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.
Catalysis ; Intramolecular Transferases ; genetics ; Mutation ; Penicillin G ; pharmacology ; Penicillin-Binding Proteins ; genetics ; Streptomyces ; enzymology ; genetics

Gene expression exhibits temporal and spatial patterns to response environmental changes and growth cycle. Gene expression is under strict control at different levels among which control at transcription level is the predominant mode, especially in prokaryotes. In this review, we summarized the new developments of methods used in transcriptional studies, including modifications and improvements of the classic methods, such as gel-shift assay, DNA foot printing, and in vivo reporter system. In addition, we introduced examples to apply new methods, such as surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) to characterize protein-DNA, ligand-protein, and ligand-protein-DNA interactions. The collection of these methods and their application could guide and accelerate relevant studies.
Calorimetry ; DNA Footprinting ; Gene Expression ; Ligands ; Proteins ; Surface Plasmon Resonance ; Transcription, Genetic

Bacterial two-hybrid system is a newly developed method for studying protein-protein interactions. However, in our studies of the interaction of regulatory proteins in Streptomyces, it was found that the bacterial two-hybrid system is not sensitive enough by the blue-and-white selection on X-gal plate. To overcome this drawback, the reason of false positive clone was firstly determined, which was the disturbance of other direct or indirect regulation on lacZ promoter. Then the disturbance was diluted by introducing multicopy lacZ promoter, which drive another reporter gene gfp. By such design, the sensitivity of the modified bacterial two-hybrid system was significantly inproved and the two different reporters also help to decrease the rate of the false positive clones. Further the evaluation of the modifiedd bacterial two-hybrid system indicated that the sensitivity was significantly improved.
Bacteria ; Genes, Reporter ; Promoter Regions, Genetic ; Protein Interaction Mapping ; methods ; Two-Hybrid System Techniques

Objective To study the mechanism of increased sensitivity to cisplatin by nm23-H1.Methods The plasmid,pCMV-BamH1-Neo-nm23-H1,was transfected to cell line Tca8113 by lipofection strategy and was selected by G418 for 5 weeks.The stable products of nm23-H1 gene were identified by Immunohistofluorescence and Western-blotting.Using MTT and flow cytometer,we detected the changes of cell mortality rate,apoptosis and mitochondrial membrane potential.Results We successfully established the cell line of stable overexpression of nm23-H1.In vitro,the cell mortality rate and apoptosis were increased significantly in stable expression cell line of nm23-H1,compared with those in Tca8113 cell line of non-transfection;this effect could be inhibited by oubain which is an inhibitor of Na(+)/K(+)-ATPase.Mitochondrial membrane potential was decreased significantly in stable expression cell line of nm23-H1.Conclusion Nm23-H1 can increase the sensitivity of cisplatin on Tca8113 cell line;The mechanism may be the mitochondrial membrane potential is decreased by nm23-H1 so that intracellular platinum are increased to finally cause the apoptosis and necrosis of cells.

A library with potential to produce six amino acids cyclic peptides was prepared using pET-28a as the starting plasmid. pVmut was used to amplify the Int(C)-dnaB-N-Int(N) fragment that was inserted into pET28a to give pEV. On pEV, DnaB split intein was expressed under the strong T7 promoter. Analyses of Escherichia coli transformed with pEV showed that DnaB split intein was produced in large quantity and the fusion protein self-spliced efficiently to produce cyclized DnaB-N. A synthesized 115 bp fragment mixture encoding 5 random amino acids was inserted into pEV to generate pEV-IS. The ligation mixture was transformed into E. coli. A library of 10(3) clones was obtained, 20 randomly picked clones were sequenced. All of them contain different sequences. Nine clones were chosen for further analysis. Split-intein-ISs were expressed in large quantity, and 90% of them self-spliced under 16 degrees C in 20 hours. After induction at 30 degrees C for 3 hours, the expressed DnaB split intein was purified using His-column, and then a molecular weight of target cyclic peptide was detected by MALDI-TOF-MS.
Amino Acids ; chemistry ; Base Sequence ; DnaB Helicases ; chemistry ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Humans ; Inteins ; genetics ; Molecular Sequence Data ; Peptide Library ; Peptides, Cyclic ; chemistry ; Protein Splicing ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Synechocystis ; chemistry

Streptomycetes produce many antibiotics and are important model microorgansims for scientific research and antibiotic production. Metabolomics is an emerging technological platform to analyze low molecular weight metabolites in a given organism qualitatively and quantitatively. Compared to other Omics platform, metabolomics has greater advantage in monitoring metabolic flux distribution and thus identifying key metabolites related to target metabolic pathway. The present work aims at establishing a rapid, accurate sample preparation protocol for metabolomics analysis in streptomycetes. In the present work, several sample preparation steps, including cell quenching time, cell separation method, conditions for metabolite extraction and metabolite derivatization were optimized. Then, the metabolic profiles of Streptomyces coelicolor during different growth stages were analyzed by GC-MS. The optimal sample preparation conditions were as follows: time of low-temperature quenching 4 min, cell separation by fast filtration, time of freeze-thaw 45 s/3 min and the conditions of metabolite derivatization at 40 degrees C for 90 min. By using this optimized protocol, 103 metabolites were finally identified from a sample of S. coelicolor, which distribute in central metabolic pathways (glycolysis, pentose phosphate pathway and citrate cycle), amino acid, fatty acid, nucleotide metabolic pathways, etc. By comparing the temporal profiles of these metabolites, the amino acid and fatty acid metabolic pathways were found to stay at a high level during stationary phase, therefore, these pathways may play an important role during the transition between the primary and secondary metabolism. An optimized protocol of sample preparation was established and applied for metabolomics analysis of S. coelicolor, 103 metabolites were identified. The temporal profiles of metabolites reveal amino acid and fatty acid metabolic pathways may play an important role in the transition from primary to secondary metabolism in S. coelicolor.
Gas Chromatography-Mass Spectrometry ; Metabolic Networks and Pathways ; Metabolome ; Metabolomics ; methods ; Streptomyces coelicolor ; metabolism

Objective To research the effect mechanism of ultrashortwave in the correlation of ultrashortwave and the tail replantation, provide the experiment basis of clinical practice of prevention and cure for the vascular crisis after micromodule anastomosis. Methods Eighty Sprague-Dawley(SD) rats of clean grade were 3-month-old,female,and were divided into four groups:control group (group 0),model group (group 1 ),contrast group (group 2),ultrashortwave (USW) group (group 3).The preparation of tail replantation model was cut off soft tissue except for caudal veins on both sides of the tail. The coccyx was not broken away from tail.At last,the audal artery under abdominal main centre ditch was anastomosed.In experiment process, the USW group was divided into high dosage group (group 3A) and low dosage group (group 3B). The caudal arterys were ligated and not anastomosed in the group 0. Caudal arterys in other groups were anastomosed.Rats in the group 0 and group 1 received no treatment,normal management after the operation. Rats in the group 2 were given abdominal cavity injection of papaverin liquid immediately,then once a day to 5 days after the tail replantation.Rats in the group 3 were immediately given USW therapy of twenty minutes on the anastomosis section,and then once a day for 5 days after the tail replantation.The USW dosage of group 3A was 3th grade and 50mA. The USW dosage of group 3B was 2th grade and 28mA.The survival rate of the rat tails was observed after the tail replantation for 10 day.Before being grouped,it was measured that the tail skin temperature diference between near and far side of anastomosis section.After the tail relpantation, the temperature diference was inspected daily for 10 postoperative days hence. Before rats were grouped and the eighth hour after the tail replantation, rats were collected blood plasma specimens and measured contents of nitric oxide with destination colorimetric mathods of nitric oxide.Results Carrying out comparison of survival rate of every group,the output weve:between tail cutting off group (group 0,0) and tail replantation group (group 1,2,3,43.94％) to compare P ＞ 0.05; between each group of the tail replantation groups (1,2,3A,3B group) to compare P＞ 0.05,group 3B ＜ 2 ＜ 1 ＜ 3A; between group 3B and group 1 to compare P ＞ 0.05; between group 2 and group 1 to compare P ＞ 0.05. Each group were compared with the change daily between postoperative and preceding operative the skin temperature diference,single-factor analysis of variance (One-Way ANOVA) analysis:Postoperative 1 day,group 3A ＜ 1,P ＜ 0.05.Postoperative 6 day:3A ＜ 3B ＜ 1 ＜ 2,P ＞ 0.05.Postoperative 7 day:group 3B ＜ 1 ＜ 3A ＜ 2,P ＜ 0.05.Each group were compared with the change of the content of nitric oxide between postoperative 8 hour and preceding operative,with rank-sum test:group 3B ＞ 3A ＞ 2 ＞ 1 ＞ 0,H =33.760,P ＜ 0.05,shows statistically significant.Conclusions USW therapy,especially USW low-dose therapy,can reduce vascular crisis and improve the survival rate of replanted rat tails,after the postoperative 1,6,7 days,reduce skin temperature,improve blood supply,improve nitric oxide at postoperative eighth hour,prevent vascular crisis.Rat tail replantation model in this experiment is feasible.

The important and diverse regulatory roles of Ca(2+) in eukaryotes are conveyed by the EF-hand containing calmodulin superfamily. However, the calcium-regulatory proteins in prokaryotes are still poorly understood. In this study, we report the three-dimensional structure of the calcium-binding protein from Streptomyces coelicolor, named CabD, which shares low sequence homology with other known helix-loop-helix EF-hand proteins. The CabD structure should provide insights into the biological role of the prokaryotic calcium-binding proteins. The unusual structural features of CabD compared with prokaryotic EF-hand proteins and eukaryotic sarcoplasmic calcium-binding proteins, including the bending conformation of the first C-terminal α-helix, unpaired ligand-binding EF-hands and the lack of the extreme C-terminal loop region, suggest it may have a distinct and significant function in calcium-mediated bacterial physiological processes, and provide a structural basis for potential calcium-mediated regulatory roles in prokaryotes.
Amino Acid Sequence ; Binding Sites ; Calcium ; physiology ; Calcium-Binding Proteins ; chemistry ; Crystallography, X-Ray ; EF Hand Motifs ; Molecular Sequence Data ; Protein Binding ; Protein Structure, Tertiary ; Sequence Alignment ; Sequence Homology, Amino Acid ; Streptomyces coelicolor ; Structural Homology, Protein ; Surface Properties

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.
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 Specificity

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.
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 ; genetics