Chemical Synthesis, Molecular Cloning, Overexpression, and Site-Directed Mutagenesis of the Gene Coding for Pumpkin (Curcubita maxima) Trypsin Inhibitor CMTI-V

1993 ◽  
Vol 4 (3) ◽  
pp. 215-222 ◽  
Author(s):  
L. Wen ◽  
S.S. Kim ◽  
T.T. Tinn ◽  
J.K. Huang ◽  
R. Krishnamoorthi ◽  
...  
Keyword(s):  
Chemical Synthesis ◽  
Molecular Cloning ◽  
Trypsin Inhibitor ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Gene Coding

scholarly journals Ribose 5-phosphate isomerase type B from Trypanosoma cruzi: kinetic properties and site-directed mutagenesis reveal information about the reaction mechanism

2006 ◽  
Vol 401 (1) ◽  
pp. 279-285 ◽  
Author(s):  
Ana L. Stern ◽  
Emmanuel Burgos ◽  
Laurent Salmon ◽  
Juan J. Cazzulo
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Pentose Phosphate ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Kinetic Properties ◽  
Type B ◽  
Nucleotide Synthesis ◽  
Gene Coding ◽  
Genes Encoding

Trypanosoma cruzi, the human parasite that causes Chagas disease, contains a functional pentose phosphate pathway, probably essential for protection against oxidative stress and also for R5P (ribose 5-phosphate) production for nucleotide synthesis. The haploid genome of the CL Brener clone of the parasite contains one gene coding for a Type B Rpi (ribose 5-phosphate isomerase), but genes encoding Type A Rpis, most frequent in eukaryotes, seem to be absent. The RpiB enzyme was expressed in Escherichia coli as a poly-His tagged active dimeric protein, which catalyses the reversible isomerization of R5P to Ru5P (ribulose 5-phos-phate) with Km values of 4 mM (R5P) and 1.4 mM (Ru5P).4-Phospho-D-erythronohydroxamic acid, an analogue to the reaction intermediate when the Rpi acts via a mechanism involving the formation of a 1,2-cis-enediol, inhibited the enzyme competi-tively, with an IC50 value of 0.7 mM and a Ki of 1.2 mM. Site-directed mutagenesis allowed the demonstration of a role for His102, but not for His138, in the opening of the ribose furanosic ring. A major role in catalysis was confirmed for Cys69, since the C69A mutant was inactive in both forward and reverse directions of the reaction. The present paper contributes to the know-ledge of the mechanism of the Rpi reaction; in addition, the absence of RpiBs in the genomes of higher animals makes this enzyme a possible target for chemotherapy of Chagas disease.

Molecular Cloning, Modeling, and Site-Directed Mutagenesis of Type III Polyketide Synthase from Sargassum binderi (Phaeophyta)

2010 ◽  
Vol 13 (5) ◽  
pp. 845-856 ◽  
Author(s):  
Hariyanti Baharum ◽  
Hiroyuki Morita ◽  
Akifumi Tomitsuka ◽  
Fong-Chin Lee ◽  
Kim-Yong Ng ◽  
...  
Keyword(s):  
Molecular Cloning ◽  
Polyketide Synthase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Type Iii Polyketide Synthase ◽  
Type Iii ◽  
Sargassum Binderi

scholarly journals Amino Acid Repetitions in the Dihydropteroate Synthase of Streptococcus pneumoniae Lead to Sulfonamide Resistance with Limited Effects on SubstrateKm

2001 ◽  
Vol 45 (3) ◽  
pp. 805-809 ◽  
Author(s):  
Ylva Haasum ◽  
Katrin Ström ◽  
Rahma Wehelie ◽  
Vicki Luna ◽  
Marilyn C. Roberts ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Washington State ◽  
Site Directed Mutagenesis ◽  
Clinical Isolates ◽  
Directed Mutagenesis ◽  
Aminobenzoic Acid ◽  
Kinetic Measurements ◽  
Dihydropteroate Synthase ◽  
Gene Coding ◽  
Sulfonamide Resistance

ABSTRACT Sulfonamide resistance in Streptococcus pneumoniae is due to changes in the chromosomal folP (sulA) gene coding for dihydropteroate synthase (DHPS). The first reported laboratory-selected sulfonamide-resistant S. pneumoniaeisolate had a 6-bp repetition, the sul-d mutation, leading to a repetition of the amino acids Ile66 and Glu67 in the gene product DHPS. More recently, clinical isolates showing this and other repetitions have been reported. WA-5, a clinical isolate from Washington State, contains a 6-bp repetition in the folP gene, identical to the sul-d mutation. The repetition was deleted by site-directed mutagenesis. Enzyme kinetic measurements showed that the deletion was associated with a 35-fold difference in Ki for sulfathiazole but changed the Km for p-aminobenzoic acid only 2.5-fold and did not significantly change theKm for 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine pyrophosphate. The enzyme characteristics of the deletion variant were identical to those of DHPS from a sulfonamide-susceptible strain. DHPS from clinical isolates with repetitions of Ser61 had very similar enzyme characteristics to the DHPS from WA-5. The results confirm that the repetitions are sufficient for development of a resistant enzyme and suggest that the fitness cost to the organism of developing resistance may be very low.

scholarly journals Chemical Synthesis of the Sec-To-Cys Homologue of Human Selenoprotein F and Elucidation of Its Disulfide-pairing Mode

2021 ◽  
Vol 9 ◽  
Author(s):  
Peisi Liao ◽  
Chunmao He
Keyword(s):  
Chemical Synthesis ◽  
The Self ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Synthetic Route ◽  
One Pot ◽  
Synthetic Protein ◽  
Disulfide Pairing ◽  
Structural Insights ◽  
Pairing Mode

Herein, we document a highly optimized synthesis of the Sec-to-Cys homologue of the human selenoprotein F (SelF) through a three-segment two-ligation semisynthesis strategy. Highlighted in this synthetic route are two one-pot manipulations, i.e. the first ligation followed by a desulfurization and the second ligation followed by the protein refolding. This way multi-milligrams of the folded synthetic protein was obtained, which set the stage for the synthesis of the natural selenoprotein. Moreover, the disulfide pairing mode of the SelF was elucidated through a combination of site-directed mutagenesis and LC-MS study. It provides not only a criterion to judge the viability of the synthetic protein, and more importantly, useful structural insights into the previously unresolved UGGT-binding domain of SelF.

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