Engineering bi‐functional enzyme complex of formate dehydrogenase and leucine dehydrogenase by peptide linker mediated fusion for accelerating cofactor regeneration

This study reports the application of peptide linker in the construction of bi‐functional formate dehydrogenase (FDH) and leucine dehydrogenase (LeuDH) enzymatic complex for efficient cofactor regeneration and L‐tert leucine (L‐tle) biotransformation. Seven FDH‐LeuDH fusion enzymes with different peptide linker were successfully developed and displayed both parental enzyme activities. The incorporation order of FDH and LeuDH was investigated by predicting three‐dimensional structures of LeuDH‐FDH and FDH‐LeuDH models using the I‐TASSER server. The enzymatic characterization showed that insertion of rigid peptide linker obtained better activity and thermal stability in comparison with flexible peptide linker. The production rate of fusion enzymatic complex with suitable flexible peptide linker was increased by 1.2 times compared with free enzyme mixture. Moreover, structural analysis of FDH and LeuDH suggested the secondary structure of the N‐, C‐terminal domain and their relative positions to functional domains was also greatly relevant to the catalytic properties of the fusion enzymatic complex. The results show that rigid peptide linker could ensure the independent folding of moieties and stabilized enzyme structure, while the flexible peptide linker was likely to bring enzyme moieties in close proximity for superior cofactor channeling.     

Purification and characterization of the m7G(5')pppN-pyrophosphatase from human placenta

A purification scheme has been developed for the m7G(5')pppN-pyrophosphatase from human placenta. The 1400-fold purified placental enzyme exhibited physical and enzymatic properties similar to those previously reported for a crude preparation of the human m7G(5')pppN-pyrophosphatase obtained from HeLa cells. Polyacrylamide gel analysis of enzyme fractions at different stages of purification revealed a Mr = 40,000 polypeptide that increased in relative concentration as the specific activity of the enzyme fractions increased. Copurification of this polypeptide with m7G(5')pppN-pyrophosphatase activity suggests the possibility that the 81,000-dalton native enzyme is a dimer composed of subunits of identical molecular weight. The highly purified placental enzyme, like the crude HeLa enzyme, failed to hydrolyze the cap moiety of intact mRNA even under conditions known to reduce mRNA secondary structure. Moreover, when a series of capped oligonucleotides that differed progressively in chain length by a factor of one nucleotide was tested as substrate, the rate of enzyme-catalyzed cap hydrolysis decreased as the chain length increased. The purified placental enzyme failed to release m7pG from oligonucleotides containing the cap and 3 or more additional nucleotides. These results are discussed in terms of the probable biological function of the m7G(5')pppN-pyrophosphatase.     

Conformational studies on the inactivation of glyceraldehyde-3-phosphate dehydrogenase from the facultative thermophile Bacillus coagulans KU

Among the various proposals that have been made in attempting to explain the ability of thermophiles to reproduce at high temperatures, there is no doubt that obligate and extreme thermophiles synthesize proteins (and other molecules) that have sufficient intrinsic molecular stability to withstand increased thermal stress. In contrast, the glyceraldehyde-3-phosphate dehydrogenase from the facultative thermophile Bacillus coagulans KU has been shown to be quite thermolabile in vitro. Thermal inactivation is not due to loss of bound NAD+. It has also been shown that the enzymatic activity can be thermostabilized in vitro by increased ionic strength. As previously reported [J. W. Crabb, A. L. Murdock, and R. E. Amelunxen (1975) Biochem. Biophys. Res. Commun. 62, 627; (1977) Biochemistry 16, 4840], the enzyme loses 94-97% of enzymatic activity after heat treatment at 55 degrees C for 5 min in 0.05 M sodium phosphate buffer (pH 7.1); however, by increasing the ionic strength to 1.8, complete protection was conferred at this temperature. Gel-filtration chromatography has been used to study the initial dissociation and subsequent aggregation of the glyceraldehyde-3-phosphate dehydrogenase after thermal inactivation. Aggregation occurs when the enzyme is heated at 50 degrees or 55 degrees C. Loss of enzymatic activity is correlated with changes in the tertiary structure as measured by the near-uv CD spectrum of the enzyme following heat inactivation, with essential disappearance of the peaks at 263 and 296 nm, and a blue shift of the far-uv spectrum, which is a measure of secondary structure. Estimation of secondary structure of the unheated protein from the far-uv CD data showed the enzyme contains approximately 26% alpha-helix, approximately 21% beta-structure, and approximately 53% disordered structure. Heat treatment at various temperatures resulted in only slight changes of the estimated secondary structure. Increased ionic strength prevents thermal alteration of the CD spectrum in both near- and far-uv regions. The data support the previous proposal that thermolabile enzymes such as the glyceraldehyde-3-phosphate dehydrogenase from the facultative thermophile B. coagulans are thermostabilized in vivo mainly by the intracellular charged macromolecular environment.     

The lysozyme from insect (Manduca sexta) is a cold-adapted enzyme

Enzymatic activity is dependent on temperature, although some proteins have evolved to retain activity at low temperatures at the expense of stability. Cold adapted enzymes are present in a variety of organisms and there is ample interest in their structure-function relationships. Lysozyme (E.C. 3.2.1.17) is one of the most studied enzymes due to its antibacterial activity against Gram positive bacteria and is also a cold adapted protein. In this work the characterization of lysozyme from the insect Manduca sexta and its activity at low temperatures is presented. Both M. sexta lysozymes natural and recombinant showed a higher content of alpha-helix secondary structure compared to that of hen egg white lysozyme and a higher specific enzymatic activity in the range of 5-30 degrees C. These results together with measured thermodynamic activation parameters support the designation of M. sexta lysozyme as a cold adapted enzyme. Therefore, the insect recombinant lysozyme is feasible as a model for structure-function studies for cold-adapted proteins.     

Mechanism of polynucleotide phosphorylase

The de novo polymerization of RNA initiated by polynucleotide phosphorylase from nucleoside diphosphates was examined. End group analysis performed under conditions designed to specifically end label the polymer revealed no evidence for a 5'-pyrophosphate-terminated polymer. However, we observed preferential incorporation of the ADP alpha S(RP) diastereomer into the 5' end (Marlier & Benkovic, 1982) in chain initiation, suggesting that the enzyme incorporates a nucleoside diphosphate specifically into the 5' end of the product, with subsequent enzymatic removal of the polyphosphate linkage. No evidence could be obtained for a covalent adduct between the enzyme and the 5' end of the polymer chain, despite the high processivity of the polymerization reaction. Gel electrophoretic analysis showed the polymer to be highly disperse, varying from 1 to 30 kb. Scanning transmission electron microscopy supported this product analysis and further suggested that (i) each subunit can produce an RNA polymer and (ii) both 5' and 3' ends of the RNA can be bound simultaneously to the same or differing enzyme molecules.     

化学添加剂提高重组α-环糊精葡萄糖基转移酶酶制剂稳定性

通过向重组α-环糊精葡萄糖基转移酶 (α-CGT酶) 液中添加化学添加剂以提高其热稳定性及贮存稳定性。在不同温度下研究了添加剂对酶液的贮存稳定性影响,并用圆二色谱 (CD) 研究了CGT酶在近紫外区和远紫外区蛋白质结构与热稳定性的变化关系。当单独加入各种添加剂在50 ℃水浴1 h和室温放置108 d后,发现含有20％甘油的酶液稳定性最好,与未加任何添加剂的对照酶液相比仍有91％和50％的酶活,对照酶液在50 ℃水浴1 h后仅有小于10％的活性,室温放置108 d后已经没有酶活。明胶、CaCl2和PEG40     

Structure-activity relationship on fungal laccase from Rigidoporus lignosus: a Fourier-transform infrared spectroscopic study

The structure and thermal stability of a laccase from Rigidoporus lignosus (Rl) was analysed by Fourier-transform infrared (FT-IR) spectroscopy. The enzyme was depleted of copper atoms, then part of the apoenzyme was re-metalled and these two forms of the protein were analysed as well. The enzymatic activity, lost by the removal of copper atoms, was restored in the re-metalled apoenzyme and resulted similar to that of native protein. The infrared data indicated that the enzyme contains a large amount of beta-sheets and a small content of alpha-helices, and it displayed a marked thermostability showing the T(m) at 92.5 degrees C. The apoenzyme and the re-metalled apoenzyme did not show remarkable differences in the secondary structure with respect to the native protein, but the thermal stability of the apoenzyme was dramatically reduced showing a T(m) close to 72 degrees C, while the re-metalled protein displayed the T(m) at 90 degrees C. These data indicate that copper atoms, beside their role in catalytic activity, play also an important role on the stabilisation of the structure of Rl laccase. About 35% of the polypeptide chain is buried and/or constitutes a particular compact structure, which, beside copper atoms, is probably involved in the high thermal stability of the protein. Another small part of the structure is particularly sensitive to high temperatures and it could be the cause of the loss of enzymatic activity when the temperature is raised above 45-50 degrees C.     

Functional characterization of a novel xylanase from a corn strain of Erwinia chrysanthemi

A beta-1,4-xylan hydrolase (xylanase A) produced by Erwinia chrysanthemi D1 isolated from corn was analyzed with respect to its secondary structure and enzymatic function. The pH and temperature optima for the enzyme were found to be pH 6.0 and 35 degrees C, with a secondary structure under those conditions that consists of approximately 10 to 15% alpha-helices. The enzyme was still active at temperatures higher than 40 degrees C and at pHs of up to 9.0. The loss of enzymatic activity at temperatures above 45 degrees C was accompanied by significant loss of secondary structure. The enzyme was most active on xylan substrates with low ratios of xylose to 4-O-methyl-D-glucuronic acid and appears to require two 4-O-methyl-D-glucuronic acid residues for substrate recognition and/or cleavage of a beta-1,4-xylosidic bond. The enzyme hydrolyzed sweetgum xylan, generating products with a 4-O-methyl-glucuronic acid-substituted xylose residue one position from the nonreducing terminus of the oligoxyloside product. No internal cleavages of the xylan backbone between substituted xylose residues were observed, giving the enzyme a unique mode of action in the hydrolysis compared to all other xylanases that have been described. Given the size of the oligoxyloside products generated by the enzyme during depolymerization of xylan substrates, the function of the enzyme may be to render substrate available for other depolymerizing enzymes instead of producing oligoxylosides for cellular metabolism and may serve to produce elicitors during the initiation of the infectious process.     

Identification of sequences within the murine granulocyte-macrophage colony-stimulating factor mRNA 3'-untranslated region that mediate mRNA stabilization induced by mitogen treatment of EL-4 thymoma cells

Phorbol esters (TPA) and concanavalin A (ConA) are known to induce granulocyte-macrophage colony-stimulating factor (GM-CSF) production in murine thymoma EL-4 cells by mRNA stabilization. The role of the 3'-untranslated region (3'-UTR) in GM-CSF mRNA stabilization induced by TPA and ConA in EL-4 cells was examined by transfection studies using chloramphenicol acetyltransferase (CAT) constructions. The GM-CSF 3'-UTR contains a 63-nucleotide region at its 3' end with repeating ATTTA motifs which is responsible for mRNA degradation in a variety of cell types (Shaw, G., and Kamen, R. (1986) Cell 46, 659-666). We produced constructs containing most of the GM-CSF 3'-UTR (303 nucleotides, pRSV-CATgm) or the 3'-terminal AT-rich region (116 nucleotides, pRSV-CATau) and measured CAT enzyme activity and CAT mRNA after transient transfection into EL-4 and NIH 3T3 cells. Low levels of CAT activity were seen in both cells with either plasmid compared with levels of CAT activity obtained with pRSV-CAT. TPA treatment caused an approximately 10-fold increase in CAT activity and mRNA in EL-4 cells transfected with pRSV-CATgm. No increases were seen in EL-4 cells transfected with pRSV-CATau or pRSV-CAT. No response to TPA was detected in transfected NIH 3T3 cells, indicating that the response to TPA is relatively cell-specific. There was no increase in CAT activity after ConA treatment in EL-4 or NIH 3T3 cells transfected with any of the constructs suggesting that the GM-CSF 3'-UTR lacks elements that can respond alone to ConA. Nuclear run-on and actinomycin D chase experiments in EL-4 cells showed that TPA induces CAT activity via mRNA stabilization. By linker-substitution mutagenesis we show that TPA inducibility depends on a 60-nucleotide region of the 3'-UTR whose 5' end is located 160 nucleotides upstream of the 5' end of the AU-rich region.     

Correlation of structural and functional thermal stability of the integral membrane protein Na,K-ATPase

The membrane-bound cation-transporting P-type Na,K-ATPase isolated from pig kidney membranes is much more resistant towards thermal inactivation than the almost identical membrane-bound Na,K-ATPase isolated from shark rectal gland membranes. The loss of enzymatic activity is correlated well with changes in protein structure as determined using synchrotron radiation circular dichroism (SRCD) spectroscopy. The enzymatic activity is lost at a 12°C higher temperature for pig enzyme than for shark enzyme, and the major changes in protein secondary structure also occur at T(m)'s that are ~10-15°C higher for the pig than for the shark enzyme. The temperature optimum for the rate of hydrolysis of ATP is about 42°C for shark and about 57°C for pig, both of which are close to the temperatures for onset of thermal unfolding. These results suggest that the active site region may be amongst the earliest parts of the structure to unfold. Detergent-solubilized Na,K-ATPases from the two sources show the similar differences in thermal stability as the membrane-bound species, but inactivation occurs at a lower temperature for both, and may reflect the stabilizing effect of a bilayer versus a micellar environment.     

Effects of sodium alginate elicitation on secondary metabolites and antioxidant activity of safflower genotypes under in vitro salinity stress

The present study was aimed at investigating the effects of different concentrations of sodium alginate (NaAlg) (0.075 and 0.15% (w/v)) on the production of secondary metabolites (SMs) and antioxidant activity of seven safflower genotypes under in vitro salinity stress. The results showed that total phenolic content (TPC), total flavonoids (TFD), total flavonols (TFL), anthocyanin (Ant), total antioxidant capacity (TAC), phenylalanine ammonia-lyase (PAL), catalase (CAT) activity, and lipid peroxidation significantly increased under salinity stress consisting of the concentration of 1.5% (w/v) of NaCl, but callus growth traits decreased. The highest amount of TPC, Ant, and callus growth traits was observed under the elicitation of the sample with the concentration of 0.075(%) NaAlg under salinity stress, but the highest amount for TFD, TFL, CAT, PAL, and TAC was observed under elicitation of the sample with the concentration of 0.15% of NaAlg under salinity stress. This indicated the superiority of NaAlg for elicitation to increase SMs in safflower under salinity stress. Overall, the results showed that genotypes of Mex.22-191 and GE₆₂₉₁₈ could be processed to produce SMs by eliciting NaCl in safflower as an important medicinal plant at cellular level. A positive and significant correlation between CAT and TPC was observed and indicates that phenolic compounds are the major contributors to the antioxidant potential in safflower. This new elicitor introduced new ways to select and exploit the best NaAlg concentration to develop SMs that are of tremendous importance in terms of commercial purposes along with medical features in safflower at cellular level.

     

High-temperature cultivation and 5' mRNA optimization are key factors for the efficient overexpression of thermostable Deinococcus geothermalis purine nucleoside phosphorylase in Escherichia coli

Overexpression of genes from thermophiles in Escherichia coli is an attractive approach towards the large-scale production of thermostable biocatalysts. However, various factors can challenge efficient heterologous protein expression--one example is the formation of stable 5' mRNA secondary structures that can impede an efficient translation initiation. In this work, we describe the expression optimization of purine nucleoside phosphorylase from the thermophilic microbe Deinococcus geothermalis in E. coli. Poor expression levels caused by stable secondary 5' mRNA structure formation were addressed by two different approaches: (i) increasing the cultivation temperature above the range used typically for recombinant protein expression and (ii) optimizing the 5' mRNA sequence for reduced secondary structures in the translation initiation region. The increase of the cultivation temperature from 30°C to 42°C allowed a more than 10-fold increase of activity per cell and optimizing the 5' mRNA gene sequence further increased the activity per cell 1.7-fold at 42°C. Thus, the combination of high-temperature cultivation and 5' sequence optimization is described as an effective approach to overcome poor expression levels resulting from stable secondary 5' mRNA structure formation. We suggest that this method is especially suitable for improving the expression of proteins derived from thermophiles in E. coli.     

Stimulation of ornithine decarboxylase synthesis and its control by polyamines in regenerating rat liver and cultured rat hepatoma cells.

Ornithine decarboxylase has been induced in log phase hepatoma cells grown in suspension culture. Induction with N6, O2'-dibutyryl cyclic adenosine 3':5'-monophosphate produced a 4-fold increase in enzyme activity by 3 hours which was followed by a return to base levels by 6 hours. Induction with dexamethasone, a potent synthetic glucocorticoid, exhibited a slow steady rate of increase in enzyme activity, reaching a plateau level of approximately 5- to 6-fold stimulation by about 12 hours. Induced cell and regenerating rat liver ornithine decarboxylase were shown to be indistinguishable by titration with antibody monospecific to the latter and by heat stability. L-[14C]Leucine incorporation into immunoprecipitable enzyme protein after induction in vitro or partial hepatectomy showed an increase which, when coupled with the increase in enzymatic activity, indicated de novo synthesis of enzyme protein. Physiological concentrations of the naturally occurring polyamines, spermidine and spermine, abolish cyclic AMP induction whereas they have no effect on dexamethasone induction. Both inductions were abolished by cycloheximide; in contrast, inhibition by actinomycin D was complete for dexamethasone induction and only partial with respect to cyclic AMP induction. The different time pattern of induction seen with cyclic AMP and dexamethasone, the partial inhibition of the cyclic AMP induction seen with actinomycin D, as well as the absence of inhibition of the dexamethasone induction by polyamines, indicate that these inducers might affect different aspects of the control of the same enzyme.