UDP-glucose Dehydrogenase Research Articles

Carbon partitioning in sugarcane (Saccharum species).

Focus has centered on C-partitioning in stems of sugarcane (Saccharum sp.) due to their high-sucrose accumulation features, relevance to other grasses, and rising economic value. Here we review how sugarcane balances between sucrose storage, respiration, and cell wall biosynthesis. The specific topics involve (1) accumulation of exceptionally high sucrose levels (up to over 500 mM), (2) a potential, turgor-sensitive system for partitioning sucrose between storage inside (cytosol and vacuole) and outside cells, (3) mechanisms to prevent back-flow of extracellular sucrose to xylem or phloem, (4) apparent roles of sucrose-P-synthase in fructose retrieval and sucrose re-synthesis, (5) enhanced importance of invertases, and (6) control of C-flux at key points in cell wall biosynthesis (UDP-glucose dehydrogenase) and respiration (ATP- and pyrophosphate-dependent phosphofructokinases). A combination of emerging technologies is rapidly enhancing our understanding of these points and our capacity to shift C-flux between sucrose, cell wall polymers, or other C-sinks.

Possible role of a peach homolog of UDP-glucose dehydrogenase in fruit development

A cDNA fragment homologous to UDP-glucose dehydrogenase (UGD), which is known to be involved in the biosynthesis of pectin and hemicellulose through the conversion of UDP-glucose to UDP-glucuronic acid, was obtained by subtractive hybridization using RNA extracted from the fruits of the commercial and Japanese native peach cultivars with divergent fruit characteristics. The higher expression of the cDNA fragment in the fruits of the commercial cultivars was confirmed by Northern blot analysis. From the nucleotide sequence of the cDNA fragment, cDNA containing the entire coding region was cloned as a peach homolog of UGD, PpUGD1, and its deduced amino acid sequence was found to contain conserved regions important for UGD activity. The level of PpUGD1 mRNA in immature leaves was much higher than that in mature leaves. A change in the PpUGD1 mRNA level during peach fruit development corresponded to changes in the amount of cell wall material and the cell wall uronic acid content. These were greater in the fruits of the commercial cultivars compared with the Japanese native peach cultivars, and the expression of PpUGD1 was higher in the fruits of the commercial cultivars. These findings suggest a possible role of PpUGD1 in cell wall biosynthesis during peach fruit development.

Rossmann-fold motifs can confer multiple functions to metabolic enzymes: RNA binding and ribonuclease activity of a UDP-glucose dehydrogenase

Metabolic enzymes are usually characterized to have one specific function, and this is the case of UDP-glucose dehydrogenase that catalyzes the twofold NAD(+)-dependent oxidation of UDP-glucose into UDP-glucuronic acid. We have determined that this enzyme is also capable of participating in other cellular processes. Here, we report that the bacterial UDP-glucose dehydrogenase (UgdG) from Sphingomonas elodea ATCC 31461, which provides UDP-glucuronic acid for the synthesis of the exopolysaccharide gellan, is not only able to bind RNA but also acts as a ribonuclease. The ribonucleolytic activity occurs independently of the presence of NAD(+) and the RNA binding site does not coincide with the NAD(+) binding region. We have also performed the kinetics of interaction between UgdG and RNA. Moreover, computer analysis reveals that the N- and C-terminal domains of UgdG share structural features with ancient mitochondrial ribonucleases named MAR. MARs are present in lower eukaryotic microorganisms, have a Rossmannoid-fold and belong to the isochorismatase superfamily. This observation reinforces that the Rossmann structural motifs found in NAD(+)-dependent dehydrogenases can have a dual function working as a nucleotide cofactor binding domain and as a ribonuclease.

Detection of genetic association and functional polymorphisms of UGDH affecting milk production trait in Chinese Holstein cattle

BackgroundWe previously localized a quantitative trait locus (QTL) on bovine chromosome 6 affecting milk production traits to a 1.5-Mb region between BMS483 and MNB-209 via genome scanning followed by fine mapping.ResultsTotally 15 genes were mapped within such linkage region through bioinformatic analysis of the cattle-human comparative map and bovine genome assembly. Of them, the UDP-glucose dehydrogenase (UGDH) was suggested as a potential positional candidate gene for milk production traits based on its corresponding physiological and biochemical functions and genetic effects. By sequencing all the coding exons and the untranslated regions in UGDH with pooled DNA of 8 sires represented the separated families detected in our previous studies, a total of ten SNPs were identified and genotyped in 1417 Holstein cows of 8 separation families. Individual SNP-based association analysis revealed 4 significant associations of SNP Ex1-1, SNP Int3-1, SNP Int5-1, and SNP Ex12-3 with milk yield (P < 0.05), and 2 significant associations of SNP Ex1-1 and SNP Ex12-3 with protein yield (P < 0.05). Furthermore, our haplotype-based association analyses indicated that haplotypes G-C-C, formed by SNP Ex12-2-SNP Int11-1-SNP Ex11-1, T-G, formed by SNP Int9-3-SNP Int9-2, and C-C, formed by SNP Int5-1-SNP Int3-1, are significantly associated with protein percentage (F=4.15; P=0.0418) and fat percentage (F=5.18~7.25; P=0.0072~0.0231). Finally, by using an in vitro expression assay, we demonstrated that the A allele of SNP Ex1-1 and T allele of SNP Ex11-1of UGDH significantly decreases the expression of UGDH by 68.0% at the RNA, and 50.1% at the protein level, suggesting that SNP Ex1-1 and Ex11-1 represent two functional polymorphisms affecting expression of UGDH and may partly contributed to the observed association of the gene with milk production traits in our samples.ConclusionsTaken together, our findings strongly indicate that UGDH gene could be involved in genetic variation underlying the QTL for milk production traits.

Production of 3-O-xylosyl quercetin in Escherichia coli

Quercetin, a flavonol aglycone, is one of the most abundant flavonoids with high medicinal value. The bioavailability and pharmacokinetic properties of quercetin are influenced by the type of sugars attached to the molecule. To efficiently diversify the therapeutic uses of quercetin, Escherichia coli was harnessed as a production factory by the installation of various plant and bacterial UDP-xylose sugar biosynthetic genes. The genes encoding for the UDP-xylose pathway enzymes phosphoglucomutase (nfa44530), glucose-1-phosphate uridylyltransferase (galU), UDP-glucose dehydrogenase (calS8), and UDP-glucuronic acid decarboxylase (calS9) were overexpressed in E. coli BL21 (DE3) along with a glycosyltransferase (arGt-3) from Arabidopsis thaliana. Furthermore, E. coli BL21(DE3)/∆pgi, E. coli BL21(DE3)/∆zwf, E. coli BL21(DE3)/∆pgi∆zwf, and E. coli BL21(DE3)/∆pgi∆zwf∆ushA mutants carrying the aforementioned UDP-xylose sugar biosynthetic genes and glycosyltransferase and the galU-integrated E. coli BL21(DE3)/∆pgi host harboring only calS8, calS9, and arGt-3 were constructed to enhance whole-cell bioconversion of exogeneously supplied quercetin into 3-O-xylosyl quercetin. Here, we report the highest production of 3-O-xylosyl quercetin with E. coli BL21 (DE3)/∆pgi∆zwf∆ushA carrying UDP-xylose sugar biosynthetic genes and glycosyltransferase. The maximum concentration of 3-O-xylosyl quercetin achieved was 23.78 mg/L (54.75 μM), representing 54.75 % bioconversion, which was an ~4.8-fold higher bioconversion than that shown by E. coli BL21 (DE3) with the same set of genes when the reaction was carried out in 5-mL culture tubes with 100 μM quercetin under optimized conditions. Bioconversion was further improved by 98 % when the reaction was scaled up in a 3-L fermentor at 36 h.

UDP-glucose Dehydrogenase Polymorphisms from Patients with Congenital Heart Valve Defects Disrupt Enzyme Stability and Quaternary Assembly

Cardiac valve defects are a common congenital heart malformation and a significant clinical problem. Defining molecular factors in cardiac valve development has facilitated identification of underlying causes of valve malformation. Gene disruption in zebrafish revealed a critical role for UDP-glucose dehydrogenase (UGDH) in valve development, so this gene was screened for polymorphisms in a patient population suffering from cardiac valve defects. Two genetic substitutions were identified and predicted to encode missense mutations of arginine 141 to cysteine and glutamate 416 to aspartate, respectively. Using a zebrafish model of defective heart valve formation caused by morpholino oligonucleotide knockdown of UGDH, transcripts encoding the UGDH R141C or E416D mutant enzymes were unable to restore cardiac valve formation and could only partially rescue cardiac edema. Characterization of the mutant recombinant enzymes purified from Escherichia coli revealed modest alterations in the enzymatic activity of the mutants and a significant reduction in the half-life of enzyme activity at 37 °C. This reduction in activity could be propagated to the wild-type enzyme in a 1:1 mixed reaction. Furthermore, the quaternary structure of both mutants, normally hexameric, was destabilized to favor the dimeric species, and the intrinsic thermal stability of the R141C mutant was highly compromised. The results are consistent with the reduced function of both missense mutations significantly reducing the ability of UGDH to provide precursors for cardiac cushion formation, which is essential to subsequent valve formation. The identification of these polymorphisms in patient populations will help identify families genetically at risk for valve defects.

Structure of a UDP-glucose dehydrogenase from the hyperthermophilic archaeon Pyrobaculum islandicum.

The crystal structure of an extremely thermostable UDP-glucose dehydrogenase (UDP-GDH) from the hyperthermophilic archaeon Pyrobaculum islandicum was determined at a resolution of 2.0 Å. The overall fold was comprised of an N-terminal NAD(+) dinucleotide binding domain and a C-terminal UDP-sugar binding domain connected by a long α-helix, and the main-chain coordinates of the enzyme were similar to those of previously studied UDP-GDHs, including the enzymes from Burkholderia cepacia, Streptococcus pyogenes and Klebsiella pneumoniae. However, the sizes of several surface loops in P. islandicum UDP-GDH were much smaller than the corresponding loops in B. cepacia UDP-GDH but were comparable to those of the S. pyogenes and K. pneumoniae enzymes. Structural comparison revealed that the presence of extensive intersubunit hydrophobic interactions, as well as the formation of an intersubunit aromatic pair network, is likely to be the main factor contributing to the hyperthermostability of P. islandicum UDP-GDH.

Production of intracellular heparosan and derived oligosaccharides by lyase expression in metabolically engineered E. coli K-12

The cluster of genes of capsular K5 heparosan is composed of three regions, involved in the synthesis and the exportation of the polysaccharide. The region 2 possesses all the necessary genes involved in the synthesis of heparosan, namely kfiA, encoding alpha-4-N-acetylglucosaminyltransferase, kfiD, encoding β-3-glucuronyl transferase, kfiC, encoding UDP-glucose dehydrogenase (UDP-glucuronic acid synthesis), and kfiB encoding a protein of unknown function. The cloning and expression of kfiADCB into Escherichia coli K-12 were found to be sufficient for the production of heparosan, which accumulates in the cells due to a lack of the exporting system. The concentration of recombinant heparosan reached one gram per liter under fed-batch cultivation. The cytoplasmic localization of heparosan inside the bacteria allowed subsequent enzymatic modifications such as a partial degradation with K5 lyase when expressed intracellularly. Under these conditions, the production of DP 2–10 oligosaccharides occurred intracellularly, at a concentration similar to that of recombinant intracellular heparosan.

Cell Wall Ingrowths in Nematode Induced Syncytia Require UGD2 and UGD3

The cyst nematode Heterodera schachtii infects roots of Arabidopsis plants and establishes feeding sites called syncytia, which are the only nutrient source for nematodes. Development of syncytia is accompanied by changes in cell wall structures including the development of cell wall ingrowths. UDP-glucuronic acid is a precursor of several cell wall polysaccharides and can be produced by UDP-glucose dehydrogenase through oxidation of UDP-glucose. Four genes in Arabidopsis encode this enzyme. Promoter::GUS analysis revealed that UGD2 and UGD3 were expressed in syncytia as early as 1 dpi while expression of UGD1 and UGD4 could only be detected starting at 2 dpi. Infection assays showed no differences between Δugd1 and Δugd4 single mutants and wild type plants concerning numbers of males and females and the size of syncytia and cysts. On single mutants of Δugd2 and Δugd3, however, less and smaller females, and smaller syncytia formed compared to wild type plants. The double mutant ΔΔugd23 had a stronger effect than the single mutants. These data indicate that UGD2 and UGD3 but not UGD1 and UGD4 are important for syncytium development. We therefore studied the ultrastructure of syncytia in the ΔΔugd23 double mutant. Syncytia contained an electron translucent cytoplasm with degenerated cellular organelles and numerous small vacuoles instead of the dense cytoplasm as in syncytia developing in wild type roots. Typical cell wall ingrowths were missing in the ΔΔugd23 double mutant. Therefore we conclude that UGD2 and UGD3 are needed for the production of cell wall ingrowths in syncytia and that their lack leads to a reduced host suitability for H. schachtii resulting in smaller syncytia, lower number of developing nematodes, and smaller females.

Glycosaminoglycan-dependent restriction of FGF diffusion is necessary for lacrimal gland development

Glycosaminoglycans (GAGs) play a central role in embryonic development by regulating the movement and signaling of morphogens. We have previously demonstrated that GAGs are the co-receptors for Fgf10 signaling in the lacrimal gland epithelium, but their function in the Fgf10-producing periocular mesenchyme is still poorly understood. In this study, we have generated a mesenchymal ablation of UDP-glucose dehydrogenase (Ugdh), an essential biosynthetic enzyme for GAGs. Although Fgf10 RNA is expressed normally in the periocular mesenchyme, Ugdh mutation leads to excessive dispersion of Fgf10 protein, which fails to elicit an FGF signaling response or budding morphogenesis in the presumptive lacrimal gland epithelium. This is supported by genetic rescue experiments in which the Ugdh lacrimal gland defect is ameliorated by constitutive Ras activation in the epithelium but not in the mesenchyme. We further show that lacrimal gland development requires the mesenchymal expression of the heparan sulfate N-sulfation genes Ndst1 and Ndst2 but not the 6-O and 2-O-sulfation genes Hs6st1, Hs6st2 and Hs2st. Taken together, these results demonstrate that mesenchymal GAG controls lacrimal gland induction by restricting the diffusion of Fgf10.

An Edwardsiella tarda mutant lacking UDP-glucose dehydrogenase shows pleiotropic phenotypes, attenuated virulence, and potential as a vaccine candidate

Edwarsiella tarda is highly resistant to the action of cationic antimicrobial peptides (CAMPs). However, the mechanism underlying CAMP resistance is not clear. The enzyme UDP-glucose dehydrogenase (Ugd) that converts UDP-glucose into UDP-glucuronic acid may be important for this resistance. In this study, a ugd gene was identified in E. tarda and its functional role was analyzed using an in-frame deletion mutant Δugd and the complemented strain ugd+. The lipopolysaccharide (LPS) produced by Δugd consisted of a truncated core oligosaccharide (OS) with no O-antigen attached. The ugd mutant was extremely sensitive to CAMPs, presumably because of alterations in LPS structure. The mutant also exhibited enhanced autoaggregation and biofilm formation and reduced hemolytic activity. Using different infection models we found that Δugd was impaired in survival within macrophages and displayed significantly attenuated virulence and an impaired ability to persist within the host. The expression of ugd was induced by polymyxin B and under the control of PhoP and RcsB, two response regulators of the bacterial two-component systems that we identified previously. Moreover, vaccination of turbot (Scophthalmus maximus) with Δugd by intraperitoneal injection elicited significant protection against the wild-type E. tarda strain, suggesting that Δugd may be promising as a potential vaccine candidate against edwardsiellosis.

Transcription analysis of hyaluronan biosynthesis genes in Streptococcus zooepidemicus and metabolically engineered Lactococcus lactis

The has operon genes in the hyaluronan (HA) producer, Streptococcus zooepidemicus, encode for some of the critical enzymes in the HA biosynthetic pathway. Heterologous expression of different combinations of multiple has genes has resulted in increasing HA production to varying degrees in different recombinant strains. In this work, a recombinant Lactococcus lactis strain (SJR6) was constructed, with insertion of three has operon genes (hasABD) from S. zooepidemicus. The SJR6 strain was found to be a better HA producer than two previously constructed recombinant L. lactis strains (SJR2 and SJR3), containing hasAB and hasABC genes, respectively, but exhibited lower HA production than the native HA producer S. zooepidemicus. To understand the differences in HA yield between the various strains, transcriptions of the HA biosynthesis genes (has genes and their homologues) were compared at different phases of exponential growth of the L. lactis and S. zooepidemicus cultures. The mRNA levels of all the heterologous has genes were expectedly far higher than their corresponding homologues in the L. lactis strains. The relative mRNA level of the hasB-homologue, viz. ugd (encoding UDP-glucose dehydrogenase), was found to be much lower than that of other homologues, corroborating earlier reports which indicate tight transcriptional regulation of the ugd gene in L. lactis. Interestingly, all the has gene homologues were found to be up-regulated in all the recombinant L. lactis strains, when compared with the corresponding genes in the untransformed strain, L. lactis NZ9000. A transcription analysis of S. zooepidemicus cultures revealed that the has operon was down-regulated in the mid-exponential growth phase in comparison to the early- and late-exponential growth phases. The transcription analyses in this study have provided insights for the design of recombinant strains with higher HA productivity.

Down-regulation of UDP-glucuronic Acid Biosynthesis Leads to Swollen Plant Cell Walls and Severe Developmental Defects Associated with Changes in Pectic Polysaccharides

UDP-glucose dehydrogenase (UGD) plays a key role in the nucleotide sugar biosynthetic pathway, as its product UDP-glucuronic acid is the common precursor for arabinose, xylose, galacturonic acid, and apiose residues found in the cell wall. In this study we characterize an Arabidopsis thaliana double mutant ugd2,3 that lacks two of the four UGD isoforms. This mutant was obtained from a cross of ugd2 and ugd3 single mutants, which do not show phenotypical differences compared with the WT. In contrast, ugd2,3 has a strong dwarfed phenotype and often develops seedlings with severe root defects suggesting that the UGD2 and UGD3 isoforms act in concert. Differences in its cell wall composition in comparison to the WT were determined using biochemical methods indicating a significant reduction in arabinose, xylose, apiose, and galacturonic acid residues. Xyloglucan is less substituted with xylose, and pectins have a reduced amount of arabinan side chains. In particular, the amount of the apiose containing side chains A and B of rhamnogalacturonan II is strongly reduced, resulting in a swollen cell wall. The alternative pathway to UDP-glucuronic acid with the key enzyme myo-inositol oxygenase is not up-regulated in ugd2,3. The pathway also does not complement the ugd2,3 mutation, likely because the supply of myo-inositol is limited. Taken together, the presented data underline the importance of UDP GlcA for plant primary cell wall formation.

Identification and Characterization of UDP-Glucose Dehydrogenase from the Hyperthermophilic Archaon,Pyrobaculum islandicum

A gene encoding a UDP-glucose dehydrogenase homologue was identified in the hyperthermophilic archaeon, Pyrobaculum islandicum. This gene was expressed in Escherichia coli, and the product was purified and characterized. The expressed enzyme is the most thermostable UDP-glucose dehydrogenase so far described, with a half-life of 10 min at 90 °C. The enzyme retained its full activity after incubating in a pH range of 5.0-10.0 for 10 min at 80 °C. The temperature dependence of the kinetic parameters for this enzyme was examined at 37-70 °C. A decrease in K(m)s for UDP-glucose and NAD was observed with decreasing temperature. This resulted in the enzyme still retaining high catalytic efficiency (V(max)/K(m)) for the substrate and cofactor, even at 37 °C. These characteristics make the enzyme potentially useful for its application at a much lower temperature such as 37 °C than the optimum growth temperature of 100 °C for P. islandicum.

Structural Basis of Cooperativity in Human UDP-Glucose Dehydrogenase

BackgroundUDP-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.MethodologyPreviously, 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.ConclusionIn 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.

Generation and analysis of expressed sequence tags (ESTs) from cambium tissue cDNA libraries of contrasting genotypes of Eucalyptus globulus Labill

In the present study, reported the generation and analysis of ESTs of cDNA libraries from cambium tissue of secondary xylem obtained of two genotypes of Eucalyptus globulus contrasting in wood density and pulp yield. The sequences were blasted and annotated to compare with genome and ESTs database of other plant species. The goal of study was to determinate wich genes was differentially expressed and compare levels of transcript, validated by qRT-PCR, in each genotype involved in wood formation, to explain the differences founding in wood traits of contrasting genotypes.Sequences obtained was 450,000 ESTs of which approximately 21,000 sequences showed homology with genes of different vascular plants, mainly Vitis vinifera, Populus sp, Eucalyptus sp, Ricinus sp . Moreover, it was determined that 265 genes differentially expressed in both genotypes, and 41 genes were directly involved in wood formation process (xylogenesis). Of the 41 differentially expressed genes could be determined that mainly correspond genes involved in lignin biosynthesis pathway which HCT, C3H, CAD, PAL, COMT and F5H and lignin polymerization like laccase and peroxidase. Otherwise we found genes involved in carbohydrate biosynthesis (cellulose and hemicellulose) among which Sussy, UDP glucose dehydrogenase, UDP-mannose dehydrogenase and β-xylosidase. Also we described genes involved in morphological characteristics of fiber such FLA, XTH and transcription factors such MYB and LIM related to fiber length, microfibrillar angle and extensibility of cell wall. Finally some of these genes were validated by the qRT-PCR technique to determine the level of transcripts in each genotype.

Divergent Sp1 Protein Levels May Underlie Differential Expression of UDP-Glucose Dehydrogenase by Fibroblasts: ROLE IN SUSCEPTIBILITY TO ORBITAL GRAVES DISEASE

UDP-glucose dehydrogenase (UGDH) catalyzes the formation of UDP-glucuronate. Glucuronate represents an integral component of the glycosaminoglycan, hyaluronan, which accumulates in orbital Graves disease. Here we report that orbital fibroblasts express higher levels of UGDH than do those from skin. This is a consequence of greater UGDH gene promoter activity and more abundant steady-state UGDH mRNA. Six Sp1 sites located in the proximal 550 bp of the UGDH gene promoter appear to determine basal promoter activity, as does a previously unrecognized 49-bp sequence spanning -1436 nucleotides (nt) and -1388 nt that negatively affects activity. Nuclear Sp1 protein is more abundant in orbital fibroblasts, and its binding to specific sites on DNA is greater than that in dermal fibroblasts. Mutating each of these Sp1 sites in a UGDH gene promoter fragment, extending from -1387 to +71 nt and fused to a luciferase reporter, results in divergent activities when transfected in orbital and dermal fibroblasts. Reducing Sp1 attenuated UGDH gene promoter activity, lowered steady-state UGDH mRNA levels, and reduced UGDH enzyme activity. Targeting Sp1 and UGDH with specific siRNAs also lowered hyaluronan synthase-1 (HAS-1) and HAS-2 levels and reduced hyaluronan accumulation in orbital fibroblasts. These findings suggest that orbital fibroblasts express high levels of UGDH in an anatomic-specific manner, apparently the result of greater constitutive Sp1. These high UGDH levels may underlie susceptibility of the orbit to localized overproduction of hyaluronan in Graves disease.

Role of Packing Defects in the Evolution of Allostery and Induced Fit in Human UDP-Glucose Dehydrogenase

Allosteric feedback inhibition is the mechanism by which metabolic end products regulate their own biosynthesis by binding to an upstream enzyme. Despite its importance in controlling metabolism, there are relatively few allosteric mechanisms understood in detail. This is because allostery does not have an identifiable structural motif, making the discovery of new allosteric enzymes a difficult process. The lack of a conserved motif implies that the evolution of each allosteric mechanism is unique. Here we describe an atypical allosteric mechanism in human UDP-α-d-glucose 6-dehydrogenase (hUGDH) based on an easily acquired and identifiable structural attribute: packing defects in the protein core. In contrast to classic allostery, the active and allosteric sites in hUGDH are present as a single, bifunctional site. Using two new crystal structures, we show that binding of the feedback inhibitor, UDP-α-d-xylose, elicits a distinct induced-fit response; a buried loop translates ∼4 Å along and rotates ∼180° about the main chain axis, requiring surrounding side chains to repack. This allosteric transition is facilitated by packing defects, which negate the steric conformational restraints normally imposed by the protein core. Sedimentation velocity studies show that this repacking favors the formation of an inactive hexameric complex with unusual symmetry. We present evidence that hUGDH and the unrelated enzyme dCTP deaminase have converged to very similar atypical allosteric mechanisms using the same adaptive strategy, the selection for packing defects. Thus, the selection for packing defects is a robust mechanism for the evolution of allostery and induced fit.

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

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.

Conformational change upon product binding to Klebsiella pneumoniae UDP-glucose dehydrogenase: A possible inhibition mechanism for the key enzyme in polymyxin resistance

Cationic modification of lipid A with 4-amino-4-deoxy-L-arabinopyranose (L-Ara4N) allows the pathogen Klebsiella pneumoniae to resist the antibiotic polymyxin and other cationic antimicrobial peptides. UDP-glucose dehydrogenase (Ugd) catalyzes the NAD⁺-dependent twofold oxidation of UDP-glucose (UPG) to produce UDP-glucuronic acid (UGA), a requisite precursor in the biosynthesis of L-Ara4N and bacterial exopolysaccharides. Here we report five crystal structures of K. pneumoniae Ugd (KpUgd) in its apo form, in complex with UPG, UPG/NADH, two UGA molecules, and finally with a C-terminal His₆-tag. The UGA-complex structure differs from the others by a 14° rotation of the N-terminal domain toward the C-terminal domain, and represents a closed enzyme conformation. It also reveals that the second UGA molecule binds to a pre-existing positively charged surface patch away from the active site. The enzyme is thus inactivated by moving the catalytically important residues C253, K256 and D257 from their original positions. Kinetic data also suggest that KpUgd has multiple binding sites for UPG, and that UGA is a competitive inhibitor. The conformational changes triggered by UGA binding to the allosteric site can be exploited in designing potent inhibitors.