ABSTRACT
The ketoreductase (KR) domain in the first extending module of the polyketide synthase (PKS) catalyzes the reductions of both an α-keto group and a β-keto group in the biosynthesis of bacillaene, suggesting the intrinsic substrate promiscuity. In order to further investigate the substrate specificity, the KR domain (BacKR1) was heterologously overexpressed in Escherichia coli. In vitro enzymatic analysis showed that only one of the four diastereomers was formed in the reduction of the racemic (±)-2-methyl-3-oxopentanoyl-N-acetylcysteamine thioester catalyzed by BacKR1. In addition, BacKR1 was revealed to catalyze the reductions of cyclohexanone and p-chloroacetophenone, indicating the potential of KR domians of PKSs as biocatalysts.
Subject(s)
Bacterial Proteins , Genetics , Metabolism , Catalysis , Cyclohexanones , Metabolism , Escherichia coli , Polyketide Synthases , Genetics , Metabolism , Protein Structure, Tertiary , Substrate Specificity , omega-Chloroacetophenone , MetabolismABSTRACT
This study was aimed to examine the expression of apoptosis-associated gene Fas in HeLa cell, explore the effects of the co-immobilized cytokines (tumor necrosis factor-alpha and interferon-gamma), and probe the potential mechanism of action. The preparation and application of the research couple IFN-gamma and TNF-alpha to the polystyrene cell culture plate were performed using the Photo-immobilization method, with different doses (20 ng/well and 200 ng/well) and synthesized optical active material. HeLa cells were treated with cytokines for two dose and 1, 3, 6 days. The result showed that the free cytokines induced HeLa apoptosis quickly, yet the HeLa apoptosis induced by co-immobilized cytokines had longer effect.