Udpglucose dehydrogenase Kinetics and their mechanistic implications

Ordman, Alfred B.; Kirkwood, S.

doi:10.1016/0005-2744(77)90133-4

Cited by 39 publications

(43 citation statements)

References 27 publications

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“…The UGD cofactor lies at an accessible cleft near the carboxy ends of strands ␤1, ␤4, and ␤5 in an extended conformation similar to several NAD ϩ -enzyme complexes (3), leaving most of its atoms exposed to the solvent. This is in agreement with a "Bi-UniUni-Bi ping-pong" mechanism, whereby the alcohol UDPGlc binds first before the acid UDP-GlcA is released (7,23,40). The cofactor molecules then readily access the enzyme, being reduced and released into the medium without requirement of major conformational changes of the enzyme molecule.…”

Section: Resultssupporting

confidence: 84%

“…The resulting oxyanion is again stabilized by electrostatic interactions with the nearby ε-amino group of Lys214 (6) pro-S hydride (6, 45) into a freshly docked NAD ϩ , and the global oxidation of C-6Ј is accomplished with the resulting thioester. The final catalytic step, the irreversible hydrolysis of the thioester (40,45), involves a second nucleophilic attack to C-6Ј, most possibly again by a water/hydroxyl activated by Asp274, which will lead to the disruption of the thioester sulfur-carbon bond and will produce the final carboxylic group at C-6Ј. This is the unique irreversible and rate-limiting step of the overall reaction (40) and requires the stabilization of the departing thiolate.…”

Section: Vol 193 2011 Bcec Structure and The Final Step Of The Ugd mentioning

confidence: 99%

“…The final catalytic step, the irreversible hydrolysis of the thioester (40,45), involves a second nucleophilic attack to C-6Ј, most possibly again by a water/hydroxyl activated by Asp274, which will lead to the disruption of the thioester sulfur-carbon bond and will produce the final carboxylic group at C-6Ј. This is the unique irreversible and rate-limiting step of the overall reaction (40) and requires the stabilization of the departing thiolate. However, as the ε-amino group of Lys214 is at the opposite side relative to C-6Ј (Fig.…”

Section: Vol 193 2011 Bcec Structure and The Final Step Of The Ugd mentioning

confidence: 99%

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Structure of Burkholderia cepacia UDP-Glucose Dehydrogenase (UGD) BceC and Role of Tyr10 in Final Hydrolysis of UGD Thioester Intermediate

et al. 2011

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Members of the Burkholderia cepacia complex (BCC) are serious respiratory pathogens in immunocompromised individuals and in patients with cystic fibrosis (CF). They are exceptionally resistant to many antimicrobial agents and have the capacity to spread between patients, leading to a decline in lung function and necrotizing pneumonia. BCC members often express a mucoid phenotype associated with the secretion of the exopolysaccharide (EPS) cepacian. There is much evidence supporting the fact that cepacian is a major virulence factor of BCC. UDP-glucose dehydrogenase (UGD) is responsible for the NAD-dependent 2-fold oxidation of UDP-glucose (UDP-Glc) to UDP-glucuronic acid (UDP-GlcA), which is a key step in cepacian biosynthesis. Here, we report the structure of BceC, determined at 1.75-Å resolution. Mutagenic studies were performed on the active sites of UGDs, and together with the crystallographic structures, they elucidate the molecular mechanism of this family of sugar nucleotide-modifying enzymes. Superposition with the structures of human and other bacterial UGDs showed an active site with high structural homology. This family contains a strictly conserved tyrosine residue (Y10 in BceC; shown in italics) within the glycine-rich motif (GXGYXG) of its N-terminal Rossmann-like domain. We constructed several BceC Y10 mutants, revealing only residual dehydrogenase activity and thus highlighting the importance of this conserved residue in the catalytic activity of BceC. Based on the literature of the UGD/GMD nucleotide sugar 6-dehydrogenase family and the kinetic and structural data we obtained for BceC, we determined Y10 as a key catalytic residue in a UGD rate-determining step, the final hydrolysis of the enzymatic thioester intermediate.

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Section: Resultssupporting

confidence: 84%

Section: Vol 193 2011 Bcec Structure and The Final Step Of The Ugd mentioning

confidence: 99%

Section: Vol 193 2011 Bcec Structure and The Final Step Of The Ugd mentioning

confidence: 99%

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Structure of Burkholderia cepacia UDP-Glucose Dehydrogenase (UGD) BceC and Role of Tyr10 in Final Hydrolysis of UGD Thioester Intermediate

et al. 2011

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“…NAD' reduction in the presence of UDPG increased linearly until the A,4o reached a value of 1. The rate of NADH formation decreased subsequently, possibly due to an inhibitory effect of the produced NADH (Ordman & Kirkwood, 1977). Indeed, addition of 40 pM NADH to the reaction mixture led to a loss of over 90% of the UDPG-DH activity.…”

Section: Udpg-dh Assay In B Subtilismentioning

confidence: 99%

Assay for UDPglucose 6-dehydrogenase in phosphate-starved cells: gene tuaD of Bacillus subtilis 168 encodes the UDPglucose 6-dehydrogenase involved in teichuronic acid synthesis

Pagni¹,

Lazarević²,

Soldo³

et al. 1999

Microbiology

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A novel assay permitting the detection of UDPglucose 6-dehydrogenase activity in cell-free extracts obtained from phosphate-starved cultures of Bacillus subtilis is described. The critical step, the separation of phosphatestarvation-induced exo-enzymes, phosphatases and phosphodiesterases from the cytoplasmic fraction containing the UDPglucose dehydrogenase, was achieved by protoplasting and removal of the periplasmic fraction by protoplast washing. Using this method, the following were unambiguously demonstrated: (i) the presence in the cytoplasm of an enzymic activity oxidizing UDPglucose to UDPglucuronic acid, and (ii) that detection of the activity in whole-cell-free extracts is prevented by the presence of With this method, several B. subtilis 168 mutants unable to synthesize teichuronic acid were examined. Strains inactivated in gene tuaD, whose product shares homology with UDPglucose 6-dehydrogenase and GDPmannose 6-dehydrogenase from other organisms, were shown to lack UDPglucose 6-dehydrogenase activity. Anion exchange chromatography revealed that mutants deficient in tuaD lacked a cytoplasmic UDPglucuronate pool.periplasmic' enzymes catalysing the degradation of the sugar nucleotides.

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“…6 UGDH catalyzes the conversion of UDP-glucose to UDPglucuronate, and the bovine enzyme and the enzyme from Streptococcus pyogenes have been extensively studied. [7][8][9][10][11][12][13] In the proposed catalytic mechanism, NAD + is reduced by accepting two electrons from the C-6' pro-R hydride of UDP-glucose to form NADH and an aldehyde intermediate. 9,13 The second NAD + is reduced by accepting two electrons from the hydride of the thiohemiacetal intermediate generated by the addition of a cysteine thiol to the aldehyde intermediate.…”

Section: Introductionmentioning

confidence: 99%

Cloning and Characterization of UDP-glucose Dehydrogenase from Sphingomonas chungbukensis DJ77

Yoon¹,

Park²,

Park³

et al. 2009

Bulletin of the Korean Chemical Society

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Sphingomonas chungbukensis DJ77 has the ability to produce large quantities of an extracellular polysaccharide that can be used as a gelling agent in the food and pharmaceutical industries. We identified, cloned and expressed the UDP-glucose dehydrogenase gene of S. chungbukensis DJ77, and characterized the resulting protein. The purified UDP-glucose dehydrogenase (UGDH), which catalyzes the reversible conversion of UDP-glucose to UDPglucuronic acid, formed a homodimer and the mass of the monomer was estimated to be 46 kDa. Kinetic analysis at the optimal pH of 8.5 indicated that the Km and Vmax for UDP-glucose were 0.18 mM and 1.59 mM/min/mg, respectively. Inhibition assays showed that UDP-glucuronic acid strongly inhibits UGDH. Site-directed mutagenesis was performed on Gly9, Gly12 Thr127, Cys264, and Lys267. Substitutions of Cys264 with Ala and of Lys267 with Asp resulted in complete loss of enzymatic activity, suggesting that Cys264 and Lys267 are essential for the catalytic activity of UGDH.

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Udpglucose dehydrogenase Kinetics and their mechanistic implications

Cited by 39 publications

References 27 publications

Structure of Burkholderia cepacia UDP-Glucose Dehydrogenase (UGD) BceC and Role of Tyr10 in Final Hydrolysis of UGD Thioester Intermediate

Structure of Burkholderia cepacia UDP-Glucose Dehydrogenase (UGD) BceC and Role of Tyr10 in Final Hydrolysis of UGD Thioester Intermediate

Assay for UDPglucose 6-dehydrogenase in phosphate-starved cells: gene tuaD of Bacillus subtilis 168 encodes the UDPglucose 6-dehydrogenase involved in teichuronic acid synthesis

Cloning and Characterization of UDP-glucose Dehydrogenase from Sphingomonas chungbukensis DJ77

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