Influence of Site-Directed Mutagenesis in Coenzyme-Binding Domain of Car-bonyl Reductase on Its Catalytic Performance for Asymmetric Reduction
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Site-directed mutagenesis
Coenzyme A
Site-directed mutagenesis
Directed mutagenesis
Saturated mutagenesis
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Site-directed mutagenesis
Directed mutagenesis
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Methyl (RS)-5-bromo-3-hydroxy-3-methyl-pentanoate was prepared by bromination of methyl mevalonate and used for the formation of 4-carboxy-3-hydroxy-3-methylbutyl thioether derivatives by reaction with N-octanoyl-cysteamine, pantetheine, phosphopantetheine and coenzyme A. These thiols were also converted to the (RS)-3-hydroxy-3-methylglutaryl thioester derivatives. The thioesters formed with pantetheine and phosphopantetheine are substrates of 3-hydroxy-3-methylglutaryl-CoA reductase; Km and V values are similar to those of the superior CoA-derivative. The corresponding thioether derivatives in which the oxygen next to sulfur of the substrates is replaced by hydrogen, are inhibitors of the reductase. The inhibition is competitive with 3-hydroxy-3-methylglutaryl-CoA varied, and noncompetitive with NADPH varied. For each of the corresponding pairs of thioester and thioether derivatives Km (substrate) is nearly identical with Ki (inhibitor). The specificity and stereospecificity of the inhibitor action are also shown.
Thioether
Thioester
Coenzyme A
Stereospecificity
Ethylenedioxy
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Coenzyme A
Hydroxymethylglutaryl-CoA reductase
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A new approach to induce directed mutations in genes of study through simple cotransfection of E. coli cells by the mixture of primer and template was developed. This method is based on the use of synthetic phosphotriester analogues of oligonucleotides as site-specific mutagenic primers. The achieved yield of mutant clones was 2-3%.
Site-directed mutagenesis
Primer (cosmetics)
Directed mutagenesis
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Coenzyme A
Thioglycolic acid
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Since genomic data are widely available, many strategies have been implemented to reveal the function of specific nucleotides or amino acids in promoter regions or proteins, respectively. One of the methods most commonly used to determine the impact of mutations is the site‐directed mutagenesis using the polymerase chain reaction (PCR). There are different published protocols to develop single or multiple site‐directed mutagenesis. In this chapter, we reviewed the enzymes commonly used in site‐directed mutagenesis, the methods for simple and multiple site‐directed mutagenesis in large constructs, mediated by insertion of restriction sites. Other methods reviewed include high‐throughput site‐directed mutagenesis using oligonucleotides synthesized on DNA chips, and those based on multi‐site‐directed mutagenesis, based on recombination. Software tools to design site‐directed mutagenesis primers are also presented.
Site-directed mutagenesis
Directed mutagenesis
Restriction site
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Objective: A convenient PCR was used for site-directed mutagenesis to modify structure of proteins or peptides, For function-structure studies of proteins or peptides. Method: the one-step PCR site-directed mutagenesis strategy can introduce mutation of gene through 5 / -end of primer, which was applied on peptide Erabutoxin B (EB) to produce three mutants of EB, the activity of mutants was detected by LD50 value of mice. Result: S8Y, R33D, K47R three mutants of EB were obtained by one-step PCR, LD 50 of mutants indicated that the activity of mutants decreased in different degree, the activity of R33D was nearly deprived. Conclusion: one-step PCR site-directed mutagenesis was convenien and efficient, it can be applied on restructuring the primary structure of proteins or peptides.
Site-directed mutagenesis
Primer (cosmetics)
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Site-directed mutagenesis is used to introduce desired mutations into target DNA sequences.Site-directed mutagenesis has become the conventional means to operate gene.Some methods of site-directed mutagenesis are reviewed and discussed in the paper.
Site-directed mutagenesis
Directed mutagenesis
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Substitute Ala for the sites of Ser 32 and Ser 36 in porcine IκBα gene,to establish the fundament for mechanism research of the mutant IκBα inhibition on delayed xenograft rejection.QuikChange Site-Directed Mutagenesis Kit was first uesd for the mutagenesis of Ser 32 site,then the kit was subsequently used for the mutagenesis of Ser 36 site,but failed.Finally OE-PCR was used for the mutagenesis of Ser 36 site.These two mutagenesis methods were combined to achieve site-specific mutagenesis at the sites of Ser 32 and Ser 36 in porcine IκBα gene.These two methods each has a unique,and each has their own shortcoming.So,in order to get better result by less work,mutation methods should be chosen flexibly according to the real need.
Site-directed mutagenesis
Directed mutagenesis
Insertional mutagenesis
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