Ferredoxin-dependent glutamate synthase moonlights in plant sulfolipid biosynthesis by forming a complex with SQD1

UDP-sulfoquinovose synthase, SQD1, catalyzes the transfer of sulfite to UDP-glucose giving rise to UDP-sulfoquinovose, which is the head group donor for the biosynthesis of the plant sulfolipid sulfoquinovosyldiacylglyerol. The native SQD1 enzyme of spinach exists as a 250 kDa heteroprotein complex with much higher affinity for the substrate sulfite than the recombinant SQD1 protein itself. The SQD1 protein co-purified with nine proteins. Likely binding partners included rubisco activase, HSP70, and ferredoxin-dependent glutamate synthase (FdGOGAT). While the first two proteins are known to interact with many other proteins, the identification of FdGOGAT was most intriguing because this 160kDa protein contains an FMN cofactor known to bind sulfite in vitro. Using different constructs expressing recombinant forms of the multidomain protein FdGOGAT, it was demonstrated that the FMN-binding domain of FdGOGAT is essential for specific binding of the protein to SQD1. A model suggests that FdGOGAT could channel sulfite to SQD1.     

Recombinant Arabidopsis SQD1 converts udp-glucose and sulfite to the sulfolipid head group precursor UDP-sulfoquinovose in vitro

The sulfolipid sulfoquinovosyldiacylglycerol is a component of plant photosynthetic membranes and represents one of the few naturally occurring sulfonic acids with detergent properties. Sulfolipid biosynthesis involves the transfer of sulfoquinovose, a 6-deoxy-6-sulfoglucose, from UDP-sulfoquinovose to diacylglycerol. The formation of the sulfonic acid precursor, UDP-sulfoquinovose, from UDP-glucose and a sulfur donor is proposed to be catalyzed by the bacterial SQDB proteins or the orthologous plant SQD1 proteins. To investigate the underlying enzymatic mechanism and to elucidate the de novo synthesis of sulfonic acids in biological systems, we developed an in vitro assay for the recombinant SQD1 protein from Arabidopsis thaliana. Among different possible sulfur donors tested, sulfite led to the formation of UDP-sulfoquinovose in the presence of UDP-glucose and SQD1. An SQD1 T145A mutant showed greatly reduced activity. The UDP-sulfoquinovose formed in this assay was identified by co-chromatography with standards and served as substrate for the sulfolipid synthase associated with spinach chloroplast membranes. Approximate K(m) values of 150 microm for UDP-glucose and 10 microm for sulfite were established for SQD1. Based on our results, we propose that SQD1 catalyzes the formation of UDP-sulfoquinovose from UDP-glucose and sulfite, derived from the sulfate reduction pathway in the chloroplast.     

A UDP-glucose:isoflavone 7-O-glucosyltransferase from the roots of soybean (glycine max) seedlings. Purification, gene cloning, phylogenetics, and an implication for an alternative strategy of enzyme catalysis

Isoflavones, a class of flavonoids, play very important roles in plant-microbe interactions in certain legumes such as soybeans (Glycine max L. Merr.). G. max UDP-glucose:isoflavone 7-O-glucosyltransferase (GmIF7GT) is a key enzyme in the synthesis of isoflavone conjugates, which accumulate in large amounts in vacuoles and serve as an isoflavonoid pool that allows for interaction with microorganisms. In this study, the 14,000-fold purification of GmIF7GT from the roots of G. max seedlings was accomplished. The purified enzyme is a monomeric protein of 46 kDa, catalyzing regiospecific glucosyl transfer from UDP-glucose to isoflavones to produce isoflavone 7-O-beta-D-glucosides (k(cat) = 0.74 s(-1), K(m) for genistein = 3.6 microM, and K(m) for UDP-glucose = 190 microM). The GmIF7GT cDNA was isolated based on the amino acid sequence of the purified enzyme. Phylogenetic analysis showed that GmIF7GT is a novel member of glycosyltransferase family 1 and is distantly related to Glycyrrhiza echinata UDP-glucose:isoflavonoid 7-O-glucosyltransferase. The purified enzyme was unexpectedly devoid of the N-terminal 49-residue segment and thus lacks the histidine residue corresponding to the proposed catalytic residue of glycosyltransferases from Medicago truncatula (UGT71G1) and Vitis vinifera (VvGT1). The results of kinetic studies of site-directed mutants of GmIF7GT showed that both His-15 and Asp-125, which correspond to the catalytic residues of UGT71G1 and VvGT1, are not important for GmIF7GT activity. The results also suggest that an acidic residue at position 392 is very important for primary catalysis of GmIF7GT. These results led to the proposal that GmIF7GT utilizes a strategy of catalysis that is distinct from those proposed for UGT71G1 and VvGT1.     

Purification, cloning, and expression of a pathogen inducible UDP-glucose:Salicylic acid glucosyltransferase from tobacco

Salicylic acid (SA) plays an important role in plant disease resistance. Inoculation of tobacco leaves with incompatible pathogens triggers the biosynthesis of SA which accumulates primarily as the SA 2-O-beta-D-glucoside (SAG) and glucosyl salicylate (GS). The tobacco UDP-glucose:salicylic acid glucosyltransferase (SA GTase) capable of forming both SAG and GS was purified, characterized, and partially sequenced. It has an apparent molecular mass of 48 kDa, a pH optimum of 7.0, and an isoelectric point at pH 4.4. UDP-glucose was the sole sugar donor for the enzyme. However, SA and several phenolics served as glucose acceptors. The apparent K(m) values for UDP-glucose and SA were 0.27 and 1-2 mM, respectively. Zn(2+) and UDP inhibited its activity. The corresponding cDNA clone which encoded a protein of 459 amino acids was isolated from an SA-induced tobacco cDNA library and overexpressed in Escherichia coli. The recombinant protein catalyzed the formation of SAG and GS, and exhibited a broad specificity to simple phenolics, similar to that of the purified enzyme. Northern blot analysis showed that the SA GTase mRNA was induced both by SA and incompatible pathogens. The rapid induction timing of the mRNA by SA indicates that it belongs to the early SA response genes.     

Synthesis and bacterial expression of a gene encoding the heme domain of assimilatory nitrate reductase

Assimilatory NADH:nitrate reductase (EC 1.6.6.1), a complex Mo-pterin-, cytochrome b(557)-, and FAD-containing protein, catalyzes the regulated and rate-limiting step in the utilization of inorganic nitrogen by higher plants. A codon-optimized gene has been synthesized for expression of the central cytochrome b(557)-containing fragment, corresponding to residues A542-E658, of spinach assimilatory nitrate reductase. While expression of the full-length synthetic gene in Escherichia coli did not result in significant heme domain production, expression of a Y647* truncated form resulted in substantial heme domain production as evidenced by the generation of "pink" cells. The histidine-tagged heme domain was purified to homogeneity using a combination of NTA-agarose and size-exclusion FPLC, resulting in a single protein band following SDS-PAGE analysis with a molecular mass of approximately 13 kDa. MALDI-TOF mass spectrometry yielded an m/z ratio of 12,435 and confirmed the presence of the heme prosthetic group (m/z=622) while cofactor analysis indicated a 1:1 heme to protein stoichiometry. The oxidized heme domain exhibited spectroscopic properties typical of a b-type cytochrome with a visible Soret maximum at 413 nm together with epr g-values of 2.98, 2.26, and 1.49, consistent with low-spin bis-histidyl coordination. Oxidation-reduction titrations of the heme domain indicated a standard midpoint potential (E(o)') of -118 mV. The isolated heme domain formed a 1:1 complex with cytochrome c with a K(A) of 7 microM (micro=0.007) and reconstituted NADH:cytochrome c reductase activity in the presence of a recombinant form of the spinach nitrate reductase flavin domain, yielding a k(cat) of 1.4 s(-1) and a K(m app) for cytochrome c of 9 microM. These results indicate the efficient expression of a recombinant form of the heme domain of spinach nitrate reductase that retained the spectroscopic and thermodynamic properties characteristic of the corresponding domain in the native spinach enzyme.     

Characterization of the kinetic, regulatory, and structural properties of ADP-glucose pyrophosphorylase from Chlamydomonas reinhardtii.

ADP-glucose pyrophosphorylase (ADP-Glc PPase) from Chlamydomonas reinhardtii cells was purified over 2000-fold to a specific activity of 81 units/mg protein, and its kinetic and regulatory properties were characterized. Inorganic orthophosphate and 3-phosphoglycerate were the most potent inhibitor and activator, respectively. Rabbit antiserum raised against the spinach leaf ADP-Glc PPase (but not the one raised against the enzyme from Escherichia coli) inhibited the activity of the purified algal enzyme, which migrated as a single protein band in native polyacrylamide gel electrophoresis. Two-dimensional and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicate that the enzyme from C. reinhardtii is composed of two subunits with molecular masses of 50 and 53 kD, respectively. The molecular mass of the native enzyme is estimated to be 210 kD. Antisera raised against the spinach leaf holoenzyme and against the 51-kD spinach subunit cross-reacted with both subunits of the algal ADP-Glc PPase in immunoblot hybridization, but the cross-reaction was stronger for the 50-kD algal subunit than for the 53-kD subunit. No cross-reaction was observed when antiserum raised against the spinach leaf pyrophosphorylase 54-kD subunit was used. These results suggest that the ADP-Glc PPase from C. reinhardtii is a heterotetrameric protein, since the enzyme from higher plants and its two subunits are structurally more related to the small subunit of the spinach leaf enzyme than to its large subunit. This information is discussed in the context of the possible evolutionary changes leading from the bacterial ADP-Glc PPase to the cyanobacterial and higher plant enzymes.     

Plant riboflavin biosynthesis. Cloning, chloroplast localization, expression, purification, and partial characterization of spinach lumazine synthase.

Lumazine synthase, which catalyzes the penultimate step of riboflavin biosynthesis, has been cloned from three higher plants (spinach, tobacco, and arabidopsis) through functional complementation of an Escherichia coli auxotroph. Whereas the three plant proteins exhibit some structural similarities to known microbial homologs, they uniquely possess N-terminal polypeptide extensions that resemble typical chloroplast transit peptides. In vitro protein import assays with intact chloroplasts and immunolocalization experiments verify that higher plant lumazine synthase is synthesized in the cytosol as a larger molecular weight precursor protein, which is post-translationally imported into chloroplasts where it is proteolytically cleaved to its mature size. The authentic spinach enzyme is estimated to constitute <0.02% of the total chloroplast protein. Recombinant "mature" spinach lumazine synthase is expressed in E. coli at levels exceeding 30% of the total soluble protein and is readily purified to homogeneity using a simple two-step procedure. Apparent V(max) and K(m) values obtained with the purified plant protein are similar to those reported for microbial lumazine synthases. Electron microscopy and hydrodynamic studies reveal that native plant lumazine synthase is a hollow capsid-like structure comprised of 60 identical 16.5-kDa subunits, resembling its icosahedral counterparts in E. coli and Bacillus subtilis.     

Purification and characterization of UDP-glucose:tetrahydrobiopterin glucosyltransferase from Synechococcus sp. PCC 7942

Tetrahydrobiopterin (BH4)-glucoside was identified from Synechococcus sp. PCC 7942 by HPLC analysis and the enzymatic activity of a glycosyltransferase producing the compound from UDP-glucose and BH4. The novel enzyme, named UDP-glucose:BH4 glucosyltransferase, has been purified 846-fold from the cytosolic fraction of Synechococcus sp. PCC 7942 to apparent homogeneity on SDS-PAGE. The native enzyme exists as a monomer having a molecular mass of 39.2 kDa on SDS-PAGE. The enzyme was active over a broad range of pH from 6.5 to 10.5 but most active at pH 10.0. The enzyme required Mn(2+) for maximal activity. Optimum temperature was 42 degrees C. Apparent K(m) values for BH4 and UDP-glucose were determined as 4.3 microM and 188 microM, respectively, and V(max) values were 16.1 and 15.1 pmol min(-1) mg(-1), respectively. The N-terminal amino acid sequence was Thr-Ala-His-Arg-Phe-Lys-Phe-Val-Ser-Thr-Pro-Val-Gly-, sharing high homology with the predicted N-terminal sequence of an unidentified open reading frame slr1166 determined in the genome of Synechocystis sp. PCC 6803, which is known to produce a pteridine glycoside cyanopterin.     

Cloning and characterization of mammalian UDP-glucose glycoprotein: glucosyltransferase and the development of a specific substrate for this enzyme

The endoplasmic reticulum enzyme UDP-glucose glycoprotein:glucosyltransferase (UGGT) has the unique property of recognizing incompletely folded glycoproteins and, if they carry an N -linked Man(9)GlcNAc(2)oligosaccharide, of catalyzing the addition of a glucose residue from UDP-glucose. Using peptide sequence information, we have isolated the complete cDNA of rat liver UGGT and expressed it in insect cells. The cDNA specifies an open reading frame which codes for a protein of 1527 residues including an 18 amino acid signal peptide. The protein has a C-terminal tetrapeptide (HEEL) characteristic of endoplasmic reticulum luminal proteins. The purified recombinant enzyme shows the same preference for unfolded polypeptides with N -linked Man(9)GlcNAc(2)glycans as the enzyme purified from rat liver. A genetically engineered Saccharomyces cerevisiae strain capable of producing glyco-proteins with Man(9)GlcNAc(2)core oligosaccharides was constructed and secreted acid phosphatase (G0-AcP) was purified. G0-AcP was used as an acceptor glycoprotein for UGGT and found to be a better substrate than the previously used soybean agglutinin and thyroglobulin. Recombinant rat UGGT has a K (m) of 44 microM for UDP-glucose. A proteolytic fragment of UGGT was found to retain enzymatic activity thus localizing the catalytic site of the enzyme to the C-terminal 37 kDa of the protein. Using site-directed mutagenesis and photoaffinity labeling, we have identified residues D1334, D1336, Q1429, and N1433 to be necessary for the catalytic activity of the enzyme.     

The nature and mode of action of the cellulolytic component C1 of Trichoderma koningii on native cellulose

1. A purified cellulolytic component C(1) was isolated free from associated activities of the cellulase complex and shown to act as a beta-1,4-glucan cellobiohydrolase on both simple and complex forms of native cellulose. 2. The enzyme releases terminal cellobiose units from cellulose, its extent of action being determined principally by the product and by the nature of the substrate. 3. Component C(x) of the cellulase system is not required for the action of component C(1) (cellobiohydrolase). The enzyme synergizes extensively with cellobiase in extending the hydrolysis of native and of less-complex forms of cellulose to at least 70% with the liberation of glucose. 4. The cellobiohydrolase is relatively unstable, with an optimum at pH5 and a K(m) of 0.05mg/ml. The enzyme is inhibited by its product, from which it is released by cellobiase. 5. Of other compounds tested against the cellobiohydrolase the metal ions Cu(2+), Zn(2+), phenylmercuric and Fe(3+) are increasingly effective inhibitors. Glucose has no action at concentrations found inhibitory with cellobiose. 6. The relationship of the enzyme to the entire cellulase complex is discussed.     

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.     

Characterization of a ferredoxin:NADP+ oxidoreductase from a nonphotosynthetic plant tissue

A flavoprotein with properties similar to those of ferredoxin:NADP+ oxidoreductases found in the leaves of higher plants has been purified to apparent homogeneity from bean sprouts, a nonphotosynthetic plant tissue. The absorbance and circular dichroism spectra of the bean sprout protein are similar to those of spinach leaf ferredoxin:NADP+ oxidoreductase and an antibody raised against the spinach enzyme recognized the bean sprout enzyme. The bean sprout enzyme catalyzed ferredoxin-dependent electron transfer from NADPH to equine cytochrome c at a high rate but, unlike the spinach enzyme, exhibited little NADPH to 2,6-dichlorophenol indophenol diaphorase activity. The bean sprout enzyme forms a 1:1 electrostatically stabilized complex with ferredoxins isolated from either bean sprouts or spinach leaves.     

Purification and kinetic properties of UDP-glucose dehydrogenase from sugarcane

In this study, UDP-glucose dehydrogenase has been purified to electrophoretic homogeneity from sugarcane (Saccharum spp. hybrid) culm. The enzyme had a pH optimum of 8.4 and a subunit molecular mass of 52 kDa. Specific activity of the final preparation was 2.17 micromol/min/mg protein. Apparent K(m) values of 18.7+/-0.75 and 72.2+/-2.7 microM were determined for UDP-glucose and NAD(+), respectively. The reaction catalyzed by UDP-glucose dehydrogenase was irreversible with two equivalents of NADH produced for each UDP-glucose oxidized. Stiochiometry was not altered in the presence of carbonyl-trapping reagents. With respect to UDP-glucose, UDP-glucuronic acid, and UDP-xylose were competitive inhibitors of UDP-glucose dehydrogenase with K(i) values of 292 and 17.1 microM, respectively. The kinetic data are consistent with a bi-uni-uni-bi substituted enzyme mechanism for sugarcane UDP-glucose dehydrogenase. Oxidation of the alternative nucleotide sugars CTP-glucose and TDP-glucose was observed with rates of 8 and 2%, respectively, compared to UDP-glucose. The nucleotide sugar ADP-glucose was not oxidized by UDP-glucose dehydrogenase. This is of significance as it demonstrates carbon, destined for starch synthesis in tissues that synthesize cytosolic AGP-glucose, will not be partitioned toward cell wall biosynthesis.     

Cloning, sequencing, and expression of UDP-glucose pyrophosphorylase gene from Acetobacter xylinum BRC5

A UDP-glucose pyrophosphorylase (UGPase) gene from Acetobacter xylinum BRC5 has been cloned, sequenced, and expressed in Escherichia coli. The gene consists of 867 nucleotides and encodes a polypeptide of 289 amino acid residues with a calculated molecular mass of 31,493 Da. The amino acid sequences of the enzyme showed an 85.8% identity to those of an enzyme from A. xilinum ATCC 23768. A polyhistidine-UGPase fusion enzyme was expressed and purified from the transformed E. coli. The enzyme showed a 35,620-Da single protein band on SDS/PAGE and an about 160,000-Da protein band on 8-16% pore-gradient polyacrylamide gel, indicating the enzyme may be a tetramer or pentamer composed of four or five identical subunits. Kinetic analysis of the enzyme showed a typical Michaelis-Menten substrate saturation pattern, from which Km and Vmax were calculated to be 3.22 mM and 175.4 micromol x min(-1) x mg(-1) for UDP-glucose and 0.24 mM and 69.4 micromol x min(-1) x mg(-1) for PPi, respectively, required Mg2+ for maximal activity, and was inhibited by free pyrophosphate. Computer-aided comparison of the Acetobacter enzyme sequence with those of other bacterial enzymes found significant similarities among them and predicted that Lys84 is a catalytically important residue. Lys84 in the enzyme, which was also conserved in other bacterial enzyme sequences, was replaced by arginine or leucine. The K84R mutant enzyme was successfully expressed in E. coli and showed enzyme activity (63% of the wild-type enzyme activity), but K84L was not isolated in stable form. These results suggest that Lys84 is significant in not only catalysis but also maintenance of the active structure.     

Posttranslational modification of 6-phosphofructo-1-kinase in Aspergillus niger

Two different enzymes exhibiting 6-phosphofructo-1-kinase (PFK1) activity were isolated from the mycelium of Aspergillus niger: the native enzyme with a molecular mass of 85 kDa, which corresponded to the calculated molecular mass of the deduced amino acid sequence of the A. niger pfkA gene, and a shorter protein of approximately 49 kDa. A fragment of identical size also was obtained in vitro by the proteolytic digestion of the partially purified native PFK1 with proteinase K. When PFK1 activity was measured during the proteolytic degradation of the native protein, it was found to be lost after 1 h of incubation, but it was reestablished after induction of phosphorylation by adding the catalytic subunit of cyclic AMP-dependent protein kinase to the system. By determining kinetic parameters, different ratios of activities measured at ATP concentrations of 0.1 and 1 mM were detected with fragmented PFK1, as with the native enzyme. Fructose-2,6-biphosphate significantly increased the Vmax of the fragmented protein, while it had virtually no effect on the native protein. The native enzyme could be purified only from the early stages of growth on a minimal medium, while the 49-kDa fragment appeared later and was activated at the time of a sudden change in the growth rate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of sequential purifications of PFK1 enzymes by affinity chromatography during the early stages of the fungal development suggested spontaneous posttranslational modification of the native PFK1 in A. niger cells, while from the kinetic parameters determined for both isolated forms it could be concluded that the fragmented enzyme might be more efficient under physiological conditions.     

Studies on the Sulfite Reducing System of Algae

Sulfite reductase using reduced methyl viologen as an electron donor was purified about 94-fold from a red alga, Porphyra yezoensis. The enzyme was ultracentrifugically homogenous and could reduce sulfite to sulfide quantitatively with an uptake of six electrons. The enzyme had a pH optimum in the vicinity of 7.5. The Km for sulfite was determined to be 6.5×l0^?4m. The purified preparation of the algal reductase showed its absorption maximum at 385 mμ and slight shoulders at 408, 456, 485, 600 and 664 mμ in addition to an intense peak at 278 mμ. Metal analysis of the purified enzyme suggested the presence of iron and copper in the molecule. NADPH, NADH or the reduced form of spinach ferredoxin could not be a direct electron donor for the purified algal sulfite reductase.     

Implications of secondary structure prediction and amino acid sequence comparison of class I and class II phosphoribosyl diphosphate synthases on catalysis, regulation, and quaternary structure

Spinach 5-phospho-D-ribosyl alpha-1-diphosphate (PRPP) synthase isozyme 4 was synthesized in Escherichia coli and purified to near homogeneity. The activity of the enzyme is independent of P(i); it is inhibited by ADP in a competitive manner, indicating a lack of an allosteric site; and it accepts ATP, dATP, GTP, CTP, and UTP as diphosphoryl donors. All of these properties are characteristic for class II PRPP synthases. K(m) values for ATP and ribose 5-phosphate are 77 and 48 microM, respectively. Gel filtration reveals a molecular mass of the native enzyme of approximately 110 kD, which is consistent with a homotrimer. Secondary structure prediction shows that spinach PRPP synthase isozyme 4 has a general folding similar to that of Bacillus subtilis class I PRPP synthase, for which the three-dimensional structure has been solved, as the position and extent of helices and beta-sheets of the two enzymes are essentially conserved. Amino acid sequence comparison reveals that residues of class I PRPP synthases interacting with allosteric inhibitors are not conserved in class II PRPP synthases. Similarly, residues important for oligomerization of the B. subtilis enzyme show little conservation in the spinach enzyme. In contrast, residues of the active site of B. subtilis PRPP synthase show extensive conservation in spinach PRPP synthase isozyme 4.     

Macrophomate synthase: characterization, sequence, and expression in Escherichia coli of the novel enzyme catalyzing unusual multistep transformation of 2-pyrones to benzoates

Macrophoma commelinae isolated from spots on leaves of Commelina communis has the ability to transform 5-acetyl-4-methoxy-6-methyl-2-pyrone (1) to 4-acetyl-3-methoxy-5-methylbenzoic acid (macrophomic acid, 2). This biotransformation includes the condensation of the 2-pyrone ring with a C3-unit precursor to form a substituted benzoic acid. We optimized conditions for induction of enzyme activity in M. commelinae, identified oxalacetate as a C3-unit precursor with cell extract, and purified the novel enzyme, macrophomate synthase. Oxalacetate inhibited the enzyme activity at a concentration higher than 5 mM, and magnesium chloride stimulated the enzyme activity. Kinetic analyses gave K(m) of 1.7 mM for 1 at 5 mM oxalacetate, K(m) of 1.2 mM for oxalacetate at 5 mM 1, and k(cat) of 0.46 s(-1) per subunit. Pyruvate was a weak substrate, with K(m) of 35.2 mM and k(cat) of 0.027 s(-1) at 5 mM 1. We cloned and sequenced a cDNA encoding the macrophomate synthase. The cDNA of 1,225 bp contained an open reading frame that encoded a polypeptide of 339 amino acid residues and 36,244 Da, the sequence of which showed no significant similarity with known proteins in a homology search with BLAST programs. Transformed E. coli cells carrying the cDNA encoding the mature protein of macrophomate synthase overproduced macrophomate synthase under the control of the T7 phage promoter induced by IPTG. The purified enzyme showed the same values of K(m) and optimum pH as the native macrophomate synthase.     

Highly purified recombinant varicella Zoster virus thymidine kinase is a homodimer

Recombinant varicella zoster virus (VZV) thymidine kinase (TK) was isolated in a fast and gentle two-step procedure from Escherichia coli. The TK was expressed as a PreScission-cleavable fusion protein and purified by glutathione and ATP affinity chromatography, yielding homogeneous, highly pure VZV TK. The purified enzyme displays enzymatic activities with K(m) values of 0.3 +/- 0.06 microM for the natural substrate thymidine and 11.6 +/- 3.2 microM for ATP, indicating the biochemical equivalence with the viral VZV TK expressed in infected cells. Determinations of the native molecular weight by size exclusion chromatography and native polyacrylamide gel electrophoresis revealed that the pure enzyme is biologically active as a homodimer.     

Characterization of the <Emphasis Type="Italic"> ugpG </Emphasis>gene encoding a UDP-glucose pyrophosphorylase from the gellan gum producer <Emphasis Type="Italic"> Sphingomonas paucimobilis </Emphasis>ATCC 31461

The ugpGgene, which codes for a UDP-glucose pyrophosphorylase (UGP) (or glucose-1-phosphate uridylyltransferase; EC 2.7.7.9) in Sphingomonas paucimobilis ATCC 31461, was cloned and sequenced. This industrial strain produces the exopolysaccharide gellan, a new commercial gelling agent, and the ugpG gene may convert glucose-1-phosphate into UDP-glucose in the gellan biosynthetic pathway. The ugpG gene is capable of restoring the capacity of an Escherichia coli galU mutant to grow on galactose by functional complementation of its deficiency for UDP-glucose pyrophosphorylase activity. As expected, the predicted gene product shows strong homology to UDP-glucose pyrophosphorylases from several bacterial species. The N-terminal region of UgpG exhibits the motif GXGTRXLPXTK, which is highly conserved among bacterial XDP-sugar pyrophosphorylases, and a lysine residue (K(192)) is located within a VEKP motif predicted to be essential for substrate binding or catalysis. UgpG was purified to homogeneity as a heterologous fusion protein from crude cell extracts prepared from IPTG-induced cells of E. coli, using affinity chromatography. Under denaturing conditions, the fusion protein S-UgpG-His(6) migrated with an estimated molecular mass of 36 kDa [corresponding to the predicted molecular mass of native UgpG (31.2 kDa) plus 5 kDa for the S and histidine tags). Kinetic analysis of UgpG in the reverse reaction (pyrophosphorolysis) showed a typical Michaelis-Menten substrate saturation pattern. The apparent K(m) and V(max) values estimated for UDP-glucose were 7.5 microM and 1275 micromol/min/g.