Purification, molecular cloning, and sequence analysis of sucrose-6F-phosphate phosphohydrolase from plants

Sucrose-6(F)-phosphate phosphohydrolase (SPP; EC ) catalyzes the final step in the pathway of sucrose biosynthesis and is the only enzyme of photosynthetic carbon assimilation for which the gene has not been identified. The enzyme was purified to homogeneity from rice (Oryza sativa L.) leaves and partially sequenced. The rice leaf enzyme is a dimer with a native molecular mass of 100 kDa and a subunit molecular mass of 50 kDa. The enzyme is highly specific for sucrose 6(F)-phosphate with a K(m) of 65 microM and a specific activity of 1250 micromol min(-1) mg(-1) protein. The activity is dependent on Mg(2+) with a remarkably low K(a) of 8-9 microM and is weakly inhibited by sucrose. Three peptides from cleavage of the purified rice SPP with endoproteinase Lys-C showed similarity to the deduced amino acid sequences of three predicted open reading frames (ORF) in the Arabidopsis thaliana genome and one in the genome of the cyanobacterium Synechocystis sp. PCC6803, as well as cDNA clones from Arabidopsis, maize, and other species in the GenBank database of expressed sequence tags. The putative maize SPP cDNA clone contained an ORF encoding a 420-amino acid polypeptide. Heterologous expression in Escherichia coli showed that this cDNA clone encoded a functional SPP enzyme. The 260-amino acid N-terminal catalytic domain of the maize SPP is homologous to the C-terminal region of sucrose-phosphate synthase. A PSI-BLAST search of the GenBank database indicated that the maize SPP is a member of the haloacid dehalogenase hydrolase/phosphatase superfamily.     

Superoxide dismutase participates in the enzymatic formation of the tyrosine radical of ribonucleotide reductase from Escherichia coli.

One of the two nonidentical subunits of Escherichia coli ribonucleotide reductase, protein B2, contains in its active form two antiferromagnetically coupled Fe(III) ions and an organic free radical that arises by the one-electron oxidation of tyrosine-122 of the polypeptide chain. Protein B2 lacking the tyrosine radical but with the iron center intact (called protein B2/HU because it is produced by treatment with hydroxyurea) is enzymatically inactive. Previously, it was found that a crude extract from E. coli transforms B2/HU into B2 in the presence of dithiothreitol, Mg2+, and oxygen. On purification of the enzyme system, we now find that radical introduction requires three separate proteins as well as NADPH and FMN. One of the proteins is superoxide dismutase. We hypothesize that the overall reaction involves a reduction of the iron center followed by the oxidation of iron and tyrosine-122. Superoxide dismutase may then be involved in the second step to protect an oxidation-sensitive intermediate. Alternatively, the enzyme might be directly involved in the oxidation step.     

The A+T-rich genome of Herpesvirus saimiri contains a highly conserved gene for thymidylate synthase.

Herpesvirus saimiri (HVS) is the prototype member of a distinctive subset of lymphotropic herpesviruses (the gamma 2 subgroup) with A+T-rich coding sequences. In this paper, we show that cells productively infected with HVS contain high concentrations of a virus-specified thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45); we identify the active polypeptide and present the sequence of the virus gene. The predicted amino acid sequence of the 294-residue subunit of the virus enzyme is 70% homologous with the sequence of the human enzyme and about 50% homologous with prokaryotic thymidylate synthases, illustrating the remarkable structural constraints imposed by the thymidylate synthase function. However, the presence of the enzyme is not a conserved property of herpesviruses. We find no evidence for a virus-encoded thymidylate synthase activity (or a homology to a thymidylate synthase sequence) in G+C-rich representatives of alpha 1 (e.g., herpes simplex viruses, 66-68% G+C), beta (i.e., human cytomegalovirus, 58-59% G+C), and gamma 1 (i.e., Epstein-Barr virus, 60% G+C) herpesvirus subgroups. The production of excess thymidylate by a virus thymidylate synthase in cells infected with an A+T-rich herpesvirus would provide one plausible source of biased mutations by the virus-encoded replicative enzymes, which we have previously suggested as the likely general cause of differences in the mean nucleotide compositions of herpesvirus genomes.     

Covalent structure of biodegradative threonine dehydratase of Escherichia coli: homology with other dehydratases.

The 987-base-pair coding region of the tdc gene of Escherichia coli K-12 encoding biodegradative threonine dehydratase [Tdc; L-threonine hydro-lyase (deaminating), EC 4.2.1.16], previously cloned in this laboratory, was sequenced. The deduced polypeptide consists of 329 amino acid residues with a calculated Mr of 35,238. Although the purified enzyme was shown to contain tryptophan, no tryptophan codon was found in the tdc reading frame. Incubation of purified Tdc with [14C]tryptophan revealed apparent "covalent" binding of tryptophan, indicating posttranslational modification of the enzyme. A heptapeptide, 54Thr-55Gly-56Ser-57Phe-58Lys-59Ile- 60Arg, was found to contain Lys-58, which binds pyridoxal phosphate coenzyme. A comparison of amino acid sequences between the Tdc polypeptide and the biosynthetic threonine dehydratases of yeast (encoded by ILV1) and E. coli (encoded by ilvA) and the E. coli D-serine dehydratase (DsdA, encoded by dsdA) revealed various extents of homology: five domains of the Tdc polypeptide were 63-93% homologous with the yeast enzyme, and three of these same regions were 80% homologous with the biosynthetic E. coli dehydratase; two different domains showed 67% and 83% homology with DsdA. In addition, two other sequences were highly conserved in all four proteins, one of which was shown to contain the conserved lysine residue that binds pyridoxal phosphate in the Tdc and DsdA polypeptides. These observations suggest that, despite their diverse origin and metabolic significance, these enzymes may have evolved from a common ancestral protein.     

AtPCS1, a phytochelatin synthase from Arabidopsis: isolation and in vitro reconstitution.

Phytochelatins, a class of posttranslationally synthesized peptides, play a pivotal role in heavy metal, primarily Cd2+, tolerance in plants and fungi by chelating these substances and decreasing their free concentrations. Derived from glutathione and related thiols by the action of gamma-glutamylcysteine dipeptidyl transpeptidases (phytochelatin synthases; EC 2.3.2.15), phytochelatins consist of repeating units of gamma-glutamylcysteine followed by a C-terminal Gly, Ser, or beta-Ala residue [poly-(gamma-Glu-Cys)n-Xaa]. Here we report the suppression cloning of a cDNA (AtPCS1) from Arabidopsis thaliana encoding a 55-kDa soluble protein that enhances heavy-metal tolerance and elicits Cd2+-activated phytochelatin accumulation when expressed in Saccharomyces cerevisiae. On the basis of these properties and the sufficiency of immunoaffinity-purified epitope-tagged AtPCS1 polypeptide for high rates of Cd2+-activated phytochelatin synthesis from glutathione in vitro, AtPCS1 is concluded to encode the enzyme phytochelatin synthase.     

Isolation and expression of the Pneumocystis carinii thymidylate synthase gene.

The thymidylate synthase (TS) gene from Pneumocystis carinii has been isolated from complementary and genomic DNA libraries and expressed in Escherichia coli. The coding sequence of TS is 891 nucleotides, encoding a 297-amino acid protein of Mr 34,269. The deduced amino acid sequence is similar to TS from other organisms and is most closely related to the enzyme from Saccharomyces cerevisiae with 65% identity. TS is found on a 330-kilobase-pair chromosome in P. carinii. While TS and dihydrofolate reductase reside on a single polypeptide chain in all protozoa studied to date, TS is not linked to dihydrofolate reductase in P. carinii. The TS gene shows the presence of four small intervening sequences, some of which interrupt the coding sequence in highly ordered structural regions of the protein. Heterologous expression of P. carinii TS in E. coli was accomplished by cloning the coding sequence into plasmid vectors under control of the lac and tac promoters. These constructs direct the synthesis of catalytically active enzyme to the extent of 2% of total soluble protein.     

The fabJ-encoded beta-ketoacyl-[acyl carrier protein] synthase IV from Escherichia coli is sensitive to cerulenin and specific for short-chain substrates.

A fourth fatty acid condensing enzyme was isolated from Escherichia coli by its ability to restore elongating activity to a protein extract which had been treated with cerulenin, a condensing enzyme-specific inhibitor. The purified beta-ketoacyl-[acyl carrier protein] (ACP) synthase IV [3-oxoacyl-ACP synthase; acyl-ACP:malonyl-ACP C-acyltransferase (decarboxylating), EC 2.3.1.41] (KAS IV) is specific for short-chain acyl-ACP substrates. The enzyme is stable at 43 degrees C and very sensitive to cerulenin (50% inhibition at 3 microM), which binds covalently. A condensing enzyme-specific antibody raised to an expressed open reading frame from barley was used to identify KAS IV protein in Western blots, and the sequence obtained for 30 amino-terminal residues. This led to the isolation of the fabJ gene located in the fab cluster at 24.8 min of the E. coli chromosome. The fabJ gene encodes a polypeptide of 413 amino acids and molecular mass 43 kDa that shows 38% identity and 64% similarity to the fabB-encoded KAS I. The amino acid sequence of KAS IV, however, is more similar to all other published condensing enzyme sequences than the KAS I sequence is. A specialized putative function for this enzyme is to supply the octanoic substrates for lipoic acid biosynthesis. We predict that an analogue of KAS IV with the same function will be found in plant mitochondria. The described complementation assay can be used to detect condensing enzymes with other substrate specificities by supplementing the cerulenin-treated extract with appropriate purified KAS enzymes.     

An intron in the thymidylate synthase gene of Bacillus bacteriophage beta 22: evidence for independent evolution of a gene, its group I intron, and the intron open reading frame.

The thymidylate synthase gene (thy) (EC 2.1.1.45) of Bacillus subtilis bacteriophage beta 22 has a self-splicing, group I intron inserted into a highly conserved region of the coding sequence. The intron is very similar to one that is inserted 21 bp further downstream in the homologous thymidylate synthase gene (td) of Escherichia coli bacteriophage T4. In contrast, the amino acid sequences of the bacteriophage thymidylate synthases are highly divergent. The beta 22 intron has a fragmentary open reading frame (ORF) that encodes a putative helix-turn-helix DNA-binding motif, similar to one at the carboxyl terminus of the homing endonuclease (I-TevI) encoded by the T4 td intron. The td ORF and the thy ORF fragments are inserted into different regions of their respective intron structures. These results suggest that the thymidylate synthase genes, their introns, and their respective intron-ORFs all have separate evolutionary histories and that the acquisition of the intron could not have occurred by a simple homing event.     

Nitric oxide synthases reveal a role for calmodulin in controlling electron transfer.

Nitric oxide (NO) is synthesized within the immune, vascular, and nervous systems, where it acts as a wide-ranging mediator of mammalian physiology. The NO synthases (EC 1.14.13.39) isolated from neurons or endothelium are calmodulin dependent. Calmodulin binds reversibly to neuronal NO synthase in response to elevated Ca2+, triggering its NO production by an unknown mechanism. Here we show that calmodulin binding allows NADPH-derived electrons to pass onto the heme group of neuronal NO synthase. Calmodulin-triggered electron transfer to heme was independent of substrate binding, caused rapid enzymatic oxidation of NADPH in the presence of O2, and was required for NO synthesis. An NO synthase isolated from cytokine-induced macrophages that contains tightly bound calmodulin catalyzed spontaneous electron transfer to its heme, consistent with bound calmodulin also enabling electron transfer within this isoform. Together, these results provide a basis for how calmodulin may regulate NO synthesis. The ability of calmodulin to trigger electron transfer within an enzyme is unexpected and represents an additional function for calcium-binding proteins in biology.     

Oxygen-sensitive ribonucleoside triphosphate reductase is present in anaerobic Escherichia coli.

Ribonucleoside diphosphate reductase from Escherichia coli and mammalian cells provides the deoxyribonucleoside triphosphates for DNA synthesis. The active enzyme contains a tyrosyl free radical whose formation requires oxygen. Earlier genetic evidence suggested that the enzyme is not required for anaerobic growth of E. coli, implicating the activity of a different enzyme or enzyme system for deoxyribonucleotide synthesis in the absence of oxygen. We now conclude from isotope incorporation experiments that E. coli during anaerobiosis obtains its deoxyribonucleotides by reduction of ribonucleotides. Extracts from anaerobically grown bacteria contain a different enzyme activity capable of reducing CTP to dCTP. To obtain an active enzyme, strict anaerobiosis must be maintained during extract preparation and during assay of the enzyme. The reaction is stimulated by NADPH, Mg2+, and ATP. Inhibition by deoxyribonucleoside triphosphates suggests that the anaerobic enzyme has allosteric properties. Antibodies raised against the aerobic enzyme do not inhibit the new activity, and hydroxyurea, a potent scavenger of the tyrosyl radical of the aerobic enzyme, only weakly inhibits the anaerobic enzyme. The anaerobic enzyme has interesting evolutionary aspects since it might reflect on an activity that in the absence of oxygen made possible the transition from an "RNA world" into a "DNA world."     

Molecular cloning and characterization of the yeast gene for squalene synthetase.

Squalene synthetase (farnesyl-diphosphate: farnesyl-diphosphate farnesyltransferase, EC 2.5.1.21) is a critical branch point enzyme of isoprenoid biosynthesis that is thought to regulate the flux of isoprene intermediates through the sterol pathway. The structural gene for this enzyme was cloned from the yeast Saccharomyces cerevisiae by functional complementation of a squalene synthetase-deficient erg9 mutant. Identification of this ERG9 clone was confirmed by genetic linkage analysis in yeast and expression of enzyme activity in Escherichia coli. The predicted squalene synthetase polypeptide of 444 amino acids (Mr, 51,753) lacks significant homology to known protein sequences, except within a region that may represent a prenyl diphosphate (substrate) binding site. The ERG9-encoded protein contains a PEST consensus motif (rich in proline, glutamic acid, serine, and threonine) present in many proteins with short cellular half-lives. Modeling of the protein suggests that it contains at least one, and possibly two, membrane-spanning domains. Disruption of the chromosomal squalene synthetase coding region by insertional mutagenesis indicates that ERG9 is a single copy gene that is essential for cell growth in yeast.     

Purification and characterization of the cytokine-induced macrophage nitric oxide synthase: an FAD- and FMN-containing flavoprotein.

A soluble nitric oxide (NO) synthase activity was purified 426-fold from a mouse macrophage cell line activated with interferon gamma and bacterial lipopolysaccharide by sequential anion-exchange, affinity, and gel filtration chromatography. SDS/PAGE of the purified NO synthase gave three closely spaced silver-staining protein bands between 125 and 135 kDa. When assayed in the presence of L-arginine, NADPH, tetrahydrobiopterin, FAD, and reduced thiol, purified NO synthase had a specific activity of 1313 nmol of NO2- plus NO3- per min per mg. The apparent Km of the enzyme for L-arginine and NADPH was 2.8 and 0.3 microM, respectively. Addition of calcium ions with or without calmodulin did not increase the activity of the purified enzyme, and NO synthesis was not altered by calmodulin inhibitors. Gel filtration chromatography indicated that the induced NO synthase was catalytically competent as a dimer of approximately 250 kDa but could be dissociated into inactive monomers of approximately 130 kDa in the absence of L-arginine, FAD, and tetrahydrobiopterin. Upon heat denaturation, NO synthase released 1.1 mol of FAD and 0.55 mol of FMN per mol of 130-kDa subunit. Thus, inducible macrophage NO synthase differs in several respects from constitutive NO synthases and is one of very few eukaryotic enzymes containing both FAD and FMN.     

Hydrogen donor system for Escherichia coli ribonucleoside-diphosphate reductase dependent upon glutathione.

E. coli B tsnC 7004, an E. coli B/1 mutant with normal phenotype unable to replicate phage T7 DNA [Chamberlin, M. (1974)J. Virol. 14,509-516], contained no detectable level of thioredoxin when assayed with ribonucleotide reductase (2'-deoxyribonucleoside-diphosphate:oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1). Gently lysed E. coli tsnC 7004 cell extracts reduced CDP when supplemented with NADPH as efficiently as the parent strain E. coli B/1 despite the lack of thioredoxin, indicating the presence of another hydrogen transport system. This could be divided into two parts by heat treatment at 85degrees; one heat-stable fraction, which was active in the presence of dithiothreitol or glutathione, and one heat-labile fraction. Addition of yeast glutathione reductase [NAD(P)H:oxidized-glutathione oxidoreductase, EC 1.6.4.2] to the heated extracts restored full activity. The results demonstrate a novel hydrogen transport system in E. coli consisting of NADPH, glutathione, glutathione reductase, and a heat-stable enzyme called "glutaredoxin". Reduced glutathione at physiological concentrations functions as hydrogen donor for ribonucleotide reduction only in the presence of glutaredoxin. Glutaredoxin was not reduced by E. coli thioredoxin reductase (NADPH:oxidized-thioredoxin oxidoreductase, EC 1.6.4.5) and showed no crossreaction with antibodies against thioredoxin. These results demonstrate the existence of two different electron transfer systems from NADPH to deoxyribonucleotides and provide a function for glutathione in DNA synthesis.     

Sulfonamide resistance mechanism in Escherichia coli: R plasmids can determine sulfonamide-resistant dihydropteroate synthases.

Several natural isolate E. coli strains highly resistant to sulfonamides and antibiotics are shown to contain a sulfonamide-resistant dihydropteroate synthase (2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine-diphosphate:4-aminobenzoate 2-amino-4-hydroxydihydropteridine-6-methenyltransferase, EC 2.5.1.15) in addition to the normal sensitive enzyme. The resistant dihydropteroate synthases examined are determined by an R plasmid and are smaller and less heat stable than the normal sulfonamide-sensitive enzyme. One synthase resistant to any sulfonamide tested, and to sulfanilic and arsanilic acids, was still inhibited by several non-sulfonamide analogs of p-aminobenzoate. Citrobacter and Klebsiella pneumoniae strains also show similar mechanisms of sulfonamide resistance.     

A functional cellulose synthase from ascidian epidermis

Among animals, urochordates (e.g., ascidians) are unique in their ability to biosynthesize cellulose. In ascidians cellulose is synthesized in the epidermis and incorporated into a protective coat know as the tunic. A putative cellulose synthase-like gene was first identified in the genome sequences of the ascidian Ciona intestinalis. We describe here a cellulose synthase gene from the ascidian Ciona savignyi that is expressed in the epidermis. The predicted C. savignyi cellulose synthase amino acid sequence showed conserved features found in all cellulose synthases, including plants, but was most similar to cellulose synthases from bacteria, fungi, and Dictyostelium discoidium. However, unlike other known cellulose synthases, the predicted C. savignyi polypeptide has a degenerate cellulase-like region near the carboxyl-terminal end. An expression construct carrying the C. savignyi cDNA was found to restore cellulose biosynthesis to a cellulose synthase (CelA) minus mutant of Agrobacterium tumefaciens, showing that the predicted protein has cellulose synthase activity. The lack of cellulose biosynthesis in all other groups of metazoans and the similarity of the C. savignyi cellulose synthase to enzymes from cellulose-producing organisms support the hypothesis that the urochordates acquired the cellulose biosynthetic pathway by horizontal transfer.     

Cloning of a chicken liver cDNA encoding 5-aminoimidazole ribonucleotide carboxylase and 5-aminoimidazole-4-N-succinocarboxamide ribonucleotide synthetase by functional complementation of Escherichia coli pur mutants.

We have used functional complementation of Escherichia coli pur mutants to clone avian cDNA encoding 5-aminoimidazole ribonucleotide (AIR) carboxylase-5-aminoimidazole-4-N-succinocarboxamide ribonucleotide (SAICAR) synthetase, the bifunctional enzyme catalyzing steps 6 and 7 in the pathway for de novo purine nucleotide synthesis. Mutational analyses have been used to establish the structure-function relationship: NH2-SAICAR synthetase-AIR carboxylase-COOH. The amino acid sequence of the SAICAR synthetase domain is homologous to that of bacterial purC-encoded enzymes, and the sequence of the following AIR carboxylase domain is homologous to that of bacterial purE-encoded enzymes. In E. coli, AIR carboxylase is the product of genes purEK with the purK subunit postulated to have a role in CO2 binding. The avian enzyme lacks sequences corresponding to purK yet functions in E. coli. Functional complementation of E. coli pur mutants can be used to clone additional avian cDNAs for de novo purine nucleotide synthesis.     

Identification of a unique isoform of 1-aminocyclopropane-1-carboxylic acid synthase by monoclonal antibody

1-Aminocyclopropane-1-carboxylic acid (ACC) synthase (EC 4.4.1.14) is a key enzyme regulating ethylene biosynthesis in higher plants. A monoclonal antibody (mAb T20C) that immunoprecipitates the ACC synthase activity from tomato pericarp tissue extracts revealed that mAb T20C immunodecorates an ≈67-kDa polypeptide. On isoelectric focusing gels, ACC synthase activity in cell-free preparations was resolved into three distinct activity peaks with pI values 5.3, 7, and 9. mAb T20C specifically recognized the pI 7 form of the enzyme on electrophoretic transfer (Western) blots. When analyzed by sodium dodecyl sulfate gel electrophoresis under reducing conditions, the eluted pI 7 form was confirmed to migrate as a polypeptide of 67 kDa. The 67-kDa pI 7 isoform is a previously undescribed form of ACC synthase.