Generation ofEscherichia coliintimin derivatives with differing biological activities using site‐directed mutagenesis of the intimin C‐terminus domain

Friedelin, a pentacyclic triterpene found in the leaves of the Celastraceae species, demonstrates numerous biological activities and is a precursor of quinonemethide triterpenes, which are promising antitumoral agents. Friedelin is biosynthesized from the cyclization of 2,3-oxidosqualene, involving a series of rearrangements to form a ketone by deprotonation of the hydroxylated intermediate, without the aid of an oxidoreductase enzyme. Mutagenesis studies among oxidosqualene cyclases (OSCs) have demonstrated the influence of amino acid residues on rearrangements during substrate cyclization: loss of catalytic activity, stabilization, rearrangement control or specificity changing. In the present study, friedelin synthase from Maytenus ilicifolia (Celastraceae) was expressed heterologously in Saccharomyces cerevisiae. Site-directed mutagenesis studies were performed by replacing phenylalanine with tryptophan at position 473 (Phe473Trp), methionine with serine at position 549 (Met549Ser) and leucine with phenylalanine at position 552 (Leu552Phe). Mutation Phe473Trp led to a total loss of function; mutants Met549Ser and Leu552Phe interfered with the enzyme specificity leading to enhanced friedelin production, in addition to α-amyrin and β-amyrin. Hence, these data showed that methionine 549 and leucine 552 are important residues for the function of this synthase.

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Ubc2, an Ortholog of the Yeast Ste50p Adaptor, Possesses a Basidiomycete-Specific Carboxy Terminal Extension Essential for Pathogenicity Independent of Pheromone Response

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-21-1-0110 ◽

2008 ◽

Vol 21 (1) ◽

pp. 110-121 ◽

Cited By ~ 20

Author(s):

Steven J. Klosterman ◽

Alfredo D. Martinez-Espinoza ◽

David L. Andrews ◽

Jeffrey R. Seay ◽

Scott E. Gold

Keyword(s):

Map Kinase ◽

Adaptor Protein ◽

Adaptor Proteins ◽

Filamentous Growth ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Sh3 Domains ◽

Additional Signal ◽

C Terminus ◽

Mitogen Activated Protein

Proteins involved in the mitogen-activated protein (MAP) kinase pathway controlling mating, morphogenesis, and pathogenicity have been identified previously in the fungus Ustilago maydis. One of these, the Ubc2 adaptor protein, possesses a basidiomycete-specific structure. In addition to containing sterile α motif (SAM) and ras association (RA) domains typical of Ste50-like adaptor proteins found in the fungal phylum Ascomycota, Ubc2 also contains two C-terminal SH3 domains. Yeast two-hybrid assays indicated that Ubc2 interacts with the MAP kinase-kinase kinase Ubc4 via the SAM domains at each of their respective N-termini. Site-directed mutagenesis of ubc2 and complementation analyses revealed that the SAM and RA domains of Ubc2 are essential for filamentous growth. These data support a role for the ascomycete-like N-terminus of Ubc2 in regulating pheromone-responsive mating and morphogenesis analogous to the role of Ste50p in Saccharomyces cerevisiae. In contrast, C-terminal deletion mutants were fully capable of filamentous growth and mating. However, surprisingly, these strains were nonpathogenic. Further, directed mutagenesis of the C-terminus revealed that both SH3 domains are required for pathogenicity. These results suggest that the Basidiomycota have retained the mating and morphogenetic functions of Ste50-type proteins in the N-terminal half of their Ubc2-type adaptors but, additionally, have integrated C-terminal SH3 domains that are critical for additional signal transduction mechanisms, including those that lead to pathogenesis.

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Crucial role of the orexin-B C-terminus in the induction of OX1receptor-mediated apoptosis: analysis by alanine scanning, molecular modelling and site-directed mutagenesis

British Journal of Pharmacology ◽

10.1111/bph.13287 ◽

2015 ◽

Vol 172 (21) ◽

pp. 5211-5223 ◽

Cited By ~ 6

Author(s):

Pascal Nicole ◽

Pierre Couvineau ◽

Nadege Jamin ◽

Thierry Voisin ◽

Alain Couvineau

Keyword(s):

Molecular Modelling ◽

Crucial Role ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Alanine Scanning ◽

C Terminus

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Functional Inactivation of the Nociceptin Receptor by Alanine Substitution of Glutamine 286 at the C Terminus of Transmembrane Segment VI: Evidence from a Site-Directed Mutagenesis Study of the ORL1 Receptor Transmembrane-Binding Domain

Molecular Pharmacology ◽

10.1124/mol.57.3.495 ◽

2000 ◽

Vol 57 (3) ◽

pp. 495-502 ◽

Cited By ~ 33

Author(s):

Lionel Mouledous ◽

Christopher M. Topham ◽

Christiane Moisand ◽

Catherine Mollereau ◽

Jean-Claude Meunier

Keyword(s):

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Transmembrane Segment ◽

Alanine Substitution ◽

Orl1 Receptor ◽

C Terminus ◽

Functional Inactivation ◽

A Site ◽

Mutagenesis Study ◽

Nociceptin Receptor

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Identification of the glycosaminoglycan-binding site on the glycoprotein Erns of bovine viral diarrhoea virus by site-directed mutagenesis

Journal of General Virology ◽

10.1099/0022-1317-83-9-2153 ◽

2002 ◽

Vol 83 (9) ◽

pp. 2153-2159 ◽

Cited By ~ 23

Author(s):

Munir Iqbal ◽

John W. McCauley

Keyword(s):

Cell Surface ◽

Binding Site ◽

Bovine Viral Diarrhoea Virus ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Bovine Viral Diarrhoea ◽

Surface Binding ◽

C Terminus ◽

Diarrhoea Virus ◽

Lysine Residues

Bovine viral diarrhoea virus (BVDV) envelope glycoprotein Erns interacts with highly sulphated heparin-like glycosaminoglycans (GAGs) located on the cell surface as an early step in virus infection of cells. Site-directed mutagenesis of recombinant Erns was undertaken and analysis of mutants by heparin-affinity chromatography and cell surface binding showed that a cluster of basic amino acids (480KKLENKSK487) near the C terminus of Erns was essential for binding. Mutants with amino acid substitutions of lysine residues 481 and 485 in Erns reduced the binding of Erns to immobilized heparin and cellular GAGs but retained ribonuclease activity. In contrast to normal Erns, Erns that was unable to bind to cells also failed to inhibit BVDV infection of cells when the cells were pre-incubated with Erns. It is proposed that the cluster of basic residues (480KKLENKSK487) localized at the C-terminal end of Erns constitutes a GAG-binding site.

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Fine Mapping of the N-Terminal Cytotoxicity Region of Clostridium perfringens Enterotoxin by Site-Directed Mutagenesis

Infection and Immunity ◽

10.1128/iai.72.12.6914-6923.2004 ◽

2004 ◽

Vol 72 (12) ◽

pp. 6914-6923 ◽

Cited By ~ 34

Author(s):

James G. Smedley ◽

Bruce A. McClane

Keyword(s):

Complex Formation ◽

Clostridium Perfringens ◽

Sodium Dodecyl ◽

Binding Activity ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Plasma Membrane Permeability ◽

Alanine Scanning Mutagenesis ◽

Large Complex ◽

C Terminus

ABSTRACT Clostridium perfringens enterotoxin (CPE) has a unique mechanism of action that results in the formation of large, sodium dodecyl sulfate-resistant complexes involving tight junction proteins; those complexes then induce plasma membrane permeability alterations in host intestinal epithelial cells, leading to cell death and epithelial desquamation. Previous deletion and point mutational studies mapped CPE receptor binding activity to the toxin's extreme C terminus. Those earlier analyses also determined that an N-terminal CPE region between residues D45 and G53 is required for large complex formation and cytotoxicity. To more finely map this N-terminal cytotoxicity region, site-directed mutagenesis was performed with recombinant CPE (rCPE). Alanine-scanning mutagenesis produced one rCPE variant, D48A, that failed to form large complexes or induce cytotoxicity, despite having normal ability to bind and form the small complex. Two saturation variants, D48E and D48N, also had a phenotype resembling that of the D48A variant, indicating that both size and charge are important at CPE residue 48. Another alanine substitution rCPE variant, I51A, was highly attenuated for large complex formation and cytotoxicity, but rCPE saturation variants I51L and I51V displayed a normal large complex formation and cytotoxicity phenotype. Collectively, these mutagenesis results identify a core CPE sequence extending from residues G47 to I51 that directly participates in large complex formation and cytotoxicity.

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Poly(A) polymerase contains multiple functional domains.

Molecular and Cellular Biology ◽

10.1128/mcb.14.5.2946 ◽

1994 ◽

Vol 14 (5) ◽

pp. 2946-2957 ◽

Cited By ~ 39

Author(s):

T Raabe ◽

K G Murthy ◽

J L Manley

Keyword(s):

Nuclear Localization ◽

Rna Binding ◽

Catalytic Domain ◽

Site Directed Mutagenesis ◽

Nuclear Proteins ◽

Directed Mutagenesis ◽

Nuclear Targeting ◽

Nuclear Localization Signals ◽

C Terminus ◽

Sequence Similarities

Poly(A) polymerase (PAP) contains regions of similarity with several known protein domains. Through site-directed mutagenesis, we provide evidence that PAP contains a functional ribonucleoprotein-type RNA binding domain (RBD) that is responsible for primer binding, making it the only known polymerase to contain such a domain. The RBD is adjacent to, and probably overlaps with, an apparent catalytic region responsible for polymerization. Despite the presence of sequence similarities, this catalytic domain appears to be distinct from the conserved polymerase module found in a large number of RNA-dependent polymerases. PAP contains two nuclear localization signals (NLSs) in its C terminus, each by itself similar to the consensus bipartite NLS found in many nuclear proteins. Mutagenesis experiments indicate that both signals, which are separated by nearly 140 residues, play important roles in directing PAP exclusively to the nucleus. Surprisingly, basic amino acids in the N-terminal-most NLS are also essential for AAUAAA-dependent polyadenylation but not for nonspecific poly(A) synthesis, suggesting that this region of PAP is involved in interactions both with nuclear targeting proteins and with nuclear polyadenylation factors. The serine/threonine-rich C terminus is multiply phosphorylated, including at sites affected by mutations in either NLS.

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Mapping structural determinants of biological activities in snake venom phospholipases A2 by sequence analysis and site directed mutagenesis

Toxicon ◽

10.1016/j.toxicon.2003.11.027 ◽

2003 ◽

Vol 42 (8) ◽

pp. 869-883 ◽

Cited By ~ 61

Author(s):

Lucimara Chioato ◽

Richard J Ward

Keyword(s):

Sequence Analysis ◽

Snake Venom ◽

Biological Activities ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Structural Determinants ◽

Phospholipases A2

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

10.1055/s-0038-1643841 ◽

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.

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