ABSTRACT
Base excision is a crucial DNA repair process mediated by endonuclease IV in nucleotide excision. In Chlamydia pneumoniae, CpendoIV is the exclusive AP endonuclease IV, exhibiting DNA replication error-proofreading capabilities, making it a promising target for anti-chlamydial drug development. Predicting the structure of CpendoIV, molecular docking with DNA was performed, analyzing complex binding sites and protein surface electrostatic potential. Comparative structural studies were conducted with E. coli EndoIV and DNA complex containing AP sites.CpendoIV was cloned, expressed in E. coli, and purified via Ni-NTA chelation and size-exclusion chromatography. Low NaCl concentrations induced aggregation during purification, while high concentrations enhanced purity.CpendoIV recognizes and cleaving AP sites on dsDNA, and Zn2+ influences the activity. Crystallization was achieved under 8% (v/v) Tacsimate pH 5.2, 25% (w/v) polyethylene glycol 3350, and 1.91 Å resolution X-ray diffraction data was obtained at 100 K. This research is significant for provides a deeper understanding of CpendoIV involvement in the base excision repair process, offering insights into Chlamydia pneumoniae.
Subject(s)
Bacterial Proteins , Chlamydophila pneumoniae , Crystallization , Chlamydophila pneumoniae/enzymology , Chlamydophila pneumoniae/genetics , Chlamydophila pneumoniae/chemistry , Crystallography, X-Ray , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Escherichia coli/genetics , Molecular Docking Simulation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Deoxyribonuclease IV (Phage T4-Induced)/chemistry , Deoxyribonuclease IV (Phage T4-Induced)/genetics , Deoxyribonuclease IV (Phage T4-Induced)/metabolism , Deoxyribonuclease IV (Phage T4-Induced)/isolation & purification , Cloning, MolecularABSTRACT
Polygonum cuspidatum, an important medicinal plant in China, is a rich source of resveratrol compounds, and its synthesis related resveratrol synthase (RS) gene is highly expressed in stems. The sequence of the resveratrol synthase was amplified with specific primers. Sequence comparison showed that it was highly homologous to the STSs. The RS gene of Polygonum cuspidatum encodes 389 amino acids and has a theoretical molecular weight of 42.4 kDa, which is called PcRS1. To reveal the molecular basis of the synthesized resveratrol activity of PcRS1, we expressed the recombinant protein of full-length PcRS1 in Escherichia coli, and soluble protein products were produced. The collected products were purified by Ni-NTA chelation chromatography and appeared as a single band on SDS-PAGE. In order to obtain higher purity PcRS1, SEC was used to purify the protein and sharp single peak, and DLS detected that the aggregation state of protein molecules was homogeneous and stable. In order to verify the enzyme activity of the high-purity PcRS1, the reaction product was detected at 303 nm. By predicting the structural information of monomer PcRS1 and PcRS1 ligand complexes, we analyzed the ligand binding pocket and protein surface electrostatic potential of the complex, and compared it with the highly homologous STSs protein structures of the iso-ligand. New structural features of protein evolution are proposed. PcRS1 obtained a more complete configuration and the optimal orientation of the active site residues, thus improving its catalytic capacity in resveratrol synthesis.
Subject(s)
Acyltransferases , Fallopia japonica/enzymology , Plant Proteins , Acyltransferases/biosynthesis , Acyltransferases/chemistry , Acyltransferases/genetics , Acyltransferases/isolation & purification , Fallopia japonica/genetics , Plant Proteins/biosynthesis , Plant Proteins/chemistry , Plant Proteins/genetics , Plant Proteins/isolation & purification , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purificationABSTRACT
Dehydrins (DHNs) belong to group II of late embryogenesis-abundant (LEA) proteins, which are up-regulated in most plants during cold, drought, heat, or salinity stress. Despite the importance of dehydrins for the plants to resist abiotic stresses, it is necessary to obtain plant-derived dehydrins from different biomass. Generally, dehydrin PicW1 from Picea wilsonii is involved in Kn-type dehydrin with five K-segments, which has a variety of biological activities. In this work, Picea wilsonii dehydrin PicW1 was expressed in Escherichia coli and purified by chitin-affinity chromatography and size-exclusion chromatography, which showed as a single band by SDS-PAGE. A cold-sensitive enzyme of lactate dehydrogenase (LDH) is used to explore the protective activities of other proteins. Temperature stress assays showed that PicW1 had an effective protective effect on LDH activity, which was better than that of bovine serum albumin (BSA). This study provides insights into the purification and protective activity of K5 DHNs for the advancement of dehydrin structure and function from biomass.