scholarly journals High throughput mass spectrometry-based characterisation of Arabidopsis thaliana group H glycosyltransferases

RSC Advances ◽  
2018 ◽  
Vol 8 (53) ◽  
pp. 30080-30086 ◽  
Author(s):  
Aishat Akere ◽  
Qian Liu ◽  
Shibo Wu ◽  
Bingkai Hou ◽  
Min Yang
Keyword(s):  
Mass Spectrometry ◽  
Arabidopsis Thaliana ◽  
Sequence Alignment ◽  
High Throughput ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Substrate Specificities

We cloned and characterised four group H glycosyltransferases by studying their substrate specificities and kinetics. Sequence alignment and site-directed mutagenesis studies showed that serine is a crucial residue for UDPGlcNAc and UDPGal activity.

scholarly journals The two cathepsin B-like proteases of Arabidopsis thaliana are closely related enzymes with discrete endopeptidase and carboxydipeptidase activities

2018 ◽  
Vol 399 (10) ◽  
pp. 1223-1235 ◽  
Author(s):  
Andreas Porodko ◽  
Ana Cirnski ◽  
Drazen Petrov ◽  
Teresa Raab ◽  
Melanie Paireder ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Active Site ◽  
Cathepsin B ◽  
Cysteine Proteinase ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Substrate Specificities ◽  
Active Site Cleft ◽  
Molecular Modeling Studies ◽  
Human Cathepsin

Abstract The genome of the model plant Arabidopsis thaliana encodes three paralogues of the papain-like cysteine proteinase cathepsin B (AtCathB1, AtCathB2 and AtCathB3), whose individual functions are still largely unknown. Here we show that a mutated splice site causes severe truncations of the AtCathB1 polypeptide, rendering it catalytically incompetent. By contrast, AtCathB2 and AtCathB3 are effective proteases which display comparable hydrolytic properties and share most of their substrate specificities. Site-directed mutagenesis experiments demonstrated that a single amino acid substitution (Gly336→Glu) is sufficient to confer AtCathB2 with the capacity to tolerate arginine in its specificity-determining S2 subsite, which is otherwise a hallmark of AtCathB3-mediated cleavages. A degradomics approach utilizing proteome-derived peptide libraries revealed that both enzymes are capable of acting as endopeptidases and exopeptidases, releasing dipeptides from the C-termini of substrates. Mutation of the carboxydipeptidase determinant His207 also affected the activity of AtCathB2 towards non-exopeptidase substrates, highlighting mechanistic differences between plant and human cathepsin B. This was also noted in molecular modeling studies which indicate that the occluding loop defining the dual enzymatic character of cathepsin B does not obstruct the active-site cleft of AtCathB2 to the same extent as in its mammalian orthologues.

Mass Spectrometry and Site-directed Mutagenesis Identify Several Autophosphorylated Residues Required for the Activity of PrkC, a Ser/Thr Kinase from Bacillus subtilis

2003 ◽  
Vol 330 (3) ◽  
pp. 459-472 ◽  
Author(s):  
Edwige Madec ◽  
Allan Stensballe ◽  
Sven Kjellström ◽  
Lionel Cladière ◽  
Michal Obuchowski ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Bacillus Subtilis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

Mutual conversion of substrate specificities of Thermoactinomyces vulgaris R-47 α-amylases TVAI and TVAII by site-directed mutagenesis

2003 ◽  
Vol 338 (15) ◽  
pp. 1553-1558 ◽  
Author(s):  
Akashi Ohtaki ◽  
Akihiro Iguchi ◽  
Masahiro Mizuno ◽  
Takashi Tonozuka ◽  
Yoshiyuki Sakano ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Substrate Specificities ◽  
Thermoactinomyces Vulgaris ◽  
Mutual Conversion

HTP-OligoDesigner: An Online Primer Design Tool for High-Throughput Gene Cloning and Site-Directed Mutagenesis

2016 ◽  
Vol 23 (1) ◽  
pp. 27-29 ◽  
Author(s):  
Cesar M. Camilo ◽  
Gustavo M.A. Lima ◽  
Fernando V. Maluf ◽  
Rafael V.C. Guido ◽  
Igor Polikarpov
Keyword(s):  
Gene Cloning ◽  
High Throughput ◽  
Primer Design ◽  
Design Tool ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis
Sign in / Sign up

Export Citation Format

Share Document