Characteristics of Nicotiana tabacum nitrate reductase protein produced in Saccharomyces cerevisiae.
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ABSTRACT:
SUBMITTER: Truong HN
PROVIDER: S-EPMC1151355 | biostudies-other | 1991 Sep
REPOSITORIES: biostudies-other
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Project description:BackgroundThis study aimed to identify the transcription factors of nitrate reductase genes (NIA1 and NIA2) promoters and hypothetical cis-element of NRE2. Based on the constructed cDNA library of Nicotiana tabacum K326, a yeast one-hybrid system was established using the Matchmaker® Gold Yeast One-Hybrid Library Screening System from Clontech. The transcription factors of NIA1 andNIA2 promoters and NRE2 cis-elements were screened.ResultsAfter sequencing and bioinformatics analysis, 15 cDNA sequences were identified: 9 for NIA1 (including XP_016503563.1 and NP_001312236.1), 3 for NIA2 (including XP_016510250.1), and 3 for NRE2 (including XM_016576899.1). XP_016503563.1 was annotated in PREDICTED: CRM-domain containing factor CFM3, and NP_001312236.1chloroplastic/mitochondrial-like in Nicotiana tabacum. NP_001312236.1 was annotated in Sulfite oxidase-like of Nicotiana tabacum. XP_016510250.1 was annotated as PREDICTED: uncharacterized protein LOC107827596 in Nicotiana tabacum. XM_016576899.1 was annotated in PREDICTED: Nicotiana tabacum RING-H2 finger protein ATL16-like (LOC107759033).ConclusionA yeast one-hybrid library was successfully constructed. The identified transcription factors may provide a theoretical basis for the study of plant nitrate reductase.
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Project description:1. Coproporphyrinogenase was extracted and purified from tobacco (Nicotiana tabacum L.). Enzyme activity was mainly located in mitochondria rather than in chloroplasts. The enzyme was purified by differential centrifugation, ammonium sulphate fractionation, calcium phosphate gel adsorption and dialysis. A 69-fold final purification was obtained. 2. An apparent K(m) value of 3.6x10(-5)m was found, the value being largely dependent on the amount of coproporphyrin III recovered after reduction with sodium amalgam to coproporphyrinogen III. Protoporphyrin formation was linear up to 3h and decreased with further incubation. The enzyme activity increased with the concentration of enzyme protein up to 30mug/ml of solution. 3. Enzyme activity was greatly enhanced by increasing Fe(2+) concentrations up to 0.5mm, beyond which inhibition occurred. Co(2+) and Mn(2+) were also found to activate at low concentrations (0.1mm) and inhibit at higher concentrations (5mm). Fe(3+) and Cu(2+), both at 0.1mm, and o-phenanthroline and EDTA, each at 1mm, were found to be inhibitory.
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Project description:Plant molecular farming (PMF) is an important growing prospective approach in plant biotechnology; it includes production of recombinant pharmaceutical and industrial proteins in large quantities from engineered plants. Elastin is a major protein component of tissues that require elasticity, it helps keep skin smooth as it stretches to allow normal. Elastin is used as a raw material for the cosmetic industry. In this work, we aimed to use plant as a bioreactor for the expression and production of the full human tropoelastin protein. Agrobacterium- mediated transient expression system into Nicotiana tabacum using syringe agroinfiltration was used to provide fast and convenient way to produce recombinant proteins with greater expression overall the plant leaf. This study aimed to establish an efficient and rapid system for transiently expression and production of human recombinant tropoelastin protein in transgenic N. tabacum plants. Modified elastin (ELN) gene was biosynthesized and cloned into pCambia1390 vector to be used into N. tabacum agroinfilteration. Optimization of codon usage for the human tropoelastin gene, without changing the primary structure of the protein was carried out to ensure high expression in tobacco plants. The obtained data proved that the 5(th) day post-infiltration is the optimum interval to obtain the maximum production of our recombinant protein. Southern blot analysis was able to detect 2175 bp fragment length representing the ELN orf (open reding frame). On the other hand, ELN -expression within plant's tissue was visualized by RT-PCR during the period 3-10 days post agroinfiltration. At the protein level, western and ELISA confirmed the expression of recombinant tropoelastin protein. Western blot analysis detected the tropoelastin protein as parent band at ?70 kDa from freshly extracted protein, while two degraded bands of ?55 and ?45 kDa, representing a pattern of tropoelastin were appeared with frozen samples. This study showed that biosynthetic ELN gene was successfully expressed into N. tabacum leaves using agroinfiltration technique.
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Project description:The application of biocatalysis for the asymmetric reduction of activated C=C is a powerful tool for the manufacture of high-value chemical commodities. The biocatalytic potential of "-ene" reductases from the Old Yellow Enzyme (OYE) family of oxidoreductases is well-known; however, the specificity of these enzymes toward mainly small molecule substrates has highlighted the need to discover "-ene" reductases from different enzymatic classes to broaden industrial applicability. Here, we describe the characterization of a flavin-free double bond reductase from Nicotiana tabacum (NtDBR), which belongs to the leukotriene B4 dehydrogenase (LTD) subfamily of the zinc-independent, medium chain dehydrogenase/reductase superfamily of enzymes. Using steady-state kinetics and biotransformation reactions, we have demonstrated the regio- and stereospecificity of NtDBR against a variety of ?,?-unsaturated activated alkenes. In addition to catalyzing the reduction of typical LTD substrates and several classical OYE-like substrates, NtDBR also exhibited complementary activity by reducing non-OYE substrates (i.e., reducing the exocyclic C=C double bond of (R)-pulegone) and in some cases showing an opposite stereopreference in comparison with the OYE family member pentaerythritol tetranitrate (PETN) reductase. This serves to augment classical OYE "-ene" reductase activity and, coupled with its aerobic stability, emphasizes the potential industrial value of NtDBR. Furthermore, we also report the X-ray crystal structures of the holo-, binary NADP(H)-bound, and ternary [NADP+ and 4-hydroxy-3-methoxycinnamaldehyde (9a)-bound] NtDBR complexes. These will underpin structure-driven site-saturated mutagenesis studies aimed at enhancing the reactivity, stereochemistry, and specificity of this enzyme.
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Project description:The phosphorylation status of a protein is highly regulated and is determined by the opposing activities of protein kinases and protein phosphatases within the cell. While much is known about the protein kinases found in Saccharomyces cerevisiae, the protein phosphatases are much less characterized. Of the 127 protein kinases in yeast, over 90% are in the same evolutionary lineage. In contrast, protein phosphatases are fewer in number (only 43 have been identified in yeast) and comprise multiple, distinct evolutionary lineages. Here we review the protein phosphatase families of yeast with regard to structure, catalytic mechanism, regulation, and signal transduction participation.
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