Cloning and sequencing of protochlorophyllide reductase.
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ABSTRACT:
SUBMITTER: Darrah PM
PROVIDER: S-EPMC1133702 | biostudies-other | 1990 Feb
REPOSITORIES: biostudies-other
Similar Datasets
Project description:A full-length protochlorophyllide reductase clone (pWR5) has been isolated from a dark-grown wheat (Triticum aestivum) cDNA library generated in the phage vector lambda gt10. Comparison of the sequence of pWR5 with published sequences indicates a high degree of conservation of the structure of the mature protein amongst species but with the structure of the transit peptide less highly conserved. Within the cereals, the structure of the complete preprotein shows a remarkable degree of sequence homology (98% between barley and wheat). In vitro expression of pWR5 generates a preprotein of the expected molecular mass, approx. 41 kDa. Isolated pea chloroplasts can import, process and locate the mature reductase to the thylakoid membranes. From analysis of the CNBr-cleavage fragments of the N-[3H]phenylmaleimide-treated enzyme, the substrate-protected cysteine group in the enzyme is tentatively identified as Cys-296.
| S-EPMC1137677 | biostudies-other
Project description:Illumination of etiolated plants effects the activity of protochlorophyllide reductase (NADPH-protochlorophyllide oxidoreductase) in the plastids. Constant illumination or a 2-min light-triggering of etiolated plants leads to an approx. 80% decrease in activity of the enzyme, a change that can be reversed by returning the plants to darkness. The change in activity results from an alteration of the Vmax. rather than Km. Despite the fact that exogenous pigments effect the activity of the enzyme in vitro, no correlation could be drawn between the concentrations of pigments in vivo and activity of the enzyme.
| S-EPMC1161924 | biostudies-other
Project description:DNA sequence analysis of a region of the Streptomyces sp. strain C5 daunomycin biosynthesis gene cluster, located just upstream of the daunomycin polyketide biosynthesis genes, revealed the presence of six complete genes. The two genes reading right to left include genes encoding the potentially translationally coupled gene products, an acyl carrier protein and a ketoreductase, and the four genes reading divergently, left to right, include two open reading frames of unknown function followed by a gene encoding an apparent glycosyltransferase and dauE, encoding aklaviketone reductase. Extracts of Streptomyces lividans TK24 containing recombinant DauE catalyzed the NADPH-specific conversion of aklaviketone, maggiemycin, and 7-oxodaunomycinone to aklavinone, epsilon-rhodomycinone, and daunomycinone, respectively. Neither the product of dauB nor that of the ketoreductase gene directly downstream of the acyl carrier protein gene demonstrated aklaviketone reductase activity.
| S-EPMC178101 | biostudies-other
Project description:1. DNA sequences hybridizing with the wheat protochlorophyllide reductase gene have been detected during genomic Southern blots of various cyanobacterial DNA samples. No such hybridization was observed with DNA from photosynthetic bacteria. 2. A fragment amplified from Phormidium laminosum DNA has been characterized and shown to be 73% similar to the corresponding wheat sequence. At the protein level the similarity is 91%. When used as a probe for Southern blotting the Phormidium DNA fragment confirmed the authenticity of some of the original signals obtained with the wheat probe. 3. Peptides of molecular mass 36, 30 and 60 kDa are immunodetected by a wheat reductase antibody during Western blotting of Phormidium preparations. These are purported to correspond to the cyanobacterial mature reductase protein, a stable proteolytic fragment and soluble dimeric forms of the latter respectively. 4. Adaptation of Phormidium to growth in red light (delta > 670 nm) or darkness led to no significant changes in the total level of immunodetected peptides or protochlorophyllide within the cells. 5. The specific activity of the reductase in Phormidium membranes has been tentatively estimated as 0.5 unit/mg of protein, a value comparable with that found in preparations from mature chloroplasts of higher plants.
| S-EPMC1136016 | biostudies-other
Project description:The biosynthesis of the tetrapyrrole end-products chlorophyll and heme depends on a multifaceted control mechanism that acts primarily at the post-translational level upon the rate-limiting step of 5-aminolevulinic acid synthesis and upon light-dependent protochlorophyllide oxidoreductase (POR). These regulatory processes require auxiliary factors that modulate the activity, stability, complex formation, and subplastidal localization of the relevant proteins. Together, they ensure optimal metabolic flow during the day and at night. As an Arabidopsis homolog of the POR-interacting tetratricopeptide-repeat protein (Pitt) first reported in Synechocystis, we characterize tetrapyrrole biosynthesis-regulating tetratricopeptide-repeat protein1 (TTP1). TTP1 is a plastid-localized, membrane-bound factor that interacts with POR, the Mg protoporphyrin monomethylester cyclase CHL27, glutamyl-tRNA reductase (GluTR), GluTR-binding protein, and FLUORESCENCE IN BLUE LIGHT. Lack of TTP1 leads to accumulation of GluTR, enhanced 5-aminolevulinic acid synthesis and lower levels of POR. Knockout mutants show enhanced sensitivity to reactive oxygen species and a slower greening of etiolated seedlings. Based on our studies, the interaction of TTP1 with GluTR and POR does not directly inhibit their enzymatic activity and contribute to the control of 5-aminolevulinic acid synthesis. Instead, we propose that TTP1 sequesters a fraction of these proteins on the thylakoid membrane, and contributes to their stability.
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Project description:The gene encoding sulfide-quinone reductase (SQR; E.C.1.8.5.'), the enzyme catalyzing the first step of anoxygenic photosynthesis in the filamentous cyanobacterium Oscillatoria limnetica, was cloned by use of amino acid sequences of tryptic peptides as well as sequences conserved in the Rhodobacter capsulatus SQR and in an open reading frame found in the genome of Aquifex aeolicus. SQR activity was also detected in the unicellular cyanobacterium Aphanothece halophytica following sulfide induction, with a V(max) of 180 micromol of plastoquinone-1 (PQ-1) reduced/mg of chlorophyll/h and apparent K(m) values of 20 and 40 microM for sulfide and quinone, respectively. Based on the conserved sequences, the gene encoding A. halophytica SQR was also cloned. The SQR polypeptides deduced from the two cyanobacterial genes consist of 436 amino acids for O. limnetica SQR and 437 amino acids for A. halophytica SQR and show 58% identity and 74% similarity. The calculated molecular mass is about 48 kDa for both proteins; the theoretical isoelectric points are 7.7 and 5.6 and the net charges at a neutral pH are 0 and -14 for O. limnetica SQR and A. halophytica SQR, respectively. A search of databases showed SQR homologs in the genomes of the cyanobacterium Anabaena PCC7120 as well as the chemolithotrophic bacteria Shewanella putrefaciens and Thiobacillus ferrooxidans. All SQR enzymes contain characteristic flavin adenine dinucleotide binding fingerprints. The cyanobacterial proteins were expressed in Escherichia coli under the control of the T7 promoter. Membranes isolated from E. coli cells expressing A. halophytica SQR performed sulfide-dependent PQ-1 reduction that was sensitive to the quinone analog inhibitor 2n-nonyl-4-hydroxyquinoline-N-oxide. The wide distribution of SQR genes emphasizes the important role of SQR in the sulfur cycle in nature.
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Project description:Ribonucleoside-triphosphate reductase (RTPR, EC 1.17.4.2) from Lactobacillus leichmannii, a monomeric adenosylcobalamin-requiring enzyme, catalyzes the conversion of nucleoside triphosphates to deoxynucleoside triphosphates. The gene for this enzyme has been cloned and sequenced. In contrast to expectations based on mechanistic considerations, there is no statistically significant sequence homology with the Escherichia coli reductase that requires a dinuclear-iron center and tyrosyl radical cofactor. The RTPR has been overexpressed and purified to homogeneity, yielding 90 mg of protein from 2.5 g of bacteria. Initial characterization of the recombinant RTPR indicates that its properties are identical to those of the RTPR isolated from L. leichmannii.
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Project description:1. The substrate specificity of the enzyme protochlorophyllide reductase in barley (Hordeum vulgare) etioplasts was investigated. 2. It was shown that naturally occurring esterified protochlorophyllide and chemically prepared protochlorophyllide methyl ester are not substrates for the enzyme, suggesting an important role for the C-7 carboxylic acid group in binding of the porphyrin to the enzyme. 3. Removal of magnesium from the protochlorophyllide leads to inactivity of the compound as a substrate for the enzyme. However, activity can be restored by replacing the magnesium with zinc, whereas nickel, copper or cobalt failed to restore substrate activity. 4. Binding of the second substrate, NADPH, to the enzyme probably occurs through the 2'-phosphate group in the coenzyme.
| S-EPMC1161527 | biostudies-other
Project description:Gene grdA, which encodes selenoprotein A of the glycine reductase complex from Clostridium sticklandii, was identified and characterized. This gene encodes a protein of 158 amino acids with a calculated M(r) of 17,142. The known sequence of 15 amino acids around the selenocysteine residue and the known carboxy terminus of the protein are correctly predicted by the nucleotide sequence. An opal termination codon (TGA) corresponding to the location of the single selenocysteine residue in the polypeptide was found in frame at position 130. The C. sticklandii grdA gene was inserted behind the tac promotor of an Escherichia coli expression vector. An E. coli strain transformed with this vector produced an 18-kDa polypeptide that was not detected in extracts of nontransformed cells. Affinity-purified anti-C. sticklandii selenoprotein A immunoglobulin G reacted specifically with this polypeptide, which was indistinguishable from authentic C. sticklandii selenoprotein A by immunological analysis. Addition of the purified expressed protein to glycine reductase protein components B and C reconstituted the active glycine reductase complex. Although synthesis of enzymically active protein A depended on the presence of selenium in the growth medium, formation of immunologically reactive protein did not. Moreover, synthesis of enzymically active protein in a transformed E. coli selD mutant strain indicated that there is a nonspecific mechanism of selenocysteine incorporation. These findings imply that mRNA secondary structures of C. sticklandii grdA are not functional for UGA-directed selenocysteine insertion in the E. coli expression system.
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Project description:Human rhinoviruses (RV) are associated with the majority of viral respiratory illnesses in infants, children and adults. Over the last several years, researchers have begun to sequence the many different species and strains of RV in order to determine if certain species were associated with increased disease severity. There are a variety of techniques employed to prepare samples for sequencing. One method utilizes plasmid-cloning, which is expensive and takes several hours to complete. Recently, some investigators have instead used direct sequencing to sequence RV strains, allowing for omission of the time- and labor-intensive cloning step. This study formally compares and contrasts the sequencing results obtained from plasmid-cloning and direct Sanger sequencing of a 500 base pair PCR product covering the VP4/VP2 region of RV. A slightly longer sequence (by 65 base pairs on average) was obtained when specimens were plasmid-cloned, and the sequences were 86% similar. After trimming the extra base pairs from the cloned sequences, the sequences were 99.7% identical. Overall success of directly sequencing samples was similar to that of cloning, 5% on average failed for each technique. Therefore, in many instances, directly sequencing samples may be considered in lieu of the more expensive and time-consuming plasmid-cloning technique.
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