Transverse relaxation-optimized NMR spectroscopy with the outer membrane protein OmpX in dihexanoyl phosphatidylcholine micelles

The 2H,13C,15N-labeled, 148-residue integral membrane protein OmpX from Escherichia coli was reconstituted with dihexanoyl phosphatidylcholine (DHPC) in mixed micelles of molecular mass of about 60 kDa. Transverse relaxation-optimized spectroscopy (TROSY)-type triple resonance NMR experiments and TROSY-type nuclear Overhauser enhancement spectra were recorded in 2 mM aqueous solutions of these mixed micelles at pH 6.8 and 30°C. Complete sequence-specific NMR assignments for the polypeptide backbone thus have been obtained. The 13C chemical shifts and the nuclear Overhauser effect data then resulted in the identification of the regular secondary structure elements of OmpX/DHPC in solution and in the collection of an input of conformational constraints for the computation of the global fold of the protein. The same type of polypeptide backbone fold is observed in the presently determined solution structure and the previously reported crystal structure of OmpX determined in the presence of the detergent n-octyltetraoxyethylene. Further structure refinement will have to rely on the additional resonance assignment of partially or fully protonated amino acid side chains, but the present data already demonstrate that relaxation-optimized NMR techniques open novel avenues for studies of structure and function of integral membrane proteins.

A novel human hexameric DNA helicase: expression, purification and characterization

We have cloned, expressed and purified a hexameric human DNA helicase (hHcsA) from HeLa cells. Sequence analysis demonstrated that the hHcsA has strong sequence homology with DNA helicase genes from Saccharomyces cerevisiae and Caenorhabditis elegans, indicating that this gene appears to be well conserved from yeast to human. The hHcsA gene was cloned and expressed in Escherichia coli and purified to homogeneity. The expressed protein had a subunit molecular mass of 116 kDa and analysis of its native molecular mass by size exclusion chromatography suggested that hHcsA is a hexameric protein. The hHcsA protein had a strong DNA-dependent ATPase activity that was stimulated ≥5-fold by single-stranded DNA (ssDNA). Human hHcsA unwinds duplex DNA and analysis of the polarity of translocation demonstrated that the polarity of DNA unwinding was in a 5′→3′ direction. The helicase activity was stimulated by human and yeast replication protein A, but not significantly by E.coli ssDNA-binding protein. We have analyzed expression levels of the hHcsA gene in HeLa cells during various phases of the cell cycle using in situ hybridization analysis. Our results indicated that the expression of the hHcsA gene, as evidenced from the mRNA levels, is cell cycle-dependent. The maximal level of hHcsA expression was observed in late G1/early S phase, suggesting a possible role for this protein during S phase and in DNA synthesis.

A Single Limit Dextrinase Gene Is Expressed Both in the Developing Endosperm and in Germinated Grains of Barley1

The single gene encoding limit dextrinase (pullulan 6-glucanohydrolase; EC 3.2.1.41) in barley (Hordeum vulgare) has 26 introns that range in size from 93 to 822 base pairs. The mature polypeptide encoded by the gene has 884 amino acid residues and a calculated molecular mass of 97,417 D. Limit dextrinase mRNA is abundant in gibberellic acid-treated aleurone layers and in germinated grain. Gibberellic acid response elements were found in the promoter region of the gene. These observations suggest that the enzyme participates in starch hydrolysis during endosperm mobilization in germinated grain. The mRNA encoding the enzyme is present at lower levels in the developing endosperm of immature grain, a location consistent with a role for limit dextrinase in starch synthesis. Enzyme activity was also detected in developing grain. The limit dextrinase has a presequence typical of transit peptides that target nascent polypeptides to amyloplasts, but this would not be expected to direct secretion of the mature enzyme from aleurone cells in germinated grain. It remains to be discovered how the enzyme is released from the aleurone and whether another enzyme, possibly of the isoamylase group, might be equally important for starch hydrolysis in germinated grain.

A Multisubunit Acetyl Coenzyme A Carboxylase from Soybean1

A multisubunit form of acetyl coenzyme A (CoA) carboxylase (ACCase) from soybean (Glycine max) was characterized. The enzyme catalyzes the formation of malonyl CoA from acetyl CoA, a rate-limiting step in fatty acid biosynthesis. The four known components that constitute plastid ACCase are biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and the α- and β-subunits of carboxyltransferase (α- and β-CT). At least three different cDNAs were isolated from germinating soybean seeds that encode BC, two that encode BCCP, and four that encode α-CT. Whereas BC, BCCP, and α-CT are products of nuclear genes, the DNA that encodes soybean β-CT is located in chloroplasts. Translation products from cDNAs for BC, BCCP, and α-CT were imported into isolated pea (Pisum sativum) chloroplasts and became integrated into ACCase. Edman microsequence analysis of the subunits after import permitted the identification of the amino-terminal sequence of the mature protein after removal of the transit sequences. Antibodies specific for each of the chloroplast ACCase subunits were generated against products from the cDNAs expressed in bacteria. The antibodies permitted components of ACCase to be followed during fractionation of the chloroplast stroma. Even in the presence of 0.5 m KCl, a complex that contained BC plus BCCP emerged from Sephacryl 400 with an apparent molecular mass greater than about 800 kD. A second complex, which contained α- and β-CT, was also recovered from the column, and it had an apparent molecular mass of greater than about 600 kD. By mixing the two complexes together at appropriate ratios, ACCase enzymatic activity was restored. Even higher ACCase activities were recovered by mixing complexes from pea and soybean. The results demonstrate that the active form of ACCase can be reassembled and that it could form a high-molecular-mass complex.

Purification and cDNA Cloning of Isochorismate Synthase from Elicited Cell Cultures of Catharanthus roseus

Isochorismate is an important metabolite formed at the end of the shikimate pathway, which is involved in the synthesis of both primary and secondary metabolites. It is synthesized from chorismate in a reaction catalyzed by the enzyme isochorismate synthase (ICS; EC 5.4.99.6). We have purified ICS to homogeneity from elicited Catharanthus roseus cell cultures. Two isoforms with an apparent molecular mass of 64 kD were purified and characterized. The Km values for chorismate were 558 and 319 μm for isoforms I and II, respectively. The isoforms were not inhibited by aromatic amino acids and required Mg2+ for enzyme activity. Polymerase chain reaction on a cDNA library from elicited C. roseus cells with a degenerated primer based on the sequence of an internal peptide from isoform II resulted in an amplification product that was used to screen the cDNA library. This led to the first isolation, to our knowledge, of a plant ICS cDNA. The cDNA encodes a protein of 64 kD with an N-terminal chloroplast-targeting signal. The deduced amino acid sequence shares homology with bacterial ICS and also with anthranilate synthases from plants. Southern analysis indicates the existence of only one ICS gene in C. roseus.

The Caenorhabditis elegans maternal-effect sterile proteins, MES-2, MES-3, and MES-6, are associated in a complex in embryos

The Caenorhabditis elegans maternal-effect sterile genes, mes-2, mes-3, mes-4, and mes-6, encode nuclear proteins that are essential for germ-line development. They are thought to be involved in a common process because their mutant phenotypes are similar. MES-2 and MES-6 are homologs of Enhancer of zeste and extra sex combs, both members of the Polycomb group of chromatin regulators in insects and vertebrates. MES-3 is a novel protein, and MES-4 is a SET-domain protein. To investigate whether the MES proteins interact and likely function as a complex, we performed biochemical analyses on C. elegans embryo extracts. Results of immunoprecipitation experiments indicate that MES-2, MES-3, and MES-6 are associated in a complex and that MES-4 is not associated with this complex. Based on in vitro binding assays, MES-2 and MES-6 interact directly, via the amino terminal portion of MES-2. Sucrose density gradient fractionation and gel filtration chromatography were performed to determine the Stokes radius and sedimentation coefficient of the MES-2/MES-3/MES-6 complex. Based on those two values, we estimate that the molecular mass of the complex is ≈255 kDa, close to the sum of the three known components. Our results suggest that the two C. elegans Polycomb group homologs (MES-2 and MES-6) associate with a novel partner (MES-3) to regulate germ-line development in C. elegans.

The chaperone GroEL is required for the final assembly of the molybdenum-iron protein of nitrogenase

It is known that an E146D site-directed variant of the Azotobacter vinelandii iron protein (Fe protein) is specifically defective in its ability to participate in iron-molybdenum cofactor (FeMoco) insertion. Molybdenum-iron protein (MoFe protein) from the strain expressing the E146D Fe protein is partially (≈45%) FeMoco deficient. The “free” FeMoco that is not inserted accumulates in the cell. We were able to insert this “free” FeMoco into the partially pure FeMoco-deficient MoFe protein. This insertion reaction required crude extract of the ΔnifHDK A. vinelandii strain CA12, Fe protein and MgATP. We used this as an assay to purify a required “insertion” protein. The purified protein was identified as GroEL, based on the molecular mass of its subunit (58.8 kDa), crossreaction with commercially available antibodies raised against E. coli GroEL, and its NH2-terminal polypeptide sequence. The NH2-terminal polypeptide sequence showed identity of up to 84% to GroEL from various organisms. Purified GroEL of A. vinelandii alone or in combination with MgATP and Fe protein did not support the FeMoco insertion into pure FeMoco-deficient MoFe protein, suggesting that there are still other proteins and/or factors missing. By using GroEL-containing extracts from a ΔnifHDK strain of A. vinelandii CA12 along with FeMoco, Fe protein, and MgATP, we were able to supply all required proteins and/or factors and obtained a fully active reconstituted E146D nifH MoFe protein. The involvement of the molecular chaperone GroEL in the insertion of a metal cluster into an apoprotein may have broad implications for the maturation of other metalloenzymes.

CML66, a broadly immunogenic tumor antigen, elicits a humoral immune response associated with remission of chronic myelogenous leukemia

This report describes a tumor-associated antigen, termed CML66, initially cloned from a chronic myelogenous leukemia (CML) cDNA expression library. CML66 encodes a 583-aa protein with a molecular mass of 66 kDa and no significant homology to other known genes. CML66 gene is localized to human chromosome 8q23, but the function of this gene is unknown. CML66 is expressed in leukemias and a variety of solid tumor cell lines. When examined by Northern blot, expression in normal tissues was restricted to testis and heart, and no expression was found in hematopoietic tissues. When examined by quantitative reverse transcription–PCR, expression in CML cells was 1.5-fold higher than in normal peripheral blood mononuclear cells. The presence of CML66-specific antibody in patient serum was confirmed by Western blot and the development of high titer IgG antibody specific for CML66 correlated with immune induced remission of CML in a patient who received infusion of normal donor lymphocytes for treatment of relapse. CML66 antibody also was found in sera from 18–38% of patients with lung cancer, melanoma, and prostate cancer. These findings suggest that CML66 may be immunogenic in a wide variety of malignancies and may be a target for antigen-specific immunotherapy.

Spermine Is a Salicylate-Independent Endogenous Inducer for Both Tobacco Acidic Pathogenesis-Related Proteins and Resistance against Tobacco Mosaic Virus Infection1

Intercellular spaces are often the first sites invaded by pathogens. In the spaces of tobacco mosaic virus (TMV)-infected and necrotic lesion-forming tobacco (Nicotiana tabacum L.) leaves, we found that an inducer for acidic pathogenesis-related (PR) proteins was accumulated. The induction activity was recovered in gel-filtrated fractions of low molecular mass with a basic nature, into which authentic spermine (Spm) was eluted. We quantified polyamines in the intercellular spaces of the necrotic lesion-forming leaves and found 20-fold higher levels of free Spm than in healthy leaves. Among several polyamines tested, exogenously supplied Spm induced acidic PR-1 gene expression. Immunoblot analysis showed that Spm treatment increased not only acidic PR-1 but also acidic PR-2, PR-3, and PR-5 protein accumulation. Treatment of healthy tobacco leaves with salicylic acid (SA) caused no significant increase in the level of endogenous Spm, and Spm did not increase the level of endogenous SA, suggesting that induction of acidic PR proteins by Spm is independent of SA. The size of TMV-induced local lesions was reduced by Spm treatment. These results indicate that Spm accumulates outside of cells after lesion formation and induces both acidic PR proteins and resistance against TMV via a SA-independent signaling pathway.

A Chloroplast DNA Helicase II from Pea That Prefers Fork-Like Replication Structures

A DNA helicase, called chloroplast DNA (ctDNA) helicase II, was purified to apparent homogeneity from pea (Pisum sativum). The enzyme contained intrinsic, single-stranded, DNA-dependent ATPase activity and an apparent molecular mass of 78 kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The DNA helicase was markedly stimulated by DNA substrates with fork-like replication structures. A 5′-tailed fork was more active than the 3′-tailed fork, which itself was more active than substrates without a fork. The direction of unwinding was 3′ to 5′ along the bound strand, and it failed to unwind blunt-ended duplex DNA. DNA helicase activity required only ATP or dATP hydrolysis. The enzyme also required a divalent cation (Mg2+>Mn2+>Ca2+) for its unwinding activity and was inhibited at 200 mm KCl or NaCl. This enzyme could be involved in the replication of ctDNA. The DNA major groove-intercalating ligands nogalamycin and daunorubicin were inhibitory to unwinding (Ki approximately 0.85 μm and 2.2 μm, respectively) and ATPase (Ki approximately 1.3 μm and 3.0 μm, respectively) activities of pea ctDNA helicase II, whereas ellipticine, etoposide (VP-16), and camptothecin had no effect on the enzyme activity. These ligands may be useful in further studies of the mechanisms of chloroplast helicase activities.

Characterization of a Granule-Bound Starch Synthase Isoform Found in the Pericarp of Wheat1

Waxy wheat (Triticum aestivum L.) lacks the waxy protein, which is also known as granule-bound starch synthase I (GBSSI). The starch granules of waxy wheat endosperm and pollen do not contain amylose and therefore stain red-brown with iodine. However, we observed that starch from pericarp tissue of waxy wheat stained blue-black and contained amylose. Significantly higher starch synthase activity was detected in pericarp starch granules than in endosperm starch granules. A granule-bound protein that differed from GBSSI in molecular mass and isoelectric point was detected in the pericarp starch granules but not in granules from endosperm. This protein was designated GBSSII. The N-terminal amino acid sequence of GBSSII, although not identical to wheat GBSSI, showed strong homology to waxy proteins or GBSSIs of cereals and potato, and contained the motif KTGGL, which is the putative substrate-binding site of GBSSI of plants and of glycogen synthase of Escherichia coli. GBSSII cross-reacted specifically with antisera raised against potato and maize GBSSI. This study indicates that GBSSI and GBSSII are expressed in a tissue-specific manner in different organs, with GBSSII having an important function in amylose synthesis in the pericarp.

Isolation and Characterization of a Histidine Biosynthetic Gene in Arabidopsis Encoding a Polypeptide with Two Separate Domains for Phosphoribosyl-ATP Pyrophosphohydrolase and Phosphoribosyl-AMP Cyclohydrolase

Phosphoribosyl-ATP pyrophosphohydrolase (PRA-PH) and phosphoribosyl-AMP cyclohydrolase (PRA-CH) are encoded by HIS4 in yeast and by hisIE in bacteria and catalyze the second and the third step, respectively, in the histidine biosynthetic pathway. By complementing a hisI mutation of Escherichia coli with an Arabidopsis cDNA library, we isolated an Arabidopsis cDNA (At-IE) that possesses these two enzyme activities. The At-IE cDNA encodes a bifunctional protein of 281 amino acids with a calculated molecular mass of 31,666 D. Genomic DNA-blot analysis with the At-IE cDNA as a probe revealed a single-copy gene in Arabidopsis, and RNA-blot analysis showed that the At-IE gene was expressed ubiquitously throughout development. Sequence comparison suggested that the At-IE protein has an N-terminal extension of about 50 amino acids with the properties of a chloroplast transit peptide. We demonstrated through heterologous expression studies in E. coli that the functional domains for the PRA-CH (hisI) and PRA-PH (hisE) resided in the N-terminal and the C-terminal halves, respectively, of the At-IE protein.

Intracellular β-Carbonic Anhydrase of the Unicellular Green Alga Coccomyxa1 : Cloning of the cDNA and Characterization of the Functional Enzyme Overexpressed in Escherichia coli

Carbonic anhydrase (CA) (EC 4.2.1.1) enzymes catalyze the reversible hydration of CO2, a reaction that is important in many physiological processes. We have cloned and sequenced a full-length cDNA encoding an intracellular β-CA from the unicellular green alga Coccomyxa. Nucleotide sequence data show that the isolated cDNA contains an open reading frame encoding a polypeptide of 227 amino acids. The predicted polypeptide is similar to β-type CAs from Escherichia coli and higher plants, with an identity of 26% to 30%. The Coccomyxa cDNA was overexpressed in E. coli, and the enzyme was purified and biochemically characterized. The mature protein is a homotetramer with an estimated molecular mass of 100 kD. The CO2-hydration activity of the Coccomyxa enzyme is comparable with that of the pea homolog. However, the activity of Coccomyxa CA is largely insensitive to oxidative conditions, in contrast to similar enzymes from most higher plants. Fractionation studies further showed that Coccomyxa CA is extrachloroplastic.

(+)-Germacrene A Biosynthesis : The Committed Step in the Biosynthesis of Bitter Sesquiterpene Lactones in Chicory

The leaves and especially the roots of chicory (Cichorium intybus L.) contain high concentrations of bitter sesquiterpene lactones such as the guianolides lactupicrin, lactucin, and 8-deoxylactucin. Eudesmanolides and germacranolides are present in smaller amounts. Their postulated biosynthesis through the mevalonate-farnesyl diphosphate-germacradiene pathway has now been confirmed by the isolation of a (+)-germacrene A synthase from chicory roots. This sesquiterpene cyclase was purified 200-fold using a combination of anion-exchange and dye-ligand chromatography. It has a Km value of 6.6 μm, an estimated molecular mass of 54 kD, and a (broad) pH optimum around 6.7. Germacrene A, the enzymatic product, proved to be much more stable than reported in literature. Its heat-induced Cope rearrangement into (−)-β-elemene was utilized to determine its absolute configuration on an enantioselective gas chromatography column. To our knowledge, until now in sesquiterpene biosynthesis, germacrene A has only been reported as an (postulated) enzyme-bound intermediate, which, instead of being released, is subjected to additional cyclization(s) by the same enzyme that generated it from farnesyl diphosphate. However, in chicory germacrene A is released from the sesquiterpene cyclase. Apparently, subsequent oxidations and/or glucosylation of the germacrane skeleton, together with a germacrene cyclase, determine whether guaiane- or eudesmane-type sesquiterpene lactones are produced.

Phosphorylated α(1→4)Glucans as Substrate for Potato Starch-Branching Enzyme I1

The possible involvement of potato (Solanum tuberosum L.) starch-branching enzyme I (PSBE-I) in the in vivo synthesis of phosphorylated amylopectin was investigated in in vitro experiments with isolated PSBE-I using 33P-labeled phosphorylated and 3H end-labeled nonphosphorylated α(1→4)glucans as the substrates. From these radiolabeled substrates PSBE-I was shown to catalyze the formation of dual-labeled (3H/33P) phosphorylated branched polysaccharides with an average degree of polymerization of 80 to 85. The relatively high molecular mass indicated that the product was the result of multiple chain-transfer reactions. The presence of α(1→6) branch points was documented by isoamylase treatment and anion-exchange chromatography. Although the initial steps of the in vivo mechanism responsible for phosphorylation of potato starch remains elusive, the present study demonstrates that the enzyme machinery available in potato has the ability to incorporate phosphorylated α(1→4)glucans into neutral polysaccharides in an interchain catalytic reaction. Potato mini tubers synthesized phosphorylated starch from exogenously supplied 33PO43− and [U-14C]Glc at rates 4 times higher than those previously obtained using tubers from fully grown potato plants. This system was more reproducible compared with soil-grown tubers and was therefore used for preparation of 33P-labeled phosphorylated α(1→4)glucan chains.