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
Penicillin expandase, also known as deacetoxycephalosporin C synthase (DAOCS), is an essential enzyme involved in cephalosporin C biosynthesis. To evaluate the catalytic behaviors of penicillin expandase under high penicillin G concentration and to identify mutants suitable for industrial applications, the specific activities of wild-type DAOCS and several mutants with increased activities toward penicillin G were determined by HPLC under high penicillin G concentrations. Their specific activity profiles were compared with theoretical predictions by different catalytic dynamics models. We evaluated the specific activities of wild-type DAOCS and previous reported high-activity mutants H4, H5, H6 and H7 at concentrations ranging from 5.6 to 500 mmol/L penicillin G. The specific activities of wild-type DAOCS and mutant H4 increased as penicillin G concentration increased, but decreased when concentrations of substrate go above 200 mmol/L. Other mutants H5, H6 and H7 showed more complex behaviors under high concentration of penicillin G. Among all tested enzymes, mutant H6 showed the highest activity when concentration of penicillin G is above 100 mmol/L. Our results revealed that the substrate inhibition to wild-type DAOCS' by penicillin G is noncompetitive. Other DAOCS mutants showed more complex trends in their specific activities at high concentration of penicillin G (>100 mmol/L), indicating more complex substrate inhibition mechanism might exist. The substrate inhibition and activity of DAOCS mutants at high penicillin G concentration provide important insight to help select proper mutants for industrial application.
Subject(s)
Catalysis , Intramolecular Transferases , Genetics , Mutation , Penicillin G , Pharmacology , Penicillin-Binding Proteins , Genetics , Streptomyces , GeneticsABSTRACT
Two component system is a signal transduction system. It typically consists of a sensor histitine kinase and a cognate response regulator (RR) component. The activity of RR is regulated by a phosphorylation dependent mechanism. In recent years, the existence of atypical response regulators (ARRs), which rely on a phosphorylation independent mechanism to regulate their activity, have been recognized. ARRs are involved in the regulation of bacterial growth and development, antibiotic biosynthesis, iron transport, among others. Here we review the recent advances in the understanding of the structure and function of atypical response regulators, by using JadR1, a regulator in jadomycin biosynthesis in Streptomyces, as an example to elucidate the novel mechanism used by ARR to fine-tune its activity.
Subject(s)
Bacterial Proteins , Genetics , Gene Expression Regulation, Bacterial , Gene Expression Regulation, Developmental , Genes, Regulator , Genetics , Histidine Kinase , Isoquinolines , Metabolism , Naphthoquinones , Metabolism , Phosphorylation , Protein Kinases , Genetics , RNA-Binding Proteins , Genetics , Signal Transduction , Streptomyces , Metabolism , Transcription Factors , GeneticsABSTRACT
JadH is a bifunctional hydoxylase/dehydrase involved in jadomycin biosynthesis; it catalyzes a post-PKS modification reaction to convert 2,3-dehydro-UWM6 to dehydrorabelomycin. To identify the key residues involved in substrate-binding and catalysis, structural modeling and multiple sequence alignments of JadH homologs were performed to predict nine residues at the proximity of substrate. Site-directed mutagenesis of the corresponding residues and in vitro evaluation of the activities of the mutant enzymes, indicate these mutations severely reduced JadH activity. Our results indicate these residues are specifically involved in substrate-binding or catalysis in JadH.
Subject(s)
Amino Acid Sequence , Catalysis , Hydro-Lyases , Genetics , Metabolism , Isoquinolines , Metabolism , Mixed Function Oxygenases , Genetics , Metabolism , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutant Proteins , Metabolism , Naphthoquinones , Metabolism , Streptomyces , Metabolism , Substrate SpecificityABSTRACT
Fresh hepatocarcinoma tissue and spleen samples were taken from patients during surgery. B cells from spleen were purified and activated. The hepatocarcinoma vaccine was made by cell fusion between hepatic tumor cells and activated B cells. PEG was used as the fusion agent. The fusion cells were cultured and deactivated. MHC Ⅱ and B7 molecules on activated B cells were determined by flow cytometry. Fusion rate and recovery rate of cells after refrigeration were determined respectively at the same time. The results showed that MHC Ⅱ and B7 molecules on the activated B cells were enhanced comparing with B cell. The fusion rates of three cases were 66 84%, 74 43%, and 76 55%, respectively. The recovery rates of cells were 95% and 97% after DMSO and glycerol refrigeration, respectively. The results suggest that the hepatocarcinoma vaccine owns high fusion rate and recovery rate of cells after refrigeration. It's easy to make and store. So the hepatocarcinoma vaccine is suitable for clinical use.
ABSTRACT
Objective: To investigate the expression difference of TRAIL and its receptors between resting and activated peripheral blood lymphocytes, and to study their role in cellular growth and proliferation. Methods: Expression of TRAIL and its receptors were detected by RT PCR and DNA sequencing. Results: Neither the expression of TRAIL nor its receptors were detected in resting peripheral blood lymphocyte. However, both expression were found in activated peripheral blood lymphocytes of various degree. Conclusion: TRAIL and its receptors may play a role in regulation and control of the activated lymphocytes in immune reaction.