Site-Directed Mutagenesis and Saturation Mutagenesis for the Functional Study of Transcription Factors Involved in Plant Secondary Metabolite Biosynthesis

2010 ◽  
pp. 47-57 ◽  
Author(s):  
Sitakanta Pattanaik ◽  
Joshua R. Werkman ◽  
Que Kong ◽  
Ling Yuan
Keyword(s):  
Transcription Factors ◽  
Secondary Metabolite ◽  
Site Directed Mutagenesis ◽  
Saturation Mutagenesis ◽  
Plant Secondary Metabolite ◽  
Functional Study ◽  
Directed Mutagenesis ◽  
Secondary Metabolite Biosynthesis

scholarly journals Alteration of Leucine Aminopeptidase from Streptomyces septatus TH-2 to Phenylalanine Aminopeptidase by Site-Directed Mutagenesis

2005 ◽  
Vol 71 (11) ◽  
pp. 7229-7235 ◽  
Author(s):  
Jiro Arima ◽  
Yoshiko Uesugi ◽  
Masaki Iwabuchi ◽  
Tadashi Hatanaka
Keyword(s):  
Methyl Ester ◽  
Leucine Aminopeptidase ◽  
Hydrolytic Activity ◽  
Site Directed Mutagenesis ◽  
Saturation Mutagenesis ◽  
Side Chain ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Peptide Substrates ◽  
Hydrolytic Activities

ABSTRACT To tailor leucine aminopeptidase from Streptomyces septatus TH-2 (SSAP) to become a convenient biocatalyst, we are interested in Phe221 of SSAP, which is thought to interact with the side chain of the N-terminal residue of the substrate. By using saturation mutagenesis, the feasibility of altering the performance of SSAP was evaluated. The hydrolytic activities of 19 mutants were investigated using aminoacyl p-nitroanilide (pNA) derivatives as substrates. Replacement of Phe221 resulted in changes in the activities of all the mutants. Three of these mutants, F221G, F221A, and F221S, specifically hydrolyzed l-Phe-pNA, and F221I SSAP exhibited hydrolytic activity with l-Leu-pNA exceeding that of the wild type. Although the hydrolytic activities with peptide substrates decreased, the hydrolytic activities with amide and methyl ester substrates were proportional to the changes in the hydrolytic activities with pNA derivatives. Furthermore, based on a comparative kinetic study, the mechanism underlying the alteration in the preference of SSAP from leucine to phenylalanine is discussed.

scholarly journals Recognition of Phosphorylated-Smad2-Containing Complexes by a Novel Smad Interaction Motif

2004 ◽  
Vol 24 (3) ◽  
pp. 1106-1121 ◽  
Author(s):  
Rebecca A. Randall ◽  
Michael Howell ◽  
Christopher S. Page ◽  
Amanda Daly ◽  
Paul A. Bates ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Growth Factor ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Hydrophobic Pocket ◽  
Winged Helix ◽  
The Common ◽  
Necessary And Sufficient

ABSTRACT Transforming growth factor β (TGF-β) superfamily members signal via complexes of activated Smads, comprising phosphorylated receptor-regulated Smads, such as Smad2 and Smad3, and Smad4. These complexes are recruited to DNA by specific transcription factors. The forkhead/winged-helix transcription factors, XFast-1/XFoxH1a and XFast-3/XFoxH1b, bind an activated Smad heterotrimer comprising two Smad2s and one Smad4. Here we identify a novel Smad2 interaction motif, the Fast/FoxH1 motif (FM), present in all known Fast/FoxH1 family members, N-terminal to the common Smad interaction motif (SIM). The FM is necessary and sufficient to bind active Smad2/Smad4 complexes. The FM differs from the SIM since it discriminates between Smad2 and Smad3, and moreover only binds phosphorylated Smad2 in the context of activated Smad complexes. It is the first Smad interaction motif with this property. Site-directed mutagenesis indicates that the binding site for the FM on a Smad2/Smad4 heterotrimer is a hydrophobic pocket that incorporates the Smad/Smad interface. We demonstrate that the presence of an FM and SIM in the Fast/FoxH1 proteins allows them to compete efficiently for activated Smad2/Smad4 complexes with transcription factors such as Mixer that only contain a SIM. This establishes a hierarchy of Smad-interacting transcription factors, determined by their affinity for active Smad complexes.

scholarly journals A Kinetic Study of the Replacement by Site Saturation Mutagenesis of Residue 119 in NDM-1 Metallo-β-Lactamase

2018 ◽  
Vol 62 (8) ◽  
Author(s):  
Francesca Marcoccia ◽  
Paola Sandra Mercuri ◽  
Moreno Galleni ◽  
Giuseppe Celenza ◽  
Gianfranco Amicosante ◽  
...  
Keyword(s):  
Kinetic Study ◽  
Broad Spectrum ◽  
Site Directed Mutagenesis ◽  
Saturation Mutagenesis ◽  
Fourth Generation ◽  
Directed Mutagenesis ◽  
New Delhi ◽  
Site Saturation

ABSTRACT New Delhi metallo-β-lactamase 1 (NDM-1) is a subclass B1 metallo-β-lactamase that exhibits a broad spectrum of activity against β-lactam antibiotics. Here we report the kinetic study of 6 Q119X variants obtained by site-directed mutagenesis of NDM-1. All Q119X variants were able to hydrolyze carbapenems, penicillins and first-, second-, third-, and fourth-generation cephalosporins very efficiently. In particular, Q119E, Q119Y, Q119V, and Q119K mutants showed improvements in kcat/Km values for penicillins, compared with NDM-1. The catalytic efficiencies of the Q119K variant for benzylpenicillin and carbenicillin were about 65- and 70-fold higher, respectively, than those of NDM-1. The Q119K and Q119Y enzymes had kcat/Km values for ceftazidime about 25- and 89-fold higher, respectively, than that of NDM-1.

scholarly journals Probing the Substrate Specificity of Aminopyrrolnitrin Oxygenase (PrnD) by Mutational Analysis

2006 ◽  
Vol 188 (17) ◽  
pp. 6179-6183 ◽  
Author(s):  
Jung-Kul Lee ◽  
Ee-Lui Ang ◽  
Huimin Zhao
Keyword(s):  
Substrate Specificity ◽  
Mutational Analysis ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Saturation Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Enzyme Substrate ◽  
Molecular Determinants

ABSTRACT Molecular modeling and mutational analysis (site-directed mutagenesis and saturation mutagenesis) were used to probe the molecular determinants of the substrate specificity of aminopyrrolnitrin oxygenase (PrnD) from Pseudomonas fluorescens Pf-5. There are 17 putative substrate-contacting residues, and mutations at two of the positions, positions 312 and 277, could modulate the enzyme substrate specificity separately or in combination. Interestingly, several of the mutants obtained exhibited higher catalytic efficiency (approximately two- to sevenfold higher) with the physiological substrate aminopyrrolnitrin than the wild-type enzyme exhibited.

scholarly journals Transcriptional Regulation of HMOX1 Gene in Hezuo Tibetan Pigs: Roles of WT1, Sp1, and C/EBPα

Genes ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 352
Author(s):  
Wei Wang ◽  
Qiaoli Yang ◽  
Kaihui Xie ◽  
Pengfei Wang ◽  
Ruirui Luo ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Transcription Factors ◽  
Promoter Region ◽  
Core Promoter ◽  
Expression Profiles ◽  
Site Directed Mutagenesis ◽  
Functional Study ◽  
Heme Oxygenase 1 ◽  
Core Promoter Region ◽  
Tibetan Pig

Heme oxygenase 1 (HMOX1) is a stress-inducing enzyme with multiple cardiovascular protective functions, especially in hypoxia stress. However, transcriptional regulation of swine HMOX1 gene remains unclear. In the present study, we first detected tissue expression profiles of HMOX1 gene in adult Hezuo Tibetan pig and analyzed the gene structure. We found that the expression level of HMOX1 gene was highest in the spleen of the Hezuo Tibetan pig, followed by liver, lung, and kidney. A series of 5’ deletion promoter plasmids in pGL3-basic vector were used to identify the core promoter region and confirmed that the minimum core promoter region of swine HMOX1 gene was located at −387 bp to −158 bp region. Then we used bioinformatics analysis to predict transcription factors in this region. Combined with site-directed mutagenesis and RNA interference assays, it was demonstrated that the three transcription factors WT1, Sp1 and C/EBPα were important transcription regulators of HMOX1 gene. In summary, our study may lay the groundwork for further functional study of HMOX1 gene.

Functional study of mnemiopsin photoprotein using site directed mutagenesis

2011 ◽  
Vol 44 (13) ◽  
pp. S91-S92
Author(s):  
Zohreh Jahani ◽  
Reza H. Sajedi ◽  
Maryam Molakarimi ◽  
Atiyeh Mahdavi ◽  
Saman Hosseinkhani ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Functional Study ◽  
Directed Mutagenesis

scholarly journals Changes in Primary and Secondary Metabolite Levels in Response to Gene Targeting-Mediated Site-Directed Mutagenesis of the Anthranilate Synthase Gene in Rice

Metabolites ◽  
2012 ◽  
Vol 2 (4) ◽  
pp. 1123-1138 ◽  
Author(s):  
Hiroaki Saika ◽  
Akira Oikawa ◽  
Ryo Nakabayashi ◽  
Fumio Matsuda ◽  
Kazuki Saito ◽  
...  
Keyword(s):  
Gene Targeting ◽  
Secondary Metabolite ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Anthranilate Synthase

scholarly journals PEST motifs are not required for rapid calpain-mediated proteolysis of c-fos protein

1996 ◽  
Vol 313 (1) ◽  
pp. 245-251 ◽  
Author(s):  
Serge CARILLO ◽  
Magali PARIAT ◽  
Ann-Muriel STEFF ◽  
Isabelle JARIEL-ENCONTRE ◽  
Francis POULAT ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Glutamic Acid ◽  
Aspartic Acid ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Step Process ◽  
Calcium Dependent ◽  
Fos Protein ◽  
A Cell ◽  
Intracellular Proteases

Cytoplasmic degradation of c-fos protein is extremely rapid. Under certain conditions, it is a multi-step process initiated by calcium-dependent and ATP-independent proteases called calpains. PEST motifs are peptide regions rich in proline, glutamic acid/aspartic acid and serine/threonine residues, commonly assumed to constitute built-in signals for rapid recognition by intracellular proteases and particularly by calpains. Using a cell-free degradation assay and site-directed mutagenesis, we report here that the three PEST motifs of c-fos are not required for rapid cleavage by calpains. Testing the susceptibility of PEST motif-bearing and non-bearing transcription factors including GATA1, GATA3, Myo D, c-erbA, Tal-1 and Sry, demonstrates that PEST sequences are neither necessary nor sufficient for specifying degradation of other proteins by calpains. This conclusion is strengthened by the observation that certain proteins, reportedly known to be cleavable by calpains, are devoid of PEST motifs

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