scholarly journals Brassica juncea 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase 1: expression and characterization of recombinant wild-type and mutant enzymes

2004 ◽  
Vol 383 (3) ◽  
pp. 517-527 ◽  
Author(s):  
Dinesh A. NAGEGOWDA ◽  
Thomas J. BACH ◽  
Mee-Len CHYE
Keyword(s):  
Brassica Juncea ◽  
Mevalonate Pathway ◽  
Specific Activity ◽  
Substrate Inhibition ◽  
Site Directed Mutagenesis ◽  
Acetyl Coa ◽  
Ph Optimum ◽  
Double Mutation ◽  
Calculated Mass

3-Hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGS; EC 2.3.3.10) is the second enzyme in the cytoplasmic mevalonate pathway of isoprenoid biosynthesis, and catalyses the condensation of acetyl-CoA with acetoacetyl-CoA (AcAc-CoA) to yield S-HMG-CoA. In this study, we have first characterized in detail a plant HMGS, Brassica juncea HMGS1 (BjHMGS1), as a His6-tagged protein from Escherichia coli. Native gel electrophoresis analysis showed that the enzyme behaves as a homodimer with a calculated mass of 105.8 kDa. It is activated by 5 mM dithioerythreitol and is inhibited by F-244 which is specific for HMGS enzymes. It has a pH optimum of 8.5 and a temperature optimum of 35 °C, with an energy of activation of 62.5 J·mol−1. Unlike cytosolic HMGS from chicken and cockroach, cations like Mg2+, Mn2+, Zn2+ and Co2+ did not stimulate His6–BjHMGS1 activity in vitro; instead all except Mg2+ were inhibitory. His6–BjHMGS1 has an apparent Km-acetyl-CoA of 43 μM and a Vmax of 0.47 μmol·mg−1·min−1, and was inhibited by one of the substrates (AcAc-CoA) and by both products (HMG-CoA and HS-CoA). Site-directed mutagenesis of conserved amino acid residues in BjHMGS1 revealed that substitutions R157A, H188N and C212S resulted in a decreased Vmax, indicating some involvement of these residues in catalytic capacity. Unlike His6–BjHMGS1 and its soluble purified mutant derivatives, the H188N mutant did not display substrate inhibition by AcAc-CoA. Substitution S359A resulted in a 10-fold increased specific activity. Based on these kinetic analyses, we generated a novel double mutation H188N/S359A, which resulted in a 10-fold increased specific activity, but still lacking inhibition by AcAc-CoA, strongly suggesting that His-188 is involved in conferring substrate inhibition on His6–BjHMGS1. Substitution of an aminoacyl residue resulting in loss of substrate inhibition has never been previously reported for any HMGS.

scholarly journals Engineering of Phytase for Improved Activity at Low pH

2002 ◽  
Vol 68 (4) ◽  
pp. 1907-1913 ◽  
Author(s):  
Andrea Tomschy ◽  
Roland Brugger ◽  
Martin Lehmann ◽  
Allan Svendsen ◽  
Kurt Vogel ◽  
...  
Keyword(s):  
Animal Feed ◽  
Specific Activity ◽  
Three Dimensional ◽  
Industrial Applications ◽  
Low Ph ◽  
Site Directed Mutagenesis ◽  
Single Mutation ◽  
Sequence Alignments ◽  
Ph Optimum ◽  
Double Mutation

ABSTRACT For industrial applications in animal feed, a phytase of interest must be optimally active in the pH range prevalent in the digestive tract. Therefore, the present investigation describes approaches to rationally engineer the pH activity profiles of Aspergillus fumigatus and consensus phytases. Decreasing the negative surface charge of the A. fumigatus Q27L phytase mutant by glycinamidylation of the surface carboxy groups (of Asp and Glu residues) lowered the pH optimum by ca. 0.5 unit but also resulted in 70 to 75% inactivation of the enzyme. Alternatively, detailed inspection of amino acid sequence alignments and of experimentally determined or homology modeled three-dimensional structures led to the identification of active-site amino acids that were considered to correlate with the activity maxima at low pH of A. niger NRRL 3135 phytase, A. niger pH 2.5 acid phosphatase, and Peniophora lycii phytase. Site-directed mutagenesis confirmed that, in A. fumigatus wild-type phytase, replacement of Gly-277 and Tyr-282 with the corresponding residues of A. niger phytase (Lys and His, respectively) gives rise to a second pH optimum at 2.8 to 3.4. In addition, the K68A single mutation (in both A. fumigatus and consensus phytase backbones), as well as the S140Y D141G double mutation (in A. fumigatus phytase backbones), decreased the pH optima with phytic acid as substrate by 0.5 to 1.0 unit, with either no change or even a slight increase in maximum specific activity. These findings significantly extend our tools for rationally designing an optimal phytase for a given purpose.

scholarly journals Engineering a bioluminescent indicator for cyclic AMP-dependent protein kinase

1991 ◽  
Vol 279 (3) ◽  
pp. 727-732 ◽  
Author(s):  
G B Sala-Newby ◽  
A K Campbell
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Specific Activity ◽  
Phosphorylation Site ◽  
Live Cells ◽  
Phosphorylation Sites ◽  
Dependent Protein Kinase ◽  
Ph Optimum ◽  
Dependent Protein

cDNA coding for the luciferase in the firefly Photinus pyralis was amplified in vitro to generate cyclic AMP-dependent protein kinase phosphorylation sites. The DNA was transcribed and translated to generate light-emitting protein. A valine at position 217 was mutated to arginine to generate a site RRFS and the heptapeptide kemptide, the phosphorylation site of the porcine pyruvate kinase, was added at the N- or C-terminus of the luciferase. The proteins carrying phosphorylation sites were characterized for their specific activity, pI, effect of pH on the colour of the light emitted and effect of the catalytic subunit of protein kinase A in the presence of ATP. Only one of the recombinant proteins (RRFS) was significantly different from wild-type luciferase. The RRFS mutant had a lower specific activity, lower pH optimum, emitted greener light at low pH and when phosphorylated it decreased its activity by up to 80%. This latter effect was reversed by phosphatase. This recombinant protein is a good candidate to measure for the first time cyclic AMP-dependent phosphorylation in live cells.

scholarly journals Structure-based design of small peptide inhibitors of protein kinase CK2 subunit interaction

2007 ◽  
Vol 408 (3) ◽  
pp. 363-373 ◽  
Author(s):  
Béatrice Laudet ◽  
Caroline Barette ◽  
Vincent Dulery ◽  
Olivier Renaudet ◽  
Pascal Dumy ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Kinase ◽  
Protein Kinase Ck2 ◽  
Peptide Inhibitors ◽  
Fluorescent Imaging ◽  
Site Directed Mutagenesis ◽  
Small Peptide ◽  
Double Mutation ◽  
Kinase Ck2

X-ray crystallography studies, as well as live-cell fluorescent imaging, have recently challenged the traditional view of protein kinase CK2. Unbalanced expression of catalytic and regulatory CK2 subunits has been observed in a variety of tissues and tumours. Thus the potential intersubunit flexibility suggested by these studies raises the likely prospect that the CK2 holoenzyme complex is subject to disassembly and reassembly. In the present paper, we show evidence for the reversible multimeric organization of the CK2 holoenzyme complex in vitro. We used a combination of site-directed mutagenesis, binding experiments and functional assays to show that, both in vitro and in vivo, only a small set of primary hydrophobic residues of CK2β which contacts at the centre of the CK2α/CK2β interface dominates affinity. The results indicate that a double mutation in CK2β of amino acids Tyr188 and Phe190, which are complementary and fill up a hydrophobic pocket of CK2α, is the most disruptive to CK2α binding both in vitro and in living cells. Further characterization of hotspots in a cluster of hydrophobic amino acids centred around Tyr188–Phe190 led us to the structure-based design of small-peptide inhibitors. One conformationally constrained 11-mer peptide (Pc) represents a unique CK2β-based small molecule that was particularly efficient (i) to antagonize the interaction between the CK2 subunits, (ii) to inhibit the assembly of the CK2 holoenzyme complex, and (iii) to strongly affect its substrate preference.

scholarly journals Characterization of a chelator-resistant proteinase from Thermus strain Rt4A2

1993 ◽  
Vol 295 (2) ◽  
pp. 463-469 ◽  
Author(s):  
S A Freeman ◽  
K Peek ◽  
M Prescott ◽  
R Daniel
Keyword(s):  
Specific Activity ◽  
Sequence Similarity ◽  
Substrate Inhibition ◽  
Gel Filtration ◽  
Serine Proteinase ◽  
Fold Increase ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Ph Optimum ◽  
High Sequence Similarity

The Thermus isolate Rt4A2 was found to produce an extracellular chelator-resistant proteinase. The proteinase was purified to homogeneity by (NH4)2SO4 precipitation, cation-exchange chromatography, gel-filtration chromatography, and weak anion-exchange chromatography. The Rt4A2 proteinase was found to have properties typical of an alkaline serine proteinase. It had a pH optimum of 9.0 and was specifically inhibited by phenylmethanesulphonyl fluoride. Its isoelectric point was greater than 10.25. Its molecular-mass was 31.6 kDa as determined by SDS/PAGE. N-terminal sequencing has shown it to have high sequence similarity with other serine proteinases from Thermus species. The proteinase hydrolysed a number of substrates including fibrin, casein, haemoglobin, collagen, albumin and the synthetic chromogenic peptide substrate Suc-Ala-Ala-Pro-Phe-NH-Np. The specific activity of the purified proteinase using azocasein as substrate was 313 units/mg. Substrate inhibition was observed above an azocasein concentration of 0.05% (w/v). Esterase activity was directed mainly towards those substrates containing the aliphatic or aromatic residues of alanine, glycine, tryptophan, tyrosine and phenylalanine. Thermostability half-lives of greater than 7 days at 70 degrees C, 43 h at 80 degrees C and 90 min at 90 degrees C were found in the presence of 5 mM CaCl2. At 90 degrees C increasing the CaCl2 concentration 100-fold (0.5 mM to 50 mM) caused a 4.3-fold increase in the half-life of the enzyme from 30 to 130 min. Half-lives of 19.4 min at 100 degrees C and 4.4 min at 105 degrees C were found in the presence of 50 mM CaCl2. The metal chelators EGTA and EDTA reduced the stability at higher temperatures but had no effect on the activity of the proteinase. Activity was not stimulated by common metal activators such as Ca2+, Mg2+ and Zn2+.

scholarly journals Modulation of Prohormone Convertase 1/3 Properties Using Site-Directed Mutagenesis

Endocrinology ◽  
2010 ◽  
Vol 151 (9) ◽  
pp. 4437-4445 ◽  
Author(s):  
Akihiko Ozawa ◽  
Juan R. Peinado ◽  
Iris Lindberg
Keyword(s):  
Catalytic Domain ◽  
Specific Activity ◽  
Site Directed Mutagenesis ◽  
Prohormone Convertase ◽  
Ph Optimum ◽  
Specific Domain ◽  
Molecular Determinant ◽  
Prohormone Convertase 1 ◽  
Domain Interactions ◽  
Endogenous Substrate

Prohormone convertase (PC)1/3 and PC2 cleave active peptide hormones and neuropeptides from precursor proteins. Compared with PC2, recombinant PC1/3 exhibits a very low specific activity against both small fluorogenic peptides and recombinant precursors, even though the catalytic domains in mouse PC1/3 and PC2 share 56% amino acid sequence identity. In this report, we have designed PC2-specific mutations into the catalytic domain of PC1/3 in order to investigate the molecular contributions of these sequences to PC1/3-specific properties. The exchange of residues RQG314 with the SY sequence present in the same location within PC2 paradoxically shifted the pH optimum of PC1/3 upward into the neutral range; other mutations in the catalytic domain had no effect. Although none of the full-length PC1/3 mutants examined exhibited increased specific activity, the 66-kDa form of the RQG314SY mutant was two to four times more active than the 66-kDa form of wild-type PC1/3. However, stable transfection of RQG314SY into PC12 cells did not result in greater activity against the endogenous substrate proneurotensin, implying unknown cellular controls of PC1/3 activity. Mutation of GIVTDA243–248 to QPFMTDI, a molecular determinant of 7B2 binding, resulted in increased zymogen expression but no propeptide cleavage or secretion, suggesting that this mutant is trapped in the endoplasmic reticulum due to an inability to cleave its own propeptide. We conclude that many convertase-specific properties are attributable less to convertase-specific catalytic cleft residues than to convertase-specific domain interactions.

ANALYSIS OF STRUCTURAL REQUIREMENTS FOR FACTOR VIII FUNCTION USING SITE-DIRECTED MUTAGENESIS

1987 ◽  
Author(s):  
Debra D Pittman ◽  
Louise C Wasley ◽  
Beth L Murray ◽  
Jack H Wang ◽  
Randal J Kaufman
Keyword(s):  
Amino Acid ◽  
Factor Viii ◽  
Protein C ◽  
Specific Activity ◽  
Site Directed Mutagenesis ◽  
Factor X ◽  
Proteolytic Activation ◽  
Thrombin Cleavage ◽  
Cofactor Activity

Factor VIII (fVIII) functions in the intrinsic pathway of coagulation as the cofactor for Factor IXa proteolytic activation of Factor X. fVIII contains multiple sites which are susceptible to cleavage by thrombin, Factor Xa, and activate) protein C. Proteolytic cleavage is required for cofactor activity and may be responsible for inactivation of cofactor activity. In order to identify the role ofthe individual cleavages of fVIII in its activation and inactivation, site-directed DNA mediated mutagenesis of fVIII was performed and the altered forms of fVIII produced and characterized. Conversionof Arg residues to lie residues at amino acid positions 740, 1648, and 1721 resulted in resistance to thrombin cleavage at those siteswith no alteration of in vitro procoagulant activity. Modification of the thrombin cleavage sites at either positions 372 or 1689 resulted in loss of cofactor activity suggesting that these sites are important for activation. Modification of the postulated activated protein C cleavage site at position 336 resulted in fVIII with a higher specific activity than wild type, possibly due to resistance toproteolytic inactivation.DNA mediated mutagenesis was also used to study the role of post-translational biosynthetic modifications of fVIII. Structural characterization of recombinant fVIII suggested the presence of sulfated tyrosine residues within two acidic regions located between amino acid residues 336-372 and 1648-1689. Individual modification of theseTyr residues to Phe had negligible effect on synthesis and in vitrocofactor activity. The effect of combinations of these mutations onsecretion, cofactor activity, and vWF interaction will be presented.

scholarly journals Exploring the role of the CTL epitope region of listeriolysin O in the pathogenesis of Listeria monocytogenes

Microbiology ◽  
2006 ◽  
Vol 152 (5) ◽  
pp. 1287-1296 ◽  
Author(s):  
Marie-Annick Lety ◽  
Claude Frehel ◽  
Catherine Raynaud ◽  
Marion Dupuis ◽  
Alain Charbit
Keyword(s):  
Listeria Monocytogenes ◽  
Opportunistic Infections ◽  
Site Directed Mutagenesis ◽  
Haemolytic Activity ◽  
Single Mutation ◽  
Listeriolysin O ◽  
Epitope Region ◽  
Double Mutation ◽  
Ctl Epitope

Listeria monocytogenes is a facultative intracellular bacterial pathogen responsible for severe opportunistic infections in humans and animals. The secreted cholesterol-dependent cytolysin, listeriolysin O (LLO), mediates phagosomal escape and allows bacterial growth in the cytosol of infected cells. In order to identify new LLO determinants participating in bacterial pathogenesis, this study focused on a major target of LLO proteolytic cleavage in vitro, the CTL epitope region (residues 91–99). Mutations were generated by site-directed mutagenesis in the epitope or in the two clusters of positive charges flanking the epitope. Two LLO mutants (a single mutation K103A and a double mutation R89G, K90G) were normally and stably secreted by L. monocytogenes. In contrast, a mutant carrying four amino acid substitutions in the epitope itself (Y92K, D94A, E97K, Y98F) was highly susceptible to proteolytic degradation. While these three LLO mutant proteins showed a reduced haemolytic activity, they all promoted efficient phagosomal escape and intracellular multiplication in different cell types, and were non-cytotoxic. The deletion of the epitope (Δ91–99), as well as the substitution of two, three or four of the four lysine residues (K103 to K106) by alanine residues did not lead to the production of a detectable protein. These results confirm the lack of correlation between haemolytic activity and phagosomal membrane disruption. They reveal the importance of the 91–99 region in the production of a stable and functional LLO. LD50 determinations in the mouse model suggest a possible link between LLO stability and virulence.

scholarly journals Human acetyl-coenzyme A:α-glucosaminide N-acetyltransferase. Kinetic characterization and mechanistic interpretation

1995 ◽  
Vol 308 (1) ◽  
pp. 327-333 ◽  
Author(s):  
P J Meikle ◽  
A M Whittle ◽  
J J Hopwood
Keyword(s):  
Ternary Complex ◽  
Gradient Centrifugation ◽  
Heparan Sulphate ◽  
Random Order ◽  
Lysosomal Membrane ◽  
Complex Mechanism ◽  
Acetyl Coa ◽  
Ph Optimum ◽  
Inhibition Studies

Acetyl-CoA: alpha-glucosaminide N-acetyltransferase (N-acetyltransferase) is an integral lysosomal membrane protein which catalyses the transfer of acetyl groups from acetyl-CoA on to the terminal glucosamine in heparin and heparan sulphate chains within the lysosome. In vitro, the enzyme is capable of acetylating a number of mono- and oligo-saccharides derived from heparin, provided that a non-reducing terminal glucosamine is present. We have prepared highly enriched lysosomal membrane fractions from human placenta by a combination of differential centrifugation and density-gradient centrifugation in Percoll. This preparation was used to investigate the kinetics of the enzyme with three acetyl-acceptor substrates, i.e. glucosamine and a disaccharide and a tetrasaccharide derived from heparin, each containing a terminal glucosamine residue. The enzyme showed a pH optimum at 6.5, extending to 8.0 for the mono- and di-saccharide substrates but falling off sharply above pH 6.5 for the tetrasaccharide substrate. We identified two distinct Km values for the glucosamine substrate at both pH 7.0 and pH 5.0, whereas the tetrasaccharide substrate displayed only a single Km value at each pH. The Km values were found to be highly pH-dependent, and at pH 5.0 the values for the acetyl-acceptor substrates showed a decreasing trend as the size of the substrate increased, suggesting that the enzyme recognizes an extended region of the non-reducing terminus of the heparin or heparan sulphate polysaccharides. Double-reciprocal analysis, isotope exchange between N-acetylglucosamine and glucosamine, and inhibition studies with desulpho-CoA indicate that the enzyme operates by a random-order ternary-complex mechanism. Product inhibition studies display a complex pattern of dead-end inhibition. Taken in context with what is known about lysosomal utilization and physiological levels of acetyl-CoA, these results suggest that in vivo the enzyme operates via a random-order ternary-complex mechanism which involves the utilization of cytosolic acetyl-CoA to transfer acetyl groups on to the terminal glucosamine residues of heparin within the lysosome.

IMPROVEMENT OF T-PA PROPERTIES BY MEANS OF SITE DIRECTED MUTAGENESIS

1987 ◽  
Author(s):  
N Haigwood ◽  
E-P Pâques ◽  
G Mullenbach ◽  
G Moore ◽  
L DesJardin ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cleavage Site ◽  
Specific Activity ◽  
Biological Properties ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Mutant Proteins ◽  
C Terminus

The clinical relevance of tissue-plasminogen-activator (t-PA) as a potent thrombolytic agent has recently been established. It has however been recognized that t-PA does not fulfill all conditions required for an ideal thrombolytic pharmaceutical agent; for example, its physiological stability and its short half life in vivo necessitate the use of very large clinical doses. We have therefore attempted to develop novel mutant t-PA proteins with improved properties by creating mutants by site-directed mutagenesis in M13 bacteriophage. Seventeen mutants were designed, cloned, and expressed in CHO cells. Modifications were of three types: alterations to glycosylation sites, truncations of the N- or C-termini, and amino acids changes at the cleavage site utilized to generate the two chain form of t-PA. The mutant proteins were analyzed in vitro for specific activity, fibrin dependence of the plasminogen activation, fibrin affinity, and susceptibility to inhibition by PAI.In brief, the results are: 1) some unglycosylated and partially glycosylated molecules obtained by mutagenesis are characterized by several-fold higher specific activity than wild type t-PA; 2) truncation at the C-terminus by three amino acids yields a molecule with increased fibrin specificity; 3) mutations at the cleavage site lead zo a decreased inhibition by PAI; and 4) recombinants of these genes have been constructed and the proteins were shown to possess multiple improved properties. The use of site directed mutagenesis has proved to be a powerful instrument to modulate the biological properties of t-PA.

scholarly journals Staphylococcus aureus Mevalonate Kinase: Isolation and Characterization of an Enzyme of the Isoprenoid Biosynthetic Pathway

2004 ◽  
Vol 186 (1) ◽  
pp. 61-67 ◽  
Author(s):  
Natalya E. Voynova ◽  
Sandra E. Rios ◽  
Henry M. Miziorko
Keyword(s):  
Staphylococcus Aureus ◽  
Mevalonate Pathway ◽  
Specific Activity ◽  
Feedback Inhibition ◽  
Polyacrylamide Gel Electrophoresis ◽  
Substrate Inhibition ◽  
Sodium Dodecyl ◽  
Farnesyl Diphosphate ◽  
Mevalonate Kinase ◽  
Isolation And Characterization

ABSTRACT It has been proposed that isoprenoid biosynthesis in several gram-positive cocci depends on the mevalonate pathway for conversion of acetyl coenzyme A to isopentenyl diphosphate. Mevalonate kinase catalyzes a key reaction in this pathway. In this study the enzyme from Staphylococcus aureus was expressed in Escherichia coli, isolated in a highly purified form, and characterized. The overall amino acid sequence of this enzyme was very heterologous compared with the sequences of eukaryotic mevalonate kinases. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical gel filtration chromatography suggested that the native enzyme is a monomer with a molecular mass of approximately 33 kDa. The specific activity was 12 U/mg, and the pH optimum was 7.0 to 8.5. The apparent Km values for R,S-mevalonate and ATP were 41 and 339 μM, respectively. There was substantial substrate inhibition at millimolar levels of mevalonate. The sensitivity to feedback inhibition by farnesyl diphosphate and its sulfur-containing analog, farnesyl thiodiphosphate, was characterized. These compounds were competitive inhibitors with respect to ATP; the Ki values were 46 and 45 μM for farnesyl diphosphate and its thio analog, respectively. Parallel measurements with heterologous eukaryotic mevalonate kinases indicated that S. aureus mevalonate kinase is much less sensitive to feedback inhibition (Ki difference, 3 orders of magnitude) than the human enzyme. In contrast, both enzymes tightly bound trinitrophenyl-ATP, a fluorescent substrate analog, suggesting that there are similarities in structural features that are important for catalytic function.

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