures of DE3/pHUGDH were used to inoculate 1 liter of Luria-Bertani (LB) containing 100 µg of ampicillin/ml. DE3/pHUGDH was grown at 37 °C until the A600 reached 1.0, and then isopropyl thio-{beta}-D-galactoside was added to a final concentration of 1 mM. After isopropyl thio-{beta}-D-galactoside induction, DE3/pHUGDH was grown for an additional 3 h at 37 °C and then harvested by centrifugation. Cell pellets were suspended in 100 ml of 100 mM Tris-HCl, pH 7.4, 1 mM EDTA, 5 mM dithiothreitol and lysed with a sonicator. Cellular debris was removed by centrifugation, and the crude extract was loaded onto a blue Sepharose CL-6B column that was equilibrated with buffer A (20 mM Tris-HCl, pH 6.5, 5 mM MgCl2, 10 mM {beta}-mercaptoethanol). The column was washed with buffer A until the breakthrough peak of protein had been eluted. The enzyme was then eluted by a gradient up to 500 mM NaCl. The fractions containing hUGDH were pooled and concentrated, and the buffer was changed to buffer B (50 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, 10 mM {beta}-mercaptoethanol) using Amicon concentrator, after which the fractions were applied to a fast protein liquid chromatography Resource-Q column equilibrated with buffer B. The enzyme was then eluted using a linear gradient made with buffer B in increasing concentrations of NaCl (from 0 to 300 mM) at 3 ml/min. The fractions containing hUGDH were pooled and concentrated. The purified enzymes were analyzed by SDS-PAGE and recognized by Western blot using monoclonal antibodies against the hUGDH. Protein concentration was determined by the method of Bradford (38) using bovine serum albumin as a standard.

Enzyme Assay and Kinetic Studies—hUGDH activity was measured with NAD+ in 100 mM sodium glycine, pH 8.7, and 1 mM UDP-glucose at 25 °C. The reaction started by the addition of NAD+ to 1 mM final concentration. One unit of enzyme was defined as the amount of enzyme required to reduce 2 µmol of NAD+/min at 25 °C. For determination of Km and Vmax values, the assays were carried out by varying the substrate under investigation while keeping the other substrate and reagents at the optimal concentration indicated above. The Km and Vmax values were calculated by fitting the data to the Michaelis-Menten equation and assuming a single binding site each for the substrate and cofactor. Catalytic efficiency was estimated by use of the equation v/[E0] = (kcat/Km)[S] (39).

Photolabeling of hUGDH with [32P]2N3NAD+ —Photolabeling of the wild-type hUGDH with [32P]2N3NAD+ was performed by the method of Yoon et al. (26) with a slight modification. For saturation studies, the wild-type hUGDH (100 µg) in 10 mM Tris acetate, pH 8.0, was incubated with various concentrations of [32P]2N3NAD+ in Eppendorf tubes for 5 min. For competition studies, 100 µg of enzyme was incubated with various concentrations of NAD+ for 10 min in the same buffer prior to the addition of 50 µM [32P]2N3NAD+ and then allowed to incubate with the photoprobe for 5 min as described above. The samples were irradiated with a handheld 254-nm UV lamp for 90 s twice at 4 °C. The reaction was quenched by the addition of ice-cold trichloroacetic acid (final 7%). The reaction mixtures were kept on the ice bath for 30 min and centrifuged at 10,000 x g for 15 min at 4 °C. The pellets were washed and resuspended with 10 mM Tris acetate, pH 8.0. The remaining free photoprobe, if any, was further removed from the protein by exhaustive washing using Centrifree (Amicon), and 32P incorporation into protein was determined by liquid scintillation counting.

Tryptic Digestion and Isolation of Photolabeled Peptide—To determine the site modified by [32P]2N3NAD+, 1.0 mg of the wild-type hUGDH in 10 mM Tris acetate, pH 8.0, was incubated with 200 µM [32P]2N3NAD+ for 5 min at 4 °C. The mixtures were irradiated for 90 s twice. The reaction was quenched by the addition of ice-cold trichloroacetic acid (final 7%) and kept at 4 °C for 30 min. The protein was precipitated by centrifugation at 10,000 x g for 15 min at 4 °C, and the pellet was resuspended in 75 mM NH4HCO3, pH 8.5, containing 2 M urea. The enzymes were proteolyzed by the addition of 15 µg of trypsin and kept at room temperature for 3 h, after which time 15 µg of trypsin was added again. After an additional 3 h at room temperature, 20 µgof trypsin was added, and the digestion mixture was kept at 25 °C overnight. To validate that the isolated peptide was specific for the NAD+-binding site and could be protected from photomodification, the hUGDH proteins were also photolyzed in the presence of 300 µM NAD+ and proteolyzed as described above. The trypsin-digested peptides were separated by the phosphopeptide isolation kit from Pierce following the manufacturer's protocol and sequenced by the Edman degradation method as described before (26).

Construction and Purification of the G13E Mutant—Single amino acid substitution of Gly-13 was constructed by cassette mutagenesis of plasmid pHUGDH. Plasmid DNA (5 µg) was digested with BstBI and SalI to remove the 98-bp fragment that encodes amino acids 3–35, and vector DNA was purified by electrophoresis using 1% low melting point agarose. The 98-bp BstBI/SalI fragment was replaced with a 98-bp synthetic DNA duplex containing a substitution on both DNA strands at positions encoding Gly-13 to make the G13E mutant protein. The mutagenic oligonucleotide was annealed, ligated, and transformed into DH5{alpha} as described above, and G13E mutant was identified by DNA sequencing using plasmid DNA as a template. G13E mutant was expressed in E. coli strain DE3 and purified to homogeneity as described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Construction of the Synthetic hUGDH Gene and Expression—The gene was initially constructed as three segments using the plasmid pUC18 as a cloning vector (Fig. 1). A total of 20 synthetic oligonucleotides that varied from 56 to 92 nucleotides in length were used to assemble the three segments. The ends of the segments were chosen from restriction sites present in the polylinker of pUC18 to allow stepwise assembly in that vector. The three segments were, respectively, a 486-bp EcoRI/PstI fragment composed of 6 oligonucleotides, a 580-bp PstI/AgeI fragment composed of 7 oligonucleotides, and a 443-bp AgeI/HindIII fragment composed of 7 oligonucleotides (Fig. 1). Several isolates of each of the gene segments were characterized by DNA sequencing. Based on the sequencing of several isolates of each gene segment, an overall mutation frequency of ~3/1000 bp synthesized was observed and corrected to the designed sequence using a standard cassette mutagenesis procedure. The designed sequence and position of 37 restriction sites in the hUGDH coding region of pHUGDH are shown in Fig. 2. High level expression of the synthetic hUGDH gene was achieved by transformation of pHUGDH into E. coli strain DE3. Upon induction with 1 mM isopropyl thio-{beta}-D-galactoside at 37 °C for 3 h, expression of hUGDH in soluble extracts was about 0.163 units/mg. SDS-PAGE analysis of crude cell extracts and measurement of specific activities indicated that pHUGDH directed hUGDH expression to a level of ~16% of total cellular protein upon induction with isopropyl thio-{beta}-D-galactoside (Fig. 3).



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  		FIG. 1.
Assembly of the synthetic hUGDH gene. A total of 40 oligonucleotides were used to assemble three gene fragments that varied from 443 to 580 bp in length. DNA fragments corresponding to the gene segments were isolated and used to create the functional hUGDH gene (pHUGDH) via stepwise ligation into pUC18 as shown. R, EcoRI; P, PstI; A, AgeI; H, HindIII.

 



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  		FIG. 2.
DNA sequence of the synthetic hUGDH gene carried in pHUGDH. Numbers on the left refer to amino acids (upper) and DNA (lower). Only the unique restriction sites are shown. Position 1 of the amino acid sequence corresponds to the first amino acid (Met) of the human UGDH (15).

 



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  		FIG. 3.
SDS-PAGE analysis of hUGDH. Lane 1, marker proteins (Bio-Rad; 97, 66, 45, 31, 21, and 14 kDa); lane 2, crude extract from DE3 transformed with pUC18 only; lane 3, crude extract from DE3 transformed with pHUGDH; lane 4, fast protein liquid chromatography-purified recombinant hUGDH; lane 5, HPLC-purified recombinant hUGDH.

 
Purification and Characterization of Synthetic hUGDH— hUGDH encoded by pHUGDH in DE3 was purified by several chromatographic methods. Because the recombinant hUGDH was readily solubilized, no detergents were required throughout the entirety of the purification steps. The purified hUGDH was estimated to be >98% pure by SDS-PAGE (Fig. 3). No additional amino acids such as N-terminal histidine tags or protease recognition sites were introduced in the synthetic hUGDH gene product (Fig. 2). N-terminal sequence analysis of the first 7 amino acids (MFEIKKI) is in agreement with the published sequence of human UGDH (15). The subunit size (57 kDa) and the native size (340 kDa) of the hexameric recombinant hUGDH were determined by SDS-PAGE (Fig. 3) and HPLC-gel filtration chromatography (data not shown), respectively. The 57-kDa protein was absent in extracts from DE3 transformed with pUC18 (Fig. 3). The highly purified enzyme was a mixture of hexamer, tetramer, and dimer as determined by HPLC gel filtration. The hUGDH was predominantly observed as a hexamer together with significant tetrameric and dimeric species, indicating some subunit dissociation. Although relative species ratios were conserved over several different preparations of the enzyme, the ratio of the oligomeric state was changed by storage conditions and periods.

UGDH catalyzes conversion of a UDP-glucose substrate to a UDP-glucuronic acid product, concomitantly reducing 2 molecules of NAD+ to NADH. The enzymatic activity of hUGDH was characterized by steady state kinetic analysis. Measurements to determine dependence of the reaction on cofactor concentration were done using purified hUGDH incubated with increasing concentrations of NAD+ in the presence of saturating UDP-glucose substrate. Similarly, dependence of reaction kinetics on substrate was measured by increasing UDP-glucose concentration in the presence of saturating NAD+. Saturation kinetic data were observed for both conditions. Data were fitted to the Michaelis-Menten equation to obtain Km and Vmax for the reaction catalyzed by the wild-type enzyme. Both sets of conditions yielded a similar Vmax of 157 ± 5 nmol of NAD+/min/mg of enzyme (Fig. 4, A and B). The Km for UDP-glucose was 17 ± 3 µM (Fig. 4A), and the Km for NAD+ was 133 ± 4 µM (Fig. 4B). For more detailed catalytic properties of hUGDH, the enzyme efficiency (kcat/Km) for the individual substrates was determined (Table I). The kcat value of hUGDH was 105 ± 4s–1, and the kcat/Km values for NAD+ and UDP-Glc were 7.9 ± 1.0 s–1·µM–1 and 6.2 ± 1.3 s–1·µM–1, respectively (Table I).



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  		FIG. 4.
Vmax and Km values of hUGDH for NAD+ (A) and UDP-glucose (B). Steady state enzyme activity was measured as conversion of NAD+ to NADH detected by absorbance at 340 nm. Purified hUGDH was incubated in 100 mM sodium glycine, pH 8.7, at 25 °C with increasing concentrations of NAD+ in the presence of 1 mM UDP-glucose (A) or with increasing concentrations of UDP-glucose in the presence of 1 mM NAD+ (B). The Km and Vmax values were calculated by linear regression analysis of double-reciprocal plots.

 


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  		TABLE I
Kinetic properties of hUGDH and G13E mutant

Km and kcat/Km values of wild-type hUGDH and the G13E mutant were obtained from the initial velocity data and linear regression analysis of double-reciprocal plots as described under "Experimental Procedures." The data represent the mean of two independent experiments with the difference expressed as n ± deviation. Duplicates of the kinetic values differed less than 5%.

 
Specific Binding of 2N3NAD+ to hUGDH—The reactive amino acid in the direct binding of NAD+ to hUGDH was identified by photoaffinity labeling with [32P]nicotinamide 2-azidoadenosine dinucleotide (2N3NAD+). To show specificity of the photoprobe-protein interaction, saturation of photoinsertion should be observed. For demonstrating saturation effects with the photoprobe, the purified enzymes were photolabeled with increasing concentrations of [32P]2N3NAD+. Under the experimental conditions described, saturation of photoinsertion with [32P]2N3NAD+ occurred at around 120 µM photoprobe (Fig. 5). In all photolabeling experiments, the ionic strength was kept low to enhance binding affinity, as we have observed in general that the lower the ionic strength is, the tighter the binding of nucleotide photoaffinity probes and the more efficient the photoinsertion are (26). The data in Fig. 5 demonstrate the apparent Kd value of ~48 µM, which is in reasonably good agreement with the recently reported Kd value (118 µM) of NAD+ with hUGDH (40). The stoichiometry of [32P]2N3NAD+ incorporation was estimated to be 3 mol of the label/hexameric enzyme. The results in Fig. 5 indicate the saturability of the NAD+-specific site of hUGDH with this photoprobe and therefore decrease the possibility of nonspecific photoinsertion. To further demonstrate specific labeling of hUGDH, the wild-type hUGDH was photolabeled with [32P]2N3NAD+ in the presence of increasing NAD+ concentrations. As shown in the results of the competition experiments (Fig. 6), NAD+ at concentrations in the range of Km values was able to protect photolabeling from [32P]2N3NAD+. Approximately 95% protection was observed with 160 µM NAD+ for the wild-type hUGDH (Fig. 6). These results show the specificity and utility of [32P]2N3NAD+ as a good probe for determining the NAD+-binding site.



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  		FIG. 5.
Saturation of photoinsertion of [32P]2N3 NAD+ into hUGDH. The wild-type hUGDH and the G13E mutant in the reaction buffer were photolyzed with the indicated concentrations of [32P]2N3NAD+. 32P incorporations into proteins were determined by liquid scintillation counting and expressed relative to each control. {blacksquare}, wild-type hUGDH; {circ}, G13E mutant.

 



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  		FIG. 6.
Effect of NAD+ on photoinsertion of [32P]2N3 NAD+ into hUGDH. The wild-type hUGDH and the G13E mutant in the reaction buffer were photolyzed with 50 µM [32P]2N3NAD+ in the presence of the indicated concentrations of NAD+. 32P incorporations into proteins were determined by liquid scintillation counting and expressed relative to each control. {blacksquare}, wild-type hUGDH; {circ}, G13E mutant.

 
Tryptic Digestion of Photolabeled Proteins—To identify the peptides modified by [32P]2N3NAD+, the wild-type hUGDH was photolabeled twice in the absence and presence of 300 µM NAD+ and digested with trypsin. To reduce any possible nonspecific labeling and at the same time to optimize the specific labeling of the enzymes, 100 µM [32P]2N3NAD+ was used, which is the concentration at which photoinsertion approaches saturation. The photolabeled proteins were separated from most of the noncovalently bound nucleotide by acid precipitation and proteolyzed by trypsin. The trypsin-digested photolabeled peptides were isolated by the phosphopeptide isolation kit from Pierce. The radioactive peptides were eluted with 100 mM ammonium bicarbonate, pH 9, containing 0.3 M ammonium hydroxide and 20% acetonitrile. Photolabeling of the peptide was prevented by the presence of NAD+ during photolysis. When 300 µM NAD+ was originally present in the incubation mixture, ~95% of the radioactivity of the eluent was eliminated. These results demonstrate a selectivity of the photoprobe and suggest that the radioactive fractions represent a peptide in the NAD+-binding domain of the hUGDH. The radioactive fractions were collected and identified by amino acid sequence analysis.

Sequence Analysis of Photolabeled Peptide—The amino acid sequence analysis revealed that the radioactive eluent contained the amino acid sequence ICCIGAXYVGGPT. As judged by comparison with the amino acid sequence of mammalian UGDHs (Table II), this site was identified as residues 7–19 of hUGDH. The symbol X indicates a position for which no phenylthiohydantoin amino acid could be assigned. The missing residue, however, can be designated as a photolabeled glycine because the sequences including the glycine residue in question have a complete identity with those of the other UGDH species known. Because the photolabeled peptides are tryptic digests, it was expected to produce a sequence ending with Arg or Lys. The amino acid composition of the photolabeled peptide revealed that the peptide had a composition that was compatible with that of the tryptic peptide spanning residues 7–31 with an exception that there was a significant reduction in glycine (Table III). On the basis of information obtained on the amino acid sequence determination and composition analysis of the isolated peptide, we suggest that the attachment site of [32P]2N3 NAD+ is Gly-13. The importance of the Gly-13 residue in the binding of NAD+ was further examined by substituting Gly into Glu, Leu, Arg, and Tyr at position 13. Because all four mutants (G13E, G13L, G13R, and G13Y) at the Gly-13 site were studied to the same extent as G13E and showed almost identical results in protein expression, purification, kinetic parameters, and photoaffinity labeling study, only the results from G13E are presented for the purpose of clarity.


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  		TABLE II
Alignment of [32P]2N3NAD+-labeled peptides with homologous sequences from various UGDHs

 


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  		TABLE III
Amino acid composition of [32P]2N3NAD+-labeled peptide

 
Construction, Expression, and Properties of the G13E Mutant—The 98-bp BstBI/SalI fragment in pHUGDH was replaced with 98-bp synthetic DNA duplexes containing a substitution on both DNA strands at positions encoding Gly-13. This substitution made mutant protein G13E. Analysis of crude cell extracts by Western blot showed that the G13E mutant plasmid directed the synthesis of a protein that interacted with monoclonal antibodies raised against hUGDH at almost identical levels as it did with wild-type hUGDH (Fig. 7). These results indicate that the mutagenesis at Gly-13 has no effects on expression or stability of the mutant. In addition, the mutant enzymes were purified homogeneously by the same method as was the wild-type enzyme, indicating that no gross conformational change of the enzyme had occurred.



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  		FIG. 7.
Western blot analysis of the G13E mutant in crude extracts. Lane 1, prestained marker proteins (NOVEX); lane 2, wild-type hUGDH; lane 3, G13E mutant; lane 4, vector only.

 
Catalytic activity of hUGDH point mutant G13E was not measurable under standard assay conditions. To evaluate the role of the Gly-13 residue in catalysis and to determine the extent of impaired catalytic function, we performed a time course over 3 h, measuring enzyme activity every 10 min by absorbance wavelength scans from 290 to 400 nm. Less than 0.05% activity relative to wild-type enzyme was observed for the G13E mutant up to 3 h, demonstrating that loss of enzymatic activity was the result of the mutagenesis at the Gly-13 site. In addition, no photoaffinity labeling of [32P]2N3NAD+ was measured for the G13E mutant in the presence and absence of NAD+ under the same experimental conditions used for the wild-type enzyme (Figs. 5 and 6). These results are consistent with the postulated role of Gly-13 in NAD+ binding and support its position in the reaction mechanism for the human enzyme.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The major functional significance of UGDH in animals is developmental, and its regulation may be related to several pathologies such as cancer progression (22). Having a detailed understanding of its structure and mechanism would be necessary to design specific targeted inhibitors for a therapeutic avenue. The majority of previous studies on UGDH have focused on the bovine liver enzyme, and until recently very little was known about the human enzyme. The structure of the enzyme is well conserved between the species and phyla. The cloned mammalian proteins from different species showed overall 97% identity (21, 41). The human enzyme cDNA codes for 494 amino acids and has 27% homology to the E. coli ortholog. The conserved amino acids are uniformly distributed along the molecule. Interestingly, the upstream NAD+-binding region (GXGXXG) is 100% identical in mammals and E. coli enzymes (42, 43). The term "fold" was introduced by Rossmann (44–47) to describe a nucleotide-binding domain found in families of oxidoreductases such as lactate dehydrogenase and flavodoxin. This fold begins with a {beta}-strand connected by a short loop to an {alpha}-helix (45) and contains a conserved sequence motif, GX1–2GXXG (48), where the glycine residues are located on the ligand-binding loop between the {beta}-strand and {alpha}-helix, although not all Rossmann folds bind to the nucleotides FAD or NADP.

The importance of the glycine residues has been explained previously (49); the first glycine allows a tight turn of the main chain from the {beta}-strand into the loop, and the second glycine permits close contact of the main chain to the pyrophosphate of the nucleotide. The third glycine allows close packing of the helix with the {beta}-strand. Mutations in the conserved glycine residues of the loop have been correlated with attenuation or elimination of enzyme activity (50–52) and also with disease (53). The conserved structure suggests that UGDH may be part of a minimal genome for multicellular species and that the rigid basal structure is necessary for its activity (14, 42). To our knowledge, the crystallographic structure of hUGDH has not been determined, and no studies by site-directed mutagenesis for the coenzyme-binding site(s) of the mammalian UGDHs have been reported yet. In the present study, hUGDH was chemically synthesized, expressed, and purified to homogeneity. A specific point mutant was generated based on homology modeling of the enzyme and used to identify the coenzyme-binding site of hUGDH.

We identified an NAD+-binding site of hUGDH using a photoaffinity probe, [32P]2N3NAD+.[32P]2N3NAD+ is a probe that, on photolysis, generates a very reactive nitrene that has the capacity to photoinsert into any residue. The data showing decreased photoinsertion with addition of NAD+ (Fig. 6) demonstrate that photoinsertion is carried out only by the bound form of [32P]2N3NAD+. This indicates that proximity controls photoinsertion and that the residues modified are within the NAD+-binding domain. In addition, prephotolysis followed by immediate addition of hUGDH did not lead to covalent labeling (data not shown), eliminating the existence of any long lived chemically reactive intermediate that could be involved in covalently modifying enzymes. The selectivity and the specificity have been successfully utilized to locate the specific base-binding domains of nucleotide-binding site peptides of many proteins (23–32).

The specificity of [32P]2N3NAD+ and the utility of this probe as a good candidate for determining the NAD+-binding site were further supported by the following. First, in the absence of activating light, 2N3NAD+ is a substrate with properties similar to NAD+ for hUGDH (data not shown). The ability to mimic a native compound before photolysis has an advantage over determination of the enzyme function after modification. Second, the photoinsertion into hUGDH shows saturation with [32P]2N3NAD+. Saturation of photoinsertion with [32P]2N3NAD+ occurred at around 120 µM photoprobe (Fig. 5). In addition, the sites of attachment of the photoaffinity label were more precisely defined by generating small peptide fragments of the labeled protein and separating the labeled peptides efficiently by one-step affinity chromatography. Third, the photolabeled peptide of the hUGDH identified is located within the proposed NAD+-binding domain of many proteins (42–48), confirming that this sequence is expected to interact with NAD+.

It has been reported that the two substrates NAD+ and UDP-glucose bind in equimolar amounts rather than in a 2:1 ratio in favor of NAD+ over UDP-glucose in view of the sequential mechanism of the two-stage oxidation (3, 40, 54). Interestingly, our data show that only 3 mol of [32P]2N3NAD+ are incorporated/hexameric hUGDH. From what is known of the subunit nature of hUGDH, it was suspected that six NAD+-binding sites would exist in the native hexameric enzyme. Therefore, these results suggest that the binding sites are located in the interfaces between pairs of subunits and support that the hUGDH quaternary structure adopts a trimer of dimers arrangement (40, 54).

There were differences in the biochemical properties of the wild-type hUGDH and the G13E mutant. Catalytic activity of hUGDH point mutant G13E was not measurable under normal assay conditions up to 3 h, measuring enzyme activity every 10 min. This result suggests the possibility that loss of enzymatic activity is due to a change in binding of NAD+ by the G13E mutant. In addition, no detectable amounts of photoaffinity labeling were observed for the G13E mutant when treated with [32P]2N3NAD+ both in the presence and absence of NAD+ under the same experimental conditions as used for the wild-type enzyme (Figs. 5 and 6), and therefore no photolabeled peptides were detected for the G13E mutant (Table II). The results from the Western blot analysis show that the mutagenesis at the Gly-13 site has no effects on expression or stability of the mutant (Fig. 4). The results of HPLC gel filtration analysis also showed no differences in their elution profiles in terms of the native molecular size (data not shown), suggesting that the mutagenesis at Gly-13 did not cause gross structural changes in hUGDH. Therefore, the results with cassette mutagenesis and photoaffinity labeling techniques suggest that the loss of enzyme activity of the G13E mutant is mainly caused by its inability to bind NAD+ and that Gly-13 is required for efficient base binding of NAD+ to hUGDH.

The construction of a synthetic gene encoding hUGDH will enable us to generate a large number of site-directed mutations at several positions in the coding region. The high level of hUGDH expression as a soluble protein in E. coli will facilitate the purification of large quantities of mutant proteins and will allow a broad range of questions to be addressed relating to the structure and function of hUGDH.


   FOOTNOTES
 
* This work was supported by Grant 03-PJ1-PG3-20900-0047 from the Korea Health 21 R&D Project, Ministry of Health and Welfare, Korea (to S.-W. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY212254 [GenBank] . Back

{ddagger} Present address: Laboratory of Cellular Oncology, Center for Cancer Research, NCI, National Institutes of Health, Bldg. 37, Rm. 4118, Bethesda, MD 20892. Back

§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Ulsan College of Medicine, 388-1 Poongnap-Dong, Songpa-Ku, Seoul 138-736, Korea. Fax: 82-2-3010-4278; E-mail: swcho{at}amc.seoul.kr.

1 The abbreviations used are: UGDH, UDP-glucose dehydrogenase; 2N3NAD+, nicotinamide 2-azidoadenosine dinucleotide; HPLC, high performance liquid chromatography; GAG, glycosaminoglycan; h-, human, as in hUGDH; NMN, nicotinamide mononucleotide. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Mathias Ziegler (Institut für Biochemie, Freie Universität Berlin) for providing NMN adenyltransferase.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Feingold, D. S., and Franzen, J. S. (1981) Trends Biochem. Sci. 6, 103–106
  2. Ordman, A. B., and Kirkwood, S. (1977) J. Biol. Chem. 252, 1320–1326[Abstract/Free Full Text]
  3. Franzen, J. S., Marchetti, P. S., and Feingold, D. S. (1980) Biochemistry 19, 6080–6089[CrossRef][Medline] [Order article via Infotrieve]
  4. Campbell, R. E., Sala, R. F., van de Rijn, I., and Tanner, M. E. (1997) J. Biol. Chem. 272, 3416–3422[Abstract/Free Full Text]
  5. Campbell, R. E., Mosimann, S. C., van De Rijn, I., Tanner, M. E., and Strynadka, N. C. (2000) Biochemistry 39, 7012–7023[CrossRef][Medline] [Order article via Infotrieve]
  6. Stewart, D. C., and Copeland, L. (1998) Plant Physiol. 116, 349–355[Abstract/Free Full Text]
  7. Turner, W., and Botha, F. C. (2002) Arch. Biochem. Biophys. 407, 209–216[CrossRef][Medline] [Order article via Infotrieve]
  8. Toyoda, H., Kinoshita-Toyoda, A., Fox, B., and Selleck, S. B. (2000) J. Biol. Chem. 275, 21856–21861[Abstract/Free Full Text]
  9. Binari, R. C., Staveley, B. E., Johnson, W. A., Godavarti, R., Sasisekharan, R., and Manoukian, A. S. (1997) Development 124, 2623–2632[Abstract]
  10. Benevolenskaya, E. V., Frolov, M. V., and Birchler, J. A. (1998) Mol. Gen. Genet. 260, 131–143[CrossRef][Medline] [Order article via Infotrieve]
  11. Walsh, E. C., and Stainier, D. Y. (2001) Science 293, 1670–1673[Abstract/Free Full Text]
  12. Hempel, J., Perozich, J., Romovacek, H., Hinich, A., Kuo, I., and Feingold, D. S. (1994) Protein Sci. 3, 1074–1080[Medline] [Order article via Infotrieve]
  13. Marcu, O., Stathakis, D. G., and Marsh, J. L. (1999) Cytogenet. Cell Genet. 86, 244–245[CrossRef][Medline] [Order article via Infotrieve]
  14. Bontemps, Y., Maquart, F. X., and Wegrowski, Y. (2000) Biochem. Biophys. Res. Commun. 275, 981–985[CrossRef][Medline] [Order article via Infotrieve]
  15. Spicer, A. P., Kaback, L. A., Smith, T. J., and Seldin, M. F. (1998) J. Biol. Chem. 273, 25117–25124[Abstract/Free Full Text]
  16. Selva, E. M., and Perrimon, N. (2001) Adv. Cancer Res. 83, 67–80[Medline] [Order article via Infotrieve]
  17. Toole, B. P., Wight, T. N., and Tammi, M. I. (2002) J. Biol. Chem. 277, 4593–4596[Free Full Text]
  18. Bassols, A., and Massague, J. (1988) J. Biol. Chem. 263, 3039–3045[Abstract/Free Full Text]
  19. Karlinsky, J. B., Rounds, S., and Farber, H. W. (1992) Circ. Res. 71, 782–789[Abstract/Free Full Text]
  20. Figueroa, J. E., Tao, Z., Sarphie, T. G., Smart, F. W., Glancy, D. L., and Vijayagopal, P. (1999) Atherosclerosis 143, 135–144[CrossRef][Medline] [Order article via Infotrieve]
  21. Iozzo, R. V., and San Antonio, J. D. (2001) J. Clin. Investig. 108, 349–355[CrossRef][Medline] [Order article via Infotrieve]
  22. Lapointe, J., and Labrie, C. (1999) Endocrinology 140, 4486–4493[Abstract/Free Full Text]
  23. Campbell, S., Kim, H., Doukas, M., and Haley, B. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1243–1246[Abstract/Free Full Text]
  24. Kim, H., and Haley, B. (1991) Bioconjugate Chem. 2, 142–147[CrossRef][Medline] [Order article via Infotrieve]
  25. Vaillancourt, R. P., Dhanasekaran, N., and Ruoho, A. E. (1995) Biochem. J. 311, 987–993
  26. Yoon, H.-Y., Cho, E. H., Kwon, H. Y., Choi, S. Y., and Cho, S.-W. (2002) J. Biol. Chem. 277, 41448–41454[Abstract/Free Full Text]
  27. Shoemaker, M. T., and Haley, B. E. (1993) Biochemistry 32, 1883–1890[CrossRef][Medline] [Order article via Infotrieve]
  28. Shoemaker, M. T., and Haley, B. E. (1996) Bioconjugate Chem. 7, 302–310[CrossRef][Medline] [Order article via Infotrieve]
  29. Lee, E.-Y., Yoon, H.-Y., Ahn, J.-Y., Choi, S. Y., and Cho, S.-W. (2001) J. Biol. Chem. 276, 47930–47936[Abstract/Free Full Text]
  30. Yoon, H.-Y., Lee, E.-Y., and Cho, S.-W. (2002) Biochemistry 41, 6817–6823[CrossRef][Medline] [Order article via Infotrieve]
  31. Gunnersen, D. J., and Haley, B. E. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11949–11953[Abstract/Free Full Text]
  32. Olcott, M., Bradly, M., and Haley, B. E. (1994) Biochemistry 33, 11835–11941
  33. Hanahan, D. (1983) J. Mol. Biol. 166, 557–580[Medline] [Order article via Infotrieve]
  34. Studier, F. W., and Moffatt, B. A. (1986) J. Mol. Biol. 189, 113–130[CrossRef][Medline] [Order article via Infotrieve]
  35. An, G., Bendiak, D. S., Mamelak, L. A., and Friesen, J. D. (1981) Nucleic Acids Res. 9, 4163–4172[Abstract/Free Full Text]
  36. Gouy, M., and Gautier, C. (1982) Nucleic Acids Res. 10, 7055–7074[Abstract/Free Full Text]
  37. Stormo, G. D. (1986) in Maximizing Gene Expression (Reznikoff, W., and Gold, L., eds) pp. 195–224, Butterworth, Stoneham, MA
  38. Bradford, M. (1976) Anal. Biochem. 72, 248–254[CrossRef][Medline] [Order article via Infotrieve]
  39. Fersht, A. (1985) Enzyme Structure and Mechanism, pp. 98–120, W. H. Freeman, New York
  40. Sommer, B. J., Barycki, J. J., and Simpson, M. A. (2004) J. Biol. Chem. 279, 23590–23596[Abstract/Free Full Text]
  41. Lind, T., Falk, E., Hjertson, E., Kusche-Gullberg, M., and Lidholt, K. (1999) Glycobiology 9, 595–600[Abstract/Free Full Text]
  42. Wegrowski, Y., Perreau, C., Bontemps, Y., and Maquart, F. X. (1998) Biochem. Biophys. Res. Commun. 250, 206–211[CrossRef][Medline] [Order article via Infotrieve]
  43. Kleiger, G., and Eisenberg, D. (2002) J. Mol. Biol. 323, 69–76[CrossRef][Medline] [Order article via Infotrieve]
  44. Rao, S. T., and Rossmann, M. G. (1973) J. Mol. Biol. 76, 241–256[CrossRef][Medline] [Order article via Infotrieve]
  45. Rossmann, M. G., Moras, D., and Olsen, K. W. (1974) Nature 250, 194–199[CrossRef][Medline] [Order article via Infotrieve]
  46. Rossmann, M. G., and Argos, P. (1976) J. Mol. Biol. 105, 75–95[CrossRef][Medline] [Order article via Infotrieve]
  47. Richardson, J. S. (1981) Adv. Protein Chem. 34, 167–339[Medline] [Order article via Infotrieve]
  48. Dym, O., and Eisenberg, D. (2001) Protein Sci. 10, 1712–1728[CrossRef][Medline] [Order article via Infotrieve]
  49. Wierenga, R. K., Terpstra, P., and Hol, W. G. (1986) J. Mol. Biol. 187, 101–107[CrossRef][Medline] [Order article via Infotrieve]
  50. Rescigno, M., and Perham, R. N. (1994) Biochemistry 33, 5721–5727[CrossRef][Medline] [Order article via Infotrieve]
  51. Nishiya, Y., and Imanaka, T. (1996) Appl. Environ. Microbiol. 62, 2405–2410[Abstract]
  52. Eschenbrenner, M., Chlumsky, L. J., Khanna, P., Strasser, F., and Jorns, M. S. (2001) Biochemistry 40, 5352–5367[CrossRef][Medline] [Order article via Infotrieve]
  53. van Grunsven, E. G., van Berkel, E., Ijlst, L., Vreken, P., de Klerk, J. B., Adamski, J., Lemonde, H., Clayton, P. T., Cuebas, D. A., and Wanders, R. J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2128–2133[Abstract/Free Full Text]
  54. Franzen, J. S., Kuo, I., Eichler, A. J., and Feingold, D. S. (1973) Biochem. Biophys. Res. Commun. 50, 517–523[CrossRef][Medline] [Order article via Infotrieve]
  55. Mak, A. S., and Jones, B. L. (1978) Anal. Biochem. 83, 432–440
  56. Dougherty, B. A., and van de Rijn, I. (1993) J. Biol. Chem. 268, 7118–7124[Abstract/Free Full Text]
  57. Arrecubieta, C., Lopez, R., and Garcia, E. (1994) J. Bacteriol. 10, 6375–6383
  58. Petit, C., Rigg, G. P., Pazzani, C., Smith, A., Sieberth, V., Stevens, M., Boulnois, G., Jann, K., and Roberts, I. S. (1995) Mol. Microbiol. 17, 611–620[CrossRef][Medline] [Order article via Infotrieve]
  59. Tenhaken, R., and Thulke, O. (1996) Plant Physiol. 112, 1127–1134[Abstract]
  60. Johansson, H., Sterky, F., Amini, B., Lundeberg, J., and Kleczkowski, L. A. (2002) Biochim. Biophys. Acta 1576, 53–58[Medline] [Order article via Infotrieve]

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