Mutagenesis of the borage Δ6 fatty acid desaturase

2000 ◽  
Vol 28 (6) ◽  
pp. 636-638 ◽  
Author(s):  
O. Sayanova ◽  
F. Beaudoin ◽  
B. Libisch ◽  
P. Shewry ◽  
J. Napier
Keyword(s):  
Fatty Acid ◽  
Consensus Sequence ◽  
Fatty Acid Desaturase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
The Third ◽  
Membrane Bound ◽  
Fusion Domain ◽  
Δ6 Desaturase

The consensus sequence of the third histidine box of a range of Δ5, Δ6, Δ8 and sphingolipid desaturases differs from that of the membrane-bound non-fusion Δ12 and Δ15 desaturases in the presence of glutamine instead of histidine. We have used site-directed mutagenesis to determine the importance of glutamine and other residues of the third histidine box and created a chimaeric enzyme to determine the ability of the Cyt b5 fusion domain from the plant sphingolipid desaturase to substitute for the endogenous domain of the Δ6 desaturase.

scholarly journals Active site labeling of fatty acid and polyketide acyl-carrier protein transacylases

2019 ◽  
Vol 17 (19) ◽  
pp. 4720-4724 ◽  
Author(s):  
Tony D. Davis ◽  
Jennifer M. Michaud ◽  
Michael D. Burkart
Keyword(s):  
Fatty Acid ◽  
Fluorescent Probe ◽  
Active Site ◽  
Polyketide Synthase ◽  
Acyl Carrier Protein ◽  
Site Directed Mutagenesis ◽  
Probe Design ◽  
Directed Mutagenesis ◽  
Carrier Protein

Fluorescent probe design and site-directed mutagenesis unveil new activity-based chemical reporters for fatty acid and polyketide synthase acyl-carrier protein transacylases.

Dissection of functional domains in Bcl-2α by site-directed mutagenesis

1994 ◽  
Vol 72 (11-12) ◽  
pp. 463-469 ◽  
Author(s):  
Christoph Borner ◽  
Reynald Olivier ◽  
Isabelle Martinou ◽  
Chantal Mattmann ◽  
Jurg Tschopp ◽  
...  
Keyword(s):  
Cell Survival ◽  
Protein Structures ◽  
Cell Types ◽  
Site Directed Mutagenesis ◽  
Functional Domains ◽  
Directed Mutagenesis ◽  
Membrane Attachment ◽  
Alternatively Spliced ◽  
Membrane Bound ◽  
Related Proteins

Bcl-2α is a mitochondrial or perinuclear-associated oncoprotein that prolongs the life span of a variety of cell types by interfering with programmed cell death. How Bcl-2 confers cell survival is unknown, although antioxidant and antiprotease functions have been proposed. In addition, protein structures of Bcl-2 that are crucial for its survival activity are still ill-defined. Bcl-2 can occur as Bcl-2α or Bcl-2β, two alternatively spliced forms which solely differ in their carboxyl termini. The finding that Bcl-2α is active and membrane bound, but Bcl-2β is inactive and cytosolic, indicates that the carboxyl terminus contributes to the survival activity of Bcl-2. This region contains two subdomains, a domain X with unknown function and a hydrophobic stretch reported to mediate membrane assocation of Bcl-2α. Recently Bcl-2-related proteins have been identified. These include Bax that heterodimerizes with Bcl-2 and, when overxpressed, counteracts Bcl-2. Bax contains two highly conserved regions of sequence homology with Bcl-2, referred to as Bcl-2 homology 1 and 2 (BH1 and BH2) domains. Site-directed mutagenesis studies have revealed that both domains are not only novel dimerization motifs for the interaction of Bax with Bcl-2 but also crucial for the survival activity of Bcl-2. Interestingly, the C-terminal end of BH2 encompasses the Bcl-2α/β splice site, as well as part of domain X in Bcl-2α. To better define the role of domain X and the hydrophobic C-terminal stretch of Bcl-2α for its survival activity, we created various deletion and truncation mutations in these regions by site-directed mutagenesis. We show here that membrane attachment and therefore the hydrophobic stretch is not required for the survival activity of Bcl-2, but part of domain X appears to be indispensable.Key words: apoptosis, Bcl-2, mutagenesis, cell survival, functional domains.

scholarly journals Sequential Autoprocessing of Marek's Disease Herpesvirus Protease Differs from That of Other Herpesviruses

2007 ◽  
Vol 81 (11) ◽  
pp. 6117-6121 ◽  
Author(s):  
S. Laurent ◽  
C. Blondeau ◽  
M. Belghazi ◽  
S. Remy ◽  
E. Esnault ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Consensus Sequence ◽  
Marek's Disease ◽  
Site Directed Mutagenesis ◽  
Viral Capsid ◽  
Directed Mutagenesis ◽  
Marek’S Disease ◽  
Polar Amino Acid ◽  
Kinetics Studies ◽  
Capsid Maturation

ABSTRACT Herpesviruses encode a unique serine protease essential for viral capsid maturation. This protease undergoes autoprocessing at two sites, R and M, at the consensus sequence (V, L, I)P3-XP2-AP1/SP1′ (where X is a polar amino acid). We observed complete autoprocessing at the R and M sites of Marek's disease virus (MDV) protease following production of the polyprotein in Escherichia coli. Site-directed mutagenesis confirmed the predicted sequence of the R and M sites, with the M site sequence being nonconsensual: MP3-NP2-AP1/SP1′. Mutagenesis and expression kinetics studies suggested that the atypical MDV M site was cleaved exclusively by the processed short protease, a feature making MDV unique among herpesviruses.

Structural significance of the GTP-binding domain of ras p21 studied by site-directed mutagenesis

1987 ◽  
Vol 7 (9) ◽  
pp. 3092-3097
Author(s):  
D J Clanton ◽  
Y Y Lu ◽  
D G Blair ◽  
T Y Shih
Keyword(s):  
Cell Lines ◽  
Consensus Sequence ◽  
Point Mutations ◽  
Soft Agar ◽  
Binding Activity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Gtp Binding ◽  
Nih 3T3 ◽  
Autokinase Activity

Point mutations of p21 proteins were constructed by oligonucleotide-directed mutagenesis of the v-rasH oncogene, which substituted amino acid residues within the nucleotide-binding consensus sequence, GXG GXGK. When the glycine residue at position 10, 13, or 15 was substituted with valine, the viral rasH product p21 lost its GTP-binding and autokinase activities. Other substitutions at position 33, 51, or 59 did not impair its binding activity. G418-resistant NIH 3T3 cell lines were derived by transfection with constructs obtained by inserting the mutant proviral DNA into the pSV2neo plasmid. Clones with a valine mutation at position 13 or 15 were incapable of transforming cells, while all other mutants with GTP-binding activity were competent. A mutant with a substitution of valine for glycine at position 10 which had lost its ability to bind GTP and its autokinase activity was fully capable of transforming NIH 3T3 cells. These cells grew in soft agar and rapidly formed tumors in nude mice. The p21 of cell lines derived from tumor explants still lacked the autokinase activity. These findings suggest that the glycine-rich consensus sequence is important in controlling p21 activities and that certain mutations may confer to p21 its active conformation without participation of ligand binding.

scholarly journals Site-directed mutagenesis of a fatty acid elongase ELO-like condensing enzyme

FEBS Letters ◽  
2013 ◽  
Vol 587 (23) ◽  
pp. 3837-3842 ◽  
Author(s):  
Selene Hernandez-Buquer ◽  
Brenda J. Blacklock
Keyword(s):  
Fatty Acid ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Fatty Acid Elongase ◽  
Condensing Enzyme

scholarly journals Identification of a Caenorhabditis elegans Δ6-fatty-acid-desaturase by heterologous expression in Saccharomyces cerevisiae

1998 ◽  
Vol 330 (2) ◽  
pp. 611-614 ◽  
Author(s):  
A. Johnathan NAPIER ◽  
J. Sandra HEY ◽  
J. Dominic LACEY ◽  
R. Peter SHEWRY
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acid ◽  
Caenorhabditis Elegans ◽  
Expressed Sequence Tag ◽  
Cytochrome B5 ◽  
Fatty Acid Desaturase ◽  
Higher Plant ◽  
Yeast Saccharomyces Cerevisiae ◽  
C Elegans ◽  
Δ6 Desaturase

We identified a cDNA expressed sequence tag from an animal (the nematode worm Caenorhabditis elegans) that showed weak similarity to a higher-plant microsomal Δ6-desaturase. A full-length cDNA clone was isolated and expressed in the yeast Saccharomyces cerevisiae. This demonstrated that the protein encoded by the C. elegans cDNA was that of a fatty acid Δ6-desaturase, as determined by the accumulation of γ-linolenic acid. The C. elegans Δ6-desaturase contained an N-terminalcytochrome b5 domain, indicating that it had a similar structure to that of the higher-plant Δ6-desaturase. The C. elegans Δ6-desaturase mapped to cosmid W08D2, a region of chromosome III. This is the first example of a Δ6-desaturase isolated from an animal and also the first example of an animal desaturase containing a cytochrome b5 domain.

scholarly journals LsbB Bacteriocin Interacts with the Third Transmembrane Domain of the YvjB Receptor

2016 ◽  
Vol 82 (17) ◽  
pp. 5364-5374 ◽  
Author(s):  
Marija Miljkovic ◽  
Gordana Uzelac ◽  
Nemanja Mirkovic ◽  
Giulia Devescovi ◽  
Dzung B. Diep ◽  
...  
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Transmembrane Helix ◽  
Sugar Transport ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Bacteriocin Activity ◽  
Binding Studies ◽  
Content Type ◽  
The Third

ABSTRACTThe Zn-dependent membrane-located protease YvjB has previously been shown to serve as a target receptor for LsbB, a class II leaderless lactococcal bacteriocin. AlthoughyvjBis highly conserved in the genusLactococcus, the bacteriocin appears to be active only against the subspeciesL. lactissubsp.lactis. Comparative analysis of the YvjB proteins of a sensitive strain (YvjBMN) and a resistant strain (YvjBMG) showed that they differ from each other in 31 positions. In this study, we applied site-directed mutagenesis and performed directed binding studies to provide biochemical evidence that LsbB interacts with the third transmembrane helix of YvjB in susceptible cells. The site-directed mutagenesis of LsbB and YvjB proteins showed that certain amino acids and the length of LsbB are responsible for the bacteriocin activity, most probably through adequate interaction of these two proteins; the essential amino acids in LsbB responsible for the activity are tryptophan (Trp25) and terminal alanine (Ala30). It was also shown that the distance between Trp25and terminal alanine is crucial for LsbB activity. The crucial region in YvjB for the interaction with LsbB is the beginning of the third transmembrane helix, particularly amino acids tyrosine (Tyr356) and alanine (Ala353).In vitroexperiments showed that LsbB could interact with both YvjBMNand YvjBMG, but the strength of interaction is significantly less with YvjBMG.In vivoexperiments with immunofluorescently labeled antibody demonstrated that LsbB specifically interacts only with cells carrying YvjBMN.IMPORTANCEThe antimicrobial activity of LsbB bacteriocin depends on the correct interaction with the corresponding receptor in the bacterial membrane of sensitive cells. Membrane-located bacteriocin receptors have essential primary functions, such as cell wall synthesis or sugar transport, and it seems that interaction with bacteriocins is suicidal for cells. This study showed that the C-terminal part of LsbB is crucial for the bacteriocin activity, most probably through adequate interaction with the third transmembrane domain of the YvjB receptor. The conserved Tyr356and Ala353residues of YvjB are essential for the function of this Zn-dependent membrane-located protease as a bacteriocin receptor.

scholarly journals A novel Delta12-fatty acid desaturase gene from methylotrophic yeast Pichia pastoris GS115.

2006 ◽  
Vol 53 (4) ◽  
pp. 753-759 ◽  
Author(s):  
D Sh Wei ◽  
M Ch Li ◽  
X X Zhang ◽  
H Zhou ◽  
L J Xing
Keyword(s):  
Fatty Acid ◽  
Functional Characterization ◽  
Fatty Acid Desaturase ◽  
Methylotrophic Yeast ◽  
Pastoris Gs115 ◽  
Desaturase Gene ◽  
Methylotrophic Yeast Pichia Pastoris ◽  
Membrane Bound ◽  
Fatty Acid Desaturase Gene

The methylotrophic yeast Pichia pastoris GS115, a widely used strain in production of various heterologous proteins, especially membrane-bound enzymes, can also produce linoleic and linolenic acids, which indicates the existence of membrane-bound Delta12 and Delta15-fatty acid desaturases. This paper describes the cloning and functional characterization of a novel Delta12-fatty acid desaturase gene from this methylotrophic yeast. The open reading frame of the gene (named Pp-FAD12) is 1263 bp in size and encodes a 420-amino-acid peptide. The deduced Pp-FAD12 protein shows high identity (50-67%) with Delta12-fatty acid desaturases from other fungi. It also shows a high identity (57%) with Delta15-fatty acid desaturase (named Sk-FAD15) from Saccharomyces kluyveri. Expression of Pp-FAD12 in polyunsaturated fatty acids non-producing yeast Saccharomyces cerevisiae demonstrated that its product converted oleic acid (18 : 1) to linoleic acid (18 : 2). This result suggests that Pp-FAD12 encodes a novel Delta12-fatty acid desaturase in P. pastoris GS115. This is the first report about the cloning and functional characterization of Delta12-fatty acid desaturase gene in methylotrophic yeast.

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