Biochemical characterization and functional analysis of UDP-glucose dehydrogenase, in the synthesis of biopolymer Ss from Sphingomonas sanxanigenens NX02.

Biopolymer Ss of Sphingomonas sanxanigenens strain NX02 is an sphingan that can be extracted using a small quantity of acid, which is a low cost extraction process. A UDP-glucose dehydrogenase gene (ugdG), related to Ss biosynthesis, was cloned from S. sanxanigenens NX02 and expressed in Escherichia coli. It encoded a 454-residue protein of 48.2 kDa. The deduced amino acid sequence had 77% identity with UDP-glucose dehydrogenase (UgdG) from Sphingomonas sp. KC8, and 73% identity with UgdG from Sphingomonas elodea ATCC31461. Purified recombinant UgdG had maximum activity at 35°C and pH 8.0, with Km values of 0.47 and 0.38 mM for UDP-glucose and NAD+, respectively. Overexpression of the ugdG gene in S. sanxanigenens resulted in increased (14.9 ± 0.5)% Ss production and higher fermentation broth viscosity. Furthermore, the weight-average molecular weight of polymer Ss from the recombinant strain was (5.3 ± 0.16)% higher and the viscosity was (74 ± 0.15)% higher than those from the WT strain at a shear rate of 1 rev/min.

UDP-glucose dehydrogenase modulates proteoglycan synthesis in articular chondrocytes: its possible involvement and regulation in osteoarthritis.

INTRODUCTION: The objective of this study was to investigate the possible role of UDP-glucose dehydrogenase (UGDH) in osteoarthritis (OA) and uncover whether, furthermore how interleukin-1beta (IL-1ß) affects UGDH gene expression. METHODS: UGDH specific siRNAs were applied to determine the role of UGDH in proteoglycan (PG) synthesis in human articular chondrocytes. Protein levels of UGDH and Sp1 in human and rat OA cartilage were detected. Then, human primary chondrocytes were treated with IL-1ß to find out whether and how IL-1ß could regulate the gene expression of UGDH and its trans-regulators, that is Sp1, Sp3 and c-Krox. Finally, p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 and stress-activated protein kinase/c-Jun N-terminal kinase (SAP/JNK) inhibitor SP600125 were used to pick out the pathway that mediated the IL-1ß-modulated PGs synthesis and gene expression of UGDH, Sp1, Sp3 and c-Krox. RESULTS: UGDH specific siRNAs markedly inhibited UGDH mRNA and protein expression, and thus led to an obvious suppression of PGs synthesis in human articular chondrocytes. UGDH protein level in human and rat OA cartilage were much lower than the corresponding controls and negatively correlated to the degree of OA. Decrease in Sp1 protein level was also observed in human and rat OA cartilage respectively. Meanwhile, IL-1ß suppressed UGDH gene expression in human articular chondrocytes in the late phase, which also modulated gene expression of Sp1, Sp3 and c-Krox and increased both Sp3/Sp1 and c-Krox/Sp1 ratio. Moreover, the inhibition of SAP/JNK and p38 MAPK pathways both resulted in an obvious attenuation of the IL-1ß-induced suppression on the UGDH gene expression. CONCLUSIONS: UGDH is essential in the PGs synthesis of articular chondrocytes, while the suppressed expression of UGDH might probably be involved in advanced OA, partly due to the modulation of p38 MAPK and SAP/JNK pathways and its trans-regulators by IL-1ß.

Metabolic engineering of Escherichia coli BL21 for biosynthesis of heparosan, a bioengineered heparin precursor.

As a precursor of bioengineered heparin, heparosan is currently produced from Escherichia coli K5, which is pathogenic bacteria potentially causing urinary tract infection. Thus, it would be advantageous to develop an alternative source of heparosan from a non-pathogeneic strain. In this work we reported the biosynthesis of heparosan via the metabolic engineering of non-pathogenic E. coli BL21 as a production host. Four genes, KfiA, KfiB, KfiC and KfiD, encoding enzymes for the biosynthesis of heparosan in E. coli K5, were cloned into inducible plasmids pETDuet-1 and pRSFDuet-1 and further transformed into E. coli BL21, yielding six recombinant strains as follows: sA, sC, sAC, sABC, sACD and sABCD. The single expression of KfiA (sA) or KfiC (sC) in E. coli BL21 did not produce heparosan, while the co-expression of KfiA and KfiC (sAC) could produce 63 mg/L heparosan in shake flask. The strain sABC and sACD could produce 100 and 120 mg/L heparosan, respectively, indicating that the expression of KfiB or KfiD was beneficial for heparosan production. The strain sABCD could produce 334 mg/L heparosan in shake flask and 652 mg/L heparosan in 3-L batch bioreactor. The heparosan yield was further increased to 1.88 g/L in a dissolved oxygen-stat fed-batch culture in 3-L bioreactor. As revealed by the nuclear magnetic resonance analysis, the chemical structure of heparosan from recombinant E. coli BL21 and E. coli K5 was identical. The weight average molecular weight of heparosan from E. coli K5, sAC, sABC, sACD, and sABCD was 51.67, 39.63, 91.47, 64.51, and 118.30 kDa, respectively. This work provides a viable process for the production of heparosan as a precursor of bioengineered heparin from a safer bacteria strain.

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.

Reduced chondrogenic matrix accumulation by 4-methylumbelliferone reveals the potential for selective targeting of UDP-glucose dehydrogenase.

4-Methylumbelliferone (4-MU) is described as a selective inhibitor of hyaluronan (HA) production. It is thought that 4-MU depletes UDP-glucuronic acid (UDP-GlcUA) substrate for HA synthesis and also suppresses HA-synthase expression. The possibility that 4-MU exerts at least some of its actions via regulation of UDP-glucose dehydrogenase (UGDH), a key enzyme required for both HA and sulphated-glycosaminoglycan (sGAG) production, remains unexplored. We therefore examined the effects of 4-MU on basal and retroviral UGDH-driven HA and sGAG release in cells derived from chick articular cartilage and its influence upon UGDH protein and mRNA expression and HA and sGAG production. We found that 4-MU: i) suppressed UGDH mRNA and protein expression and chondrogenic matrix accumulation in chick limb bud micromass culture, ii) significantly reduced both HA and sGAG production and iii) more selectively reversed the potentiating effects of UGDH overexpression on the production of HA than sGAG. Understanding how GAG synthesis is controlled and the mechanism of 4-MU action may inform its future clinical success.

Androgen-stimulated UDP-glucose dehydrogenase expression limits prostate androgen availability without impacting hyaluronan levels.

UDP-glucose dehydrogenase (UGDH) oxidizes UDP-glucose to UDP-glucuronate, an essential precursor for production of hyaluronan (HA), proteoglycans, and xenobiotic glucuronides. High levels of HA turnover in prostate cancer are correlated with aggressive progression. UGDH expression is high in the normal prostate, although HA accumulation is virtually undetectable. Thus, its normal role in the prostate may be to provide precursors for glucuronosyltransferase enzymes, which inactivate and solubilize androgens by glucuronidation. In this report, we quantified androgen dependence of UGDH, glucuronosyltransferase, and HA synthase expression. Androgen-dependent and androgen-independent human prostate cancer cell lines were used to test the effects of UGDH manipulation on tumor cell growth, HA production, and androgen glucuronidation. Dihydrotestosterone (DHT) increased UGDH expression approximately 2.5-fold in androgen-dependent cells. However, up-regulation of UGDH did not affect HA synthase expression or enhance HA production. Mass spectrometric analysis showed that DHT was converted to a glucuronide, DHT-G, at a 6-fold higher level in androgen-dependent cells relative to androgen-independent cells. The increased solubilization and elimination of DHT corresponded to slower cellular growth kinetics, which could be reversed in androgen-dependent cells by treatment with a UDP-glucuronate scavenger. Collectively, these results suggest that dysregulated expression of UGDH could promote the development of androgen-independent tumor cell growth by increasing available levels of intracellular androgen.

Epstein-Barr virus latent membrane protein 2A upregulates UDP-glucose dehydrogenase gene expression via ERK and PI3K/Akt pathway.

The Epstein-Barr virus latent membrane protein 2A (LMP2A) is frequently detected in nasopharyngeal carcinoma (NPC), a tumour of high metastatic capacity. A recent microarray assay notes that expression of the UDP-glucose dehydrogenase (UGDH) gene, participating in glycosaminoglycan synthesis, shows high correlation with LMP2A levels in NPC biopsies. This study extends the finding and demonstrates that the UGDH transcript and protein quantities, the enzyme activity, and glycosaminoglycan contents increase in LMP2A overexpressed human embryonic kidney 293 (HEK293) cells. The luciferase reporter gene assay demarcates that a region from 630 to 486 bp upstream of the transcription start is critical for LMP2A-mediated gene expression. Moreover, a specificity protein 1 (Sp1) binding site mutation in this region reduces the LMP2A-responsive expression of the UGDH gene. Consistent with these findings, cell motility enhancement by LMP2A diminishes by treating the cells with Sp1-specific inhibitor and small interference RNA (siRNA). Using a signalling pathway-specific inhibitor, it is revealed that phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK), not c-Jun N-terminal kinase (JNK) and p38, participate in LMP2A-induced UGDH expression. This study provides a model for molecular mechanism participating in LMP2A-mediated UGDH gene activation.

Expression of UDP-glucose dehydrogenase reduces cell-wall polysaccharide concentration and increases xylose content in alfalfa stems.

The primary cell-wall matrix of most higher plants is composed of large amounts of uronic acids, primarily D-galacturonic acid residues in the backbone of pectic polysaccharides. Uridine diphosphate (UDP)-glucose dehydrogenase is a key enzyme in the biosynthesis of uronic acids. We produced transgenic alfalfa (Medicago sativa) plants expressing a soybean UDP-glucose dehydrogenase cDNA under the control of two promoters active in alfalfa vascular tissues. In initial greenhouse experiments, enzyme activity in transgenic lines was up to seven-fold greater than in nontransformed control plants; however, field-grown transgenic plants had only a maximum of 1.9-fold more activity than the control. Cell-wall polysaccharide content was lower and Klason lignin content was higher in transgenics compared to the nontransformed control. No significant increase in pectin or uronic acids in the polysaccharide fraction was observed in any line. Xylose increased 15% in most transgenic lines and mannose concentration decreased slightly in all lines. Because of the complexity of pectic polysaccharides and sugar biosynthesis, it may be necessary to manipulate multiple steps in carbohydrate metabolism to alter the pectin content of alfalfa.

The deafness gene dfna5 is crucial for ugdh expression and HA production in the developing ear in zebrafish.

Over 30 genes responsible for human hereditary hearing loss have been identified during the last 10 years. The proteins encoded by these genes play roles in a diverse set of cellular functions ranging from transcriptional regulation to K(+) recycling. In a few cases, the genes are novel and do not give much insight into the cellular or molecular cause for the hearing loss. Among these poorly understood deafness genes is DFNA5. How the truncation of the encoded protein DFNA5 leads to an autosomal dominant form of hearing loss is not clear. In order to understand the biological role of Dfna5, we took a reversegenetic approach in zebrafish. Here we show that morpholino antisense nucleotide knock-down of dfna5 function in zebrafish leads to disorganization of the developing semicircular canals and reduction of pharyngeal cartilage. This phenotype closely resembles previously isolated zebrafish craniofacial mutants including the mutant jekyll. jekyll encodes Ugdh [uridine 5'-diphosphate (UDP)-glucose dehydrogenase], an enzyme that is crucial for production of the extracellular matrix component hyaluronic acid (HA). In dfna5 morphants, expression of ugdh is absent in the developing ear and pharyngeal arches, and HA levels are strongly reduced in the outgrowing protrusions of the developing semicircular canals. Previous studies suggest that HA is essential for differentiating cartilage and directed outgrowth of the epithelial protrusions in the developing ear. We hypothesize that the reduction of HA production leads to uncoordinated outgrowth of the canal columns and impaired facial cartilage differentiation.

Essential role of glycosaminoglycans in Fgf signaling during mouse gastrulation.

In vitro studies have suggested that proteoglycans facilitate signaling by mammalian growth factors, but genetic evidence supporting this role has been lacking. Here, we characterize the ENU-induced mutation lazy mesoderm (lzme), which disrupts the single mouse gene encoding UDP-glucose dehydrogenase (Ugdh), an enzyme required for the synthesis of the glycosaminoglycan (GAG) side chains of proteoglycans. lzme mutants arrest during gastrulation with defects in migration of mesoderm and endoderm, a phenotype similar to that of mutants in the fibroblast growth factor (Fgf) pathway. Analysis of the expression of molecular markers indicates that Fgf signaling is blocked in lzme mutant embryos. In contrast, signaling by the growth factors Nodal and Wnt3, which are also essential during mouse gastrulation, appears to be normal in lzme embryos. The results demonstrate that proteoglycans are required during mouse gastrulation specifically to promote Fgf signaling.

Overexpression of UDP-glucose dehydrogenase in Escherichia coli results in decreased biosynthesis of K5 polysaccharide.

The Escherichia coli K5 capsular polysaccharide (glycosaminoglycan) chains are composed of the repeated disaccharide structure: -GlcAbeta1,4-GlcNAcalpha1,4-(where GlcA is glucuronic acid and GlcNAc is N-acetyl-D-glucosamine). The GlcA, present in most glycosaminoglycans, is donated from UDP-GlcA, which, in turn, is generated from UDP-glucose by the enzyme UDP-glucose dehydrogenase (UDPGDH). The formation of UDP-GlcA is critical for the biosynthesis of glycosaminoglycans. To investigate the role of UDPGDH in glycosaminoglycan biosynthesis, we used K5 polysaccharide biosynthesis as a model. E. coli was transformed with the complete gene cluster for K5 polysaccharide production. Additional transformation with an extra copy of UDPGDH resulted in an approx. 15-fold increase in the in vitro UDPGDH enzyme activity compared with the strain lacking extra UDPGDH. UDP-GlcA levels were increased 3-fold in overexpressing strains. However, metabolic labelling with [14C]glucose showed, unexpectedly, that overexpression of UDPGDH lead to decreased formation of K5 polysaccharide. No significant difference in the K5 polysaccharide chain length was observed between control and overexpressing strains, indicating that the decrease in K5-polysaccharide production most probably was due to synthesis of fewer chains. Our results suggest that K5-polysaccharide biosynthesis is strictly regulated such that increasing the amount of available UDP-GlcA results in diminished K5-polysaccharide production.

Specific protein-1 is a universal regulator of UDP-glucose dehydrogenase expression: its positive involvement in transforming growth factor-beta signaling and inhibition in hypoxia.

UDP-glucose dehydrogenase (UGDH) is a key enzyme of the unique pathway for the synthesis of UDP-glucuronate, the substrate for the numerous glucuronosyl transferases, which act on the synthesis of glycosaminoglycans and glucuronidation reaction of xeno- and endobiotics. Using the bacterial artificial chromosome approach, we have cloned and characterized the human UGDH promoter. The core promoter of -644 nucleotides conferred reporter gene activity in transient transfection assay of a variety of cell types, including MRC5 fibroblasts and the HepG2 hepatoma cell line. The minimal promoter of -100 nucleotides contains a functional inverted TATA box. No consensus CAAT sequence was found up to -2133 nucleotides. The expression of UGDH was up- and down-regulated by transforming growth factor (TGF)-beta and hypoxia, respectively. TGF-beta enhanced the activity of all the deletion constructs, except the minimal promoter. Hypoxia slightly increased the activity of the short promoter-containing constructs but decreased that of the -374 nucleotides and core promoter constructs. The core promoter contained numerous GC-rich sequences for the binding of Sp1 transcription factor. Bisanthracycline, an anti-Sp1 compound, decreased UGDH mRNA expression and inhibited the core promoter constructs activity. Gel mobility shift and supershift assays after TGF-beta stimulation demonstrated an increased DNA binding of the nuclear extract proteins to the two Sp1 sequences located in the -374-bp promoter. By contrast, nuclear extract proteins from hypoxia-treated cells demonstrated a decreased binding of the consensus Sp1 sequence. These results indicate that numerous Sp1 cis-acting sequences of the UGDH core promoter are responsible for up- and down-regulation of the gene after TGF-beta stimulation and in hypoxic conditions, respectively.

Molecular cloning and characterization of a cDNA encoding poplar UDP-glucose dehydrogenase, a key gene of hemicellulose/pectin formation.

Plant UDP-glucose dehydrogenase (UGDH) is an important enzyme in the formation of hemicellulose and pectin, the components of newly formed cell walls. A cDNA clone (Ugdh) corresponding to UGDH was isolated from a cDNA library prepared from cambial zone of poplar (Populus tremula x tremuloides). Within the 1824-nucleotide (nt)-long clone, an open reading frame encoded a protein of 481 amino acids (aa), with a calculated molecular weight of 53.1 kDa. The derived aa sequence showed 90% and 63% identity with UGDHs from soybean and bovine liver, respectively, and had highly conserved aa motifs believed to be of importance for nt binding and catalytic efficiency. In poplar, the Ugdh corresponds to one or two genes, as found by genomic Southern analysis. The gene was expressed predominantly in differentiating xylem and young leaves, with little expression in the phloem zone of the stem. The expression pattern matched that of UGDH protein, as found by immunoblotting. In leaves, the Ugdh expression was upregulated by a short-term feeding with sucrose, sorbitol and polyethylene glycol, and this effect was to some extent mimicked by light exposure. The data suggest that Ugdh is regulated via an osmoticum-dependent pathway, possibly related to the availability of osmotically active carbohydrate precursors to UDP-glucose, a substrate of UGDH.

Enhanced hyaluronan synthesis in the MRL-Fas(lpr) kidney: role of cytokines.

BACKGROUND/AIM: Pathological accumulation of the extracellular matrix component hyaluronan (HA) occurs in the kidney cortex in immune-system mediated tissue injury. The purpose of the present study was to examine the pattern of HA deposition and the mechanisms of HA synthesis in the MRL-Fas(lpr) mouse model of lupus nephritis. METHODS: Kidneys from normal and autoimmune mice were examined for HA content by immunofluorescence staining. Steady state mRNA levels for key enzymes involved in HA synthesis - uridine diphosphate-glucose dehydrogenase (UDPGDH) and HA synthases (HAS) 1, 2 and 3 - were assessed by reverse-transcriptase polymerase chain reaction (RT-PCR). Using cultured mouse tubular epithelial cells, the regulation of the HA production in vitro in response to tumor necrosis factor alpha and interferon gamma was also examined. RESULTS: By immunofluorescence staining, large amounts of HA were detected in the cortical interstitium of MRL-Fas(lpr) mice with autoimmune renal injury, but not in congenic MRL-++ mice. By RT-PCR the presence of transcripts for several genes involved in the synthesis of HA in normal and autoimmune kidneys could be demonstrated, including mRNA for UDPGDH and HAS1 and HAS2, but not for HAS3. Except for HAS2, steady state mRNA levels for these enzymes did not correlate with disease activity. Analyzing a kidney tubular epithelial cell line in vitro, it was found that tumor necrosis factor alpha and interferon gamma, and particularly the combination of these two cytokines, markedly enhanced the synthesis of HA. The expression of HAS2 mRNA was also enhanced in response to cytokine treatment. CONCLUSIONS: HA deposition is prominent in MRL-Fas(lpr) mice with renal disease and could be mediated by local synthesis through HAS1 and HAS2. We hypothesize that the enhanced synthesis of HA could be promoted by proinflammatory cytokines in vivo. The functional significance of HA accumulation in autoimmune renal injury remains to be determined.

cDNA cloning and expression analysis of the human UDPglucose dehydrogenase.

A cDNA clone encoding the human UDPglucose dehydrogenase was isolated from a liver cDNA library. The cDNA is 2,355 bp in length with an open reading frame which is capable of encoding a protein of 494 residues. The predicted primary sequence of the gene product is in good agreement with that of the bovine enzyme determined previously found by means of protein sequencing. Two major transcripts of the UDPglucose dehydrogenase gene with sizes of 2.8 and 2.35 kb, respectively, were observed by Northern analysis. The gene was found to be expressed in a variety of tissues with the highest level in liver, consistent with the physiological function of the enzyme in excretion of endo- and xenobiotics compounds.

Cloning of an enzyme that synthesizes a key nucleotide-sugar precursor of hemicellulose biosynthesis from soybean: UDP-glucose dehydrogenase.

Hemicellulose is a major component of primary plant cell walls. Many of the glycosyl residues found in hemicellulose are derived from the sugar precursor UDP-glucuronic acid, which can be converted into UDP-arabinose, UDP-apiose, UDP-galacturonic acid, and UDP-xylose. The enzyme controlling the biosynthesis of UDP-glucuronic acid, UDP-glucose dehydrogenase (EC 1.1.1.22), was cloned from soybean (Glycine max [L.] Merr.) by an antibody screening procedure. This enzyme is surprisingly homologous to the bovine sequence, which is the only other eukaryotic UDP-glucose dehydrogenase sequence known. The characteristic motifs of the enzyme, the catalytic center, a NAD-binding site, and two proline residues for main chain bends, are conserved within the prokaryotic and eukaryotic sequences. The soybean full-length cDNA clone encodes a protein of 480 amino acids with a predicted size of 52.9 kD. The enzyme is highly expressed in young roots, but lower expression levels were observed in expanding tissues of the epicotyl and in young leaves. The expression pattern of the enzyme in different developmental stages strengthens the argument that UDP-glucose dehydrogenase is a key regulator for the availability of hemicellulose precursors.

Inducible UDP-glucose dehydrogenase from French bean (Phaseolus vulgaris L.) locates to vascular tissue and has alcohol dehydrogenase activity.

UDP-glucose dehydrogenase is responsible for channelling UDP-glucose into the pool of UDP-sugars utilized in the synthesis of wall matrix polysaccharides and glycoproteins. It has been purified to homogeneity from suspension-cultured cells of French bean by a combination of hydrophobic-interaction chromatography, gel filtration and dye-ligand chromatography. The enzyme had a subunit of Mr 40,000. Km values were measured for UDP-glucose as 5.5 +/- 1.4 mM and for NAD+ as 20 +/- 3 microM. It was subject to inhibition by UDP-xylose. UDP-glucose dehydrogenase activity co-purified with alcohol dehydrogenase activity from suspension-cultured cells, elicitor-treated cells and elongating hypocotyls, even when many additional chromatographic steps were employed subsequently. The protein from each source was resolved into virtually identical patterns of isoforms on two-dimensional isoelectric focusing/PAGE. However, a combination of peptide mapping and sequence analysis, gel analysis using activity staining and kinetic analysis suggests that both activities are a function of the same protein. An antibody was raised and used to immunolocalize UDP-glucose dehydrogenase to developing xylem and phloem of French bean hypocotyl. Together with data published previously, these results are consistent with an important role in the regulation of carbon flux into wall matrix polysaccharides.

Overexpression, one-step purification and characterization of UDP-glucose dehydrogenase and UDP-N-acetylglucosamine pyrophosphorylase.

Two enzymes of the Leloir pathway, UDP-GlcNAc pyrophosphorylase and UDP-Glc dehydrogenase, which are involved in the synthesis of activated sugar nucleotides have been cloned, overexpressed in Escherichia coli, and purified to homogeneity in only one step by chelation-affinity chromatography. The gene KfaC of E. coli K5 was thus demonstrated to encode UDP-Glc DH. Some properties of the cloned enzymes, such as stability, pH dependence, and substrate kinetics, were studied in order to facilitate the use of these enzymes in carbohydrate synthesis, especially in the synthesis of hyaluronic acid.

Expression and characterization of UDPGlc dehydrogenase (KfiD), which is encoded in the type-specific region 2 of the Escherichia coli K5 capsule genes.

Region 2 of the Escherichia coli K5 capsule gene cluster contains four genes (kfiA through -D) which encode proteins involved in the synthesis of the K5 polysaccharide. A DNA fragment containing kfiD was amplified by PCR and cloned into the gene fusion vector pGEX-2T to generate a GST-KfiD fusion protein. The fusion protein was isolated from the cytoplasms of IPTG (isopropyl-beta-D-thiogalactopyranoside)-induced recombinant bacteria by affinity chromatography and cleaved with thrombin. The N-terminal amino acid sequence of the cleavage product KfiD' corresponded to the predicted amino acid sequence of KfiD with an N-terminal glycyl-seryl extension from the cleavage site of the fusion protein. Anti-KfiD antibodies obtained with KfiD' were used to isolate the intact KfiD protein from the cytoplasms of E. coli organisms overexpressing the kfiD gene. The fusion protein, its cleavage product (KfiD'), and overexpressed KfiD converted UDPGlc to UDPGlcA. The KfiD protein could thus be characterized as a UDPglucose dehydrogenase.

Nucleotide sequence and expression of UDP-glucose dehydrogenase gene required for the synthesis of xanthan gum in Xanthomonas campestris.

Xanthomonas campestris pv. campestris, producing large amounts of exopolysaccharide xanthan gum, has a mucoid phenotype. Strain SD7 was a non-mucoid mutant deficient in UDP-glucose dehydrogenase. A DNA fragment able to complement the mutation of SD7 was cloned from the parental wild-type strain Xc11. Sequence analysis of the region required for the complementation revealed an open reading frame which could encode a polypeptide of 445 amino acids with a calculated molecular weight of 48,432, a size similar to that of the product produced by maxicell. The amino acid sequence had significant homology to that of the GDP-mannose dehydrogenase from Pseudomonas aeruginosa.