scholarly journals Expression of the Schwanniomyces occidentalis SWA2 amylase in Saccharomyces cerevisiae: role of N-glycosylation on activity, stability and secretion

1998 ◽  
Vol 329 (1) ◽  
pp. 65-71 ◽  
Author(s):  
Esther YÁÑEZ ◽  
A. Teresa CARMONA ◽  
Mercedes TIEMBLO ◽  
Antonio JIMÉNEZ ◽  
María FERNÁNDEZ-LOBATO
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Catalytic Efficiency ◽  
Extracellular Enzyme ◽  
Site Directed Mutagenesis ◽  
Activity Levels ◽  
Wild Type ◽  
Schwanniomyces Occidentalis ◽  
Type Protein ◽  
Wild Type Protein

The role of N-linked glycosylation on the biological activity of Schwanniomyces occidentalis SWA2 α-amylase, as expressed in Saccharomyces cerevisiae, was analysed by site-directed mutagenesis of the two potential N-glycosylation sites, Asn-134 and Asn-229. These residues were replaced by Ala or Gly individually or in various combinations and the effects on the activity, secretion and thermal stability of the enzyme were studied. Any Asn-229 substitution caused a drastic decrease in activity levels of the extracellular enzyme. In contrast, substitutions of Asn-134 had little or no effect. The use of antibodies showed that α-amylase was secreted in all the mutants tested, although those containing substitutions at Asn-229 seemed to have a lower rate of synthesis and/or higher degradation than the wild-type strain. α-Amylases with substitution at Asn-229 had a 2 kDa lower molecular mass than the wild-type protein, as did the wild-type protein itself after treatment with endoglycosidase F. These findings indicate that Asn-229 is the single glycosylated residue in SWA2. Thermostability analysis of both purified wild-type (T50 = 50 °C, where T50 is the temperature resulting in 50% loss of activity) and mutant enzymes indicated that removal of carbohydrate from the 229 position results in a decrease of approx. 3 °C in the T50 of the enzyme. The Gly-229 mutation does not change the apparent affinity of the enzyme for starch (Km) but decreases to 1/22 its apparent catalytic efficiency (kcat/Km). These results therefore indicate that glycosylation at the 229 position has an important role in the extracellular activity levels, kinetics and stability of the Sw. occidentalis SWA2 α-amylase in both its wild-type and mutant forms.

scholarly journals Importance of the two tryptophan residues in the Streptomyces R61 exocellular dd-peptidase

1992 ◽  
Vol 282 (2) ◽  
pp. 361-367 ◽  
Author(s):  
C Bourguignon-Bellefroid ◽  
J M Wilkin ◽  
B Joris ◽  
R T Aplin ◽  
C Houssier ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Beta Lactams ◽  
Beta Lactamases ◽  
Type Protein ◽  
Wild Type Protein ◽  
Tryptophan Residues

Modification of the Streptomyces R61 DD-peptidase by N-bromosuccinimide resulted in a rapid loss of enzyme activity. In consequence, the role of the enzyme's two tryptophan residues was investigated by site-directed mutagenesis. Trp271 was replaced by Leu. The modification yielded a stable enzyme whose structural and catalytic properties were similar to those of the wild-type protein. Thus the Trp271 residue, though almost invariant among the beta-lactamases of classes A and C and the low-Mr penicillin-binding proteins, did not appear to be essential for enzyme activity. Mutations of the Trp233 into Leu and Ser strongly decreased the enzymic activity, the affinity for beta-lactams and the protein stability. Surprisingly, the benzylpenicilloyl-(W233L)enzyme deacylated at least 300-fold more quickly than the corresponding acyl-enzyme formed with the wild-type protein and gave rise to benzylpenicilloate instead of phenylacetylglycine. This mutant DD-peptidase thus behaved as a weak beta-lactamase.

scholarly journals A 13C-NMR study of the role of Asn-155 in stabilizing the oxyanion of a subtilisin tetrahedral adduct

1997 ◽  
Vol 326 (3) ◽  
pp. 861-866 ◽  
Author(s):  
Timothy P. O'CONNELL ◽  
Regina M. DAY ◽  
Ekaterina V. TORCHILIN ◽  
William W. BACHOVCHIN ◽  
J. Paul G. MALTHOUSE
Keyword(s):  
13C Nmr ◽  
Chemical Shifts ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Imidazolium Cation ◽  
Nmr Study ◽  
In The Wild ◽  
Main Factor

By removing one of the hydrogen-bond donors in the oxyanion hole of subtilisin BPN, we have been able to determine how it affects the catalytic efficiency of the enzyme and the pKa of the oxyanion formed in a choloromethane inhibitor derivative. Variant 8397 of subtilisin BPN contains five mutations which enhance its stability. Site-directed mutagenesis was used to prepare the N155A mutant of this variant. The catalytic efficiencies of wild-type and variant 8397 are similar, but replacing Asn-155 with alanine reduces catalytic efficiency approx. 300-fold. All three forms of subtilisin were alkylated using benzyloxycarbonylglycylglycyl[2-13C]phenylalanylchloromethane and examined by 13C-NMR. A single signal due to the 13C-enriched carbon was detected in all the derivatives and it was assigned to the hemiketal carbon of a tetrahedral adduct formed between the hydroxy group of Ser-221 and the inhibitor. This signal had chemical shifts in the range 98.3–103.6 p.p.m., depending on the pH. The titration shift of 4.7–4.8 p.p.m. was assigned to oxyanion formation. The oxyanion pKa values in the wild-type and 8397 variants were 6.92 and 7.00 respectively. In the N155A mutant of the 8397 variant the oxyanion pKa increased to 8.09. We explain why such a small increase is observed and we conclude that it is the interaction between the oxyanion and the imidazolium cation of the active-site histidine that is the main factor responsible for lowering the oxyanion pKa.

scholarly journals Revisiting the role of PML protein targeting and disruption of PML bodies in human cytomegalovirus infection

2020 ◽  
Vol 2 (7A) ◽  
Author(s):  
Christina Paulus ◽  
Thomas Harwardt ◽  
Bernadette Walter ◽  
Andrea Marxreiter ◽  
Michael Nevels
Keyword(s):  
Gene Expression ◽  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Critical Role ◽  
Wild Type ◽  
Early Protein ◽  
Type Protein ◽  
Wild Type Protein ◽  
Pml Bodies

Promyelocytic leukaemia (PML) bodies are nuclear organelles implicated in post-translational modification by small ubiquitin-like modifier (SUMO) proteins and in the antiviral host cell response to infection. The 72-kDa immediate-early protein 1 (IE1) is considered the principal antagonist of PML bodies encoded by the human cytomegalovirus, one of eight human herpesviruses. Previous work has suggested that the interaction between IE1 and PML proteins, the central organisers of PML bodies, and the subsequent disruption of these organelles serve a critical role in viral replication by counteracting intrinsic antiviral immunity and the induction of interferon (IFN)-stimulated genes. However, this picture has emerged largely from studying mutant IE1 proteins known or predicted to be globally misfolded und metabolically unstable. We systematically screened for stable IE1 mutants by clustered charge-to-alanine scanning. We identified a mutant protein (IE1cc172-176) selectively defective for PML interaction. Functional comparisons between the mutant and wild-type protein revealed that IE1 can undergo modification by mixed polymeric SUMO chains and that it targets PML and Sp100, the two main constituents of PML bodies, via distinct mechanisms. Unexpectedly, IE1cc172-176 supported viral replication almost as efficiently as wild-type IE1. Moreover, lower instead of higher (as expected) levels of tumor necrosis factor alpha, IFN-beta, IFN-lambda and IFN-stimulated gene expression were observed with the mutant compared to the wild-type protein and virus. These results suggest that the disruption of PML bodies is linked to induction rather than inhibition of antiviral gene expression. Our findings challenge current views regarding the role of PML bodies in viral infection.

scholarly journals Kinetic Studies on CphA Mutants Reveal the Role of the P158-P172 Loop in Activity versus Carbapenems

2016 ◽  
Vol 60 (5) ◽  
pp. 3123-3126 ◽  
Author(s):  
Carlo Bottoni ◽  
Mariagrazia Perilli ◽  
Francesca Marcoccia ◽  
Alessandra Piccirilli ◽  
Cristina Pellegrini ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Zinc Ion ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
The Stability

ABSTRACTSite-directed mutagenesis of CphA indicated that prolines in the P158-P172 loop are essential for the stability and the catalytic activity of subclass B2 metallo-β-lactamases against carbapenems. The sequential substitution of proline led to a decrease of the catalytic efficiency of the variant compared to the wild-type (WT) enzyme but also to a higher affinity for the binding of the second zinc ion.

scholarly journals Biochemical and Mutational Analyses of a Multidomain Cellulase/Mannanase from Caldicellulosiruptor bescii

2012 ◽  
Vol 78 (7) ◽  
pp. 2230-2240 ◽  
Author(s):  
Xiaoyun Su ◽  
Roderick I. Mackie ◽  
Isaac K. O. Cann
Keyword(s):  
Catalytic Efficiency ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Wild Type ◽  
Ph Optimum ◽  
Content Type ◽  
Caldicellulosiruptor Bescii ◽  
Type Protein ◽  
Wild Type Protein ◽  
Carbohydrate Binding Modules

ABSTRACTThermophilic cellulases and hemicellulases are of significant interest to the biofuel industry due to their perceived advantages over their mesophilic counterparts. We describe here biochemical and mutational analyses ofCaldicellulosiruptor besciiCel9B/Man5A (CbCel9B/Man5A), a highly thermophilic enzyme. As one of the highly secreted proteins ofC. bescii, the enzyme is likely to be critical to nutrient acquisition by the bacterium. CbCel9B/Man5A is a modular protein composed of three carbohydrate-binding modules flanked at the N terminus and the C terminus by a glycoside hydrolase family 9 (GH9) module and a GH5 module, respectively. Based on truncational analysis of the polypeptide, the cellulase and mannanase activities within CbCel9B/Man5A were assigned to the N- and C-terminal modules, respectively. CbCel9B/Man5A and its truncational mutants, in general, exhibited a pH optimum of ∼5.5 and a temperature optimum of 85°C. However, at this temperature, thermostability was very low. After 24 h of incubation at 75°C, the wild-type protein maintained 43% activity, whereas a truncated mutant, TM1, maintained 75% activity. The catalytic efficiency with phosphoric acid swollen cellulose as a substrate for the wild-type protein was 7.2 s−1ml/mg, and deleting the GH5 module led to a mutant (TM1) with a 2-fold increase in this kinetic parameter. Deletion of the GH9 module also increased the apparentkcatof the truncated mutant TM5 on several mannan-based substrates; however, a concomitant increase in theKmled to a decrease in the catalytic efficiencies on all substrates. These observations lead us to postulate that the two catalytic activities are coupled in the polypeptide.

Role of tryptophan 135 of Chandipura virus phosphoprotein P in dimerization and complex formation with leader RNA: structural aspect using time resolved anisotropy and simulation

RSC Advances ◽  
2015 ◽  
Vol 5 (126) ◽  
pp. 104582-104593
Author(s):  
Manini Mukherjee ◽  
Aditya Sarkar ◽  
Arunava Roy ◽  
Pinki Saha Sardar ◽  
Ansuman Lahiri ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Structural Change ◽  
Complex Formation ◽  
Protein Concentration ◽  
Structural Aspect ◽  
Wild Type ◽  
Time Resolved ◽  
Type Protein ◽  
Wild Type Protein

The nanosecond and picosecond dynamics of wild type protein and its tryptophan mutants have been used to study structural change as a function of protein concentration and binding with leader RNA by time resolved anisotropy and molecular dynamics.

A model system for studying membrane biogenesis. Overexpression of cytochrome b5 in yeast results in marked proliferation of the intracellular membrane

1993 ◽  
Vol 106 (1) ◽  
pp. 249-259 ◽  
Author(s):  
G. Vergeres ◽  
T.S. Yen ◽  
J. Aggeler ◽  
J. Lausier ◽  
L. Waskell
Keyword(s):  
Cytochrome B5 ◽  
Alpha Helix ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Membrane Biogenesis ◽  
Wild Type ◽  
Membrane Anchor ◽  
Type Protein ◽  
Wild Type Protein ◽  
Membrane Bound

Cytochrome b5 is an amphipathic microsomal protein that is anchored to the endoplasmic reticulum by a single hydrophobic transmembrane alpha-helix located near the carboxyl terminus of the protein. In yeast, cytochrome b5 provides electrons for fatty acid desaturation and ergosterol biosynthesis. High level expression of cytochrome b5 in Saccharomyces cerevisiae was achieved using the yeast metallothionein promoter and a synthetic cytochrome b5 gene. In order to accommodate the markedly increased amount of the membrane-bound cytochrome b5, the yeast cell proliferated its nuclear membrane. As many as 20 pairs of stacked membranes could be observed to partially encircle the nucleus. This morphological arrangement of membrane around the nucleus is known as a karmella. In an effort to understand which part of the cytochrome b5 molecule, i.e. the membrane anchor or the soluble heme domain, which is competent in electron transfer, provided the signal for the de novo membrane biogenesis, a series of studies, including site-directed mutagenesis, was undertaken. The results of these experiments demonstrated that the inactive hemedeficient apo form of the membrane-bound protein stimulates membrane proliferation to the same extent as the holo wild-type protein, whereas cytosolic forms of cytochrome b5 did not induce membrane synthesis. These data demonstrate that membrane proliferation is a consequence of the cell's ability to monitor the level of membrane proteins and to compensate for alterations in these levels rather than the result of the ability of the extra cytochrome b5 to catalyze synthesis of extra lipid that had to be accommodated in new membrane. Site-directed mutagenesis studies of the membrane binding domain of cytochrome b5 provided additional clues about the nature of the signal for membrane proliferation. Replacement of the membrane anchor by a non-physiological nonsense sequence of 22 leucines gave rise to a mutant protein that triggered membrane biosynthesis. The conclusion from these experiments is clear; the signal for membrane proliferation does not reside in some specific amino acid sequence but instead in the hydrophobic properties of the proliferant. Interestingly, these membranes are somewhat diminished in quantity and have a slightly altered morphology compared to those induced by the wild-type protein. It was also observed that disruption of the putative alpha helix of the membrane anchor by an Ala116Pro mutation, which gives rise to two sequential prolines at positions 115 and 116 results in a protein with diminished capacity to induce membrane formation.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals The Griffithsin Dimer Is Required for High-Potency Inhibition of HIV-1: Evidence for Manipulation of the Structure of gp120 as Part of the Griffithsin Dimer Mechanism

2013 ◽  
Vol 57 (8) ◽  
pp. 3976-3989 ◽  
Author(s):  
Jie Xue ◽  
Bart Hoorelbeke ◽  
Ioannis Kagiampakis ◽  
Borries Demeler ◽  
Jan Balzarini ◽  
...  
Keyword(s):  
Protein A ◽  
Carbohydrate Binding ◽  
Wild Type ◽  
Subtype C ◽  
Subtype B ◽  
Type Protein ◽  
Wild Type Protein ◽  
Protein Dimer ◽  
Anti Hiv

ABSTRACTGriffithsin (Grft) is a protein lectin derived from red algae that tightly binds the HIV envelope protein gp120 and effectively inhibits virus infection. This inhibition is due to the binding by Grft of high-mannose saccharides on the surface of gp120. Grft has been shown to be a tight dimer, but the role of the dimer in Grft's anti-HIV function has not been fully explored. To investigate the role of the Grft dimer in anti-HIV function, an obligate dimer of Grft was designed by expressing the protein with a peptide linker between the two subunits. This “Grft-linker-Grft” is a folded protein dimer, apparently nearly identical in structural properties to the wild-type protein. A “one-armed” obligate dimer was also designed (Grft-linker-Grft OneArm), with each of the three carbohydrate binding sites of one subunit mutated while the other subunit remained intact. While both constructed dimers retained the ability to bind gp120 and the viral surface, Grft-linker-Grft OneArm was 84- to 1,010-fold less able to inhibit HIV than wild-type Grft, while Grft-linker-Grft had near-wild-type antiviral potency. Furthermore, while the wild-type protein demonstrated the ability to alter the structure of gp120 by exposing the CD4 binding site, Grft-linker-Grft OneArm largely lost this ability. In experiments to investigate gp120 shedding, it was found that Grft has different effects on gp120 shedding for strains from subtype B and subtype C, and this might correlate with Grft function. Evidence is provided that the dimer form of Grft is critical to the function of this protein in HIV inhibition.

scholarly journals Prediction and experimental testing of Bacillus acidocaldarius thioredoxin stability1

1999 ◽  
Vol 339 (2) ◽  
pp. 309-317 ◽  
Author(s):  
Emilia PEDONE ◽  
Raffaele CANNIO ◽  
Michele SAVIANO ◽  
Mosè ROSSI ◽  
Simonetta BARTOLUCCI
Keyword(s):  
Protein Stability ◽  
Molecular Dynamic ◽  
Experimental Testing ◽  
Ion Pairs ◽  
Cd Spectroscopy ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Type Protein ◽  
Wild Type Protein ◽  
Reference Protein

In order to investigate further the determinants of protein stability, four mutants of thioredoxin from Bacillus acidocaldarius were designed: K18G, R82E, K18G/R82E, and D102X, in which the last four amino acids were deleted. The mutants were constructed on the basis of molecular dynamic studies and the prediction of the structure of thioredoxin from B. acidocaldarius, performed by a comparative molecular modelling technique using Escherichia coli thioredoxin as the reference protein. The mutants obtained by PCR strategy were expressed in E. coli and then characterized. CD spectroscopy, spectrofluorimetry and thermodynamic comparative studies permitted comparison of the relative physicochemical behaviour of the four proteins with that of the wild-type protein. As predicted for the molecular dynamic analysis at 500 K in vacuo, the wild-type structure was more stable than that of the mutants; in fact the Tm of the four proteins showed a decrease of about 15 °C for the double and the truncated mutants, and a decrease of about 12 °C for the single mutants. A difference in the resistance of the proteins to denaturants such as guanidine HCl and urea was revealed; the wild-type protein always proved to be the most resistant. The results obtained show the importance of hydrogen bonds and ion pairs in determining protein stability and confirm that simulation methods are able to direct protein engineering in site-directed mutagenesis.

scholarly journals Expression and characterization of Pseudomonas aeruginosa cytochrome c-551 and two site-directed mutants: role of tryptophan 56 in the modulation of redox properties

1997 ◽  
Vol 322 (1) ◽  
pp. 35-42 ◽  
Author(s):  
Francesca CUTRUZZOLÀ ◽  
Ilaria CIABATTI ◽  
Gabriella ROLLI ◽  
Sabrina FALCINELLI ◽  
Marzia ARESE ◽  
...  
Keyword(s):  
Hydrogen Bonding ◽  
Cytochrome C ◽  
Ph Dependence ◽  
Redox Properties ◽  
Expression Vectors ◽  
Wild Type ◽  
Type Protein ◽  
Wild Type Protein ◽  
Structural And Functional Properties

The gene coding for Pseudomonas aeruginosacytochrome c-551 was expressed in Pseudomonas putidaunder aerobic conditions, using two different expression vectors; the more efficient proved to be pNM185, induced by m-toluate. Mature holo-(cytochrome c-551) was produced in high yield by this expression system, and was purified to homogeneity. Comparison of the recombinant wild-type protein with that purified from Ps. aeruginosashowed no differences in structural and functional properties. Trp56, an internal residue in cytochrome c-551, is located at hydrogen-bonding distance from haem propionate-17, together with Arg47. Ionization of propionate-17 was related to the observed pH-dependence of redox potential. The role of Trp56 in determining the redox properties of Ps. aeruginosacytochrome c-551 was assessed by site-directed mutagenesis, by substitution with Tyr (W56Y) and Phe (W56F). The W56Y mutant is similar to the wild-type cytochrome. On the other hand, the W56F mutant, although similar to the wild-type protein in spectral properties and electron donation to azurin, is characterized by a weakening of the FeŐMet61 bond, as shown in the oxidized protein by the loss of the 695 nm band approx. 2 pH units below the wild-type. Moreover, in W56F, the midpoint potential and its pH-dependence are both different from the wild-type. These results are consistent with the hypothesis that hydrogen-bonding to haem propionate-17 is important in modulation of the redox properties of Ps. aeruginosacytochrome c-551.

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