Archaea ; enzymology ; genetics ; Archaeal Proteins ; chemistry ; genetics ; Deoxyribonucleases ; chemistry ; genetics ; Gene Editing ; methods

Animals ; Archaeal Proteins ; genetics ; metabolism ; Deoxyribonuclease I ; genetics ; metabolism ; Gene Editing ; methods ; Humans ; Natronobacterium ; enzymology ; genetics

Group II chaperonins, which assemble as double-ring complexes, assist in the refolding of nascent peptides or denatured proteins in an ATP-dependent manner. The molecular mechanism of group II chaperonin assembly and thermal stability is yet to be elucidated. Here, we selected the group II chaperonins (cpn-α and cpn-β), also called thermosomes, from Acidianus tengchongensis and investigated their assembly and thermal stability. We found that the binding of ATP or its analogs contributed to the successful assembly of thermosomes and enhanced their thermal stabilities. Cpn-β is more thermally stable than cpn-α, while the thermal stability of the hetero thermosome cpn-αβ is intermediate. Cryo-electron microscopy reconstructions of cpn-α and cpn-β revealed the interwoven densities of their non-conserved flexible N/C-termini around the equatorial planes. The deletion or swapping of their termini and pH-dependent thermal stability assays revealed the key role of the termini electrostatic interactions in the assembly and thermal stability of the thermosomes.
Acidianus ; metabolism ; Adenosine Triphosphate ; metabolism ; Amino Acid Sequence ; Cryoelectron Microscopy ; Hydrogen-Ion Concentration ; Molecular Sequence Data ; Mutation ; Nucleotides ; metabolism ; Protein Binding ; Protein Folding ; Protein Stability ; Protein Structure, Quaternary ; Sequence Alignment ; Static Electricity ; Temperature ; Thermosomes ; chemistry ; genetics ; metabolism

The inherent evolvability of promiscuous enzymes endows them with great potential to be artificially evolved for novel functions. Previously, we succeeded in transforming a promiscuous acylaminoacyl peptidase (apAAP) from the hyperthermophilic archaeon Aeropyrum pernix K1 into a specific carboxylesterase by making a single mutation. In order to fulfill the urgent requirement of thermostable lipolytic enzymes, in this paper we describe how the substrate preference of apAAP can be further changed from p-nitrophenyl caprylate (pNP-C8) to p-nitrophenyl laurate (pNP-C12) by protein and solvent engineering. After one round of directed evolution and subsequent saturation mutagenesis at selected residues in the active site, three variants with enhanced activity towards pNP-C12 were identified. Additionally, a combined mutant W474V/F488G/R526V/T560W was generated, which had the highest catalytic efficiency (k (cat)/K (m)) for pNP-C12, about 71-fold higher than the wild type. Its activity was further increased by solvent engineering, resulting in an activity enhancement of 280-fold compared with the wild type in the presence of 30% DMSO. The structural basis for the improved activity was studied by substrate docking and molecular dynamics simulation. It was revealed that W474V and F488G mutations caused a significant change in the geometry of the active center, which may facilitate binding and subsequent hydrolysis of bulky substrates. In conclusion, the combination of protein and solvent engineering may be an effective approach to improve the activities of promiscuous enzymes and could be used to create naturally rare hyperthermophilic enzymes.
Aeropyrum ; chemistry ; enzymology ; Archaeal Proteins ; genetics ; metabolism ; Binding Sites ; Biocatalysis ; Caprylates ; metabolism ; Cloning, Molecular ; Dimethyl Sulfoxide ; chemistry ; Escherichia coli ; Hot Temperature ; Industrial Microbiology ; methods ; Kinetics ; Laurates ; metabolism ; Molecular Dynamics Simulation ; Mutagenesis, Site-Directed ; methods ; Peptide Hydrolases ; genetics ; metabolism ; Protein Binding ; Protein Conformation ; Recombinant Proteins ; genetics ; metabolism ; Solvents ; chemistry ; Substrate Specificity

OBJECTIVETo identify the interaction partners of a new splicing product of LMO2 gene (LMO2-C), and study its function in K562 cells.

METHODSMaltose binding protein (MBP) pull down and mammalian two-hybrid assay (MTHA) were used to identify the interaction partners of LMO2-C in K562 cells. Semiquantitative RT-PCR was used to detect the expression of hematopoietic specific gene glycoprotein (GPA) in K562 cells.

RESULTSMBP-LMO2-C fusion protein was expressed and purified in soluble form successfully. Endogenous GATA1 and LDB1 proteins were confirmed to bind to LMO2-C by MBP pull down analysis. The MTHA also showed that LMO2-C had comparable binding affinities to LDB1 with LMO2-L, and over expression of LMO2-C prevented LMO2-L from binding to LDB1, the inhibition rate being (81.13 +/- 0.68)%. RT-PCR results showed that the expression level of GPA was reduced [(51.00 +/- 1.58)%] in K562 cells while LMO2-C overexpressed.

CONCLUSIONLMO2-C can bind endogenous GATA1 and LDB1 protein in K562 cells and down regulates the expression of GPA.

Adaptor Proteins, Signal Transducing ; DNA-Binding Proteins ; genetics ; metabolism ; GATA1 Transcription Factor ; metabolism ; Humans ; K562 Cells ; LIM Domain Proteins ; Maltose-Binding Proteins ; Metalloproteins ; genetics ; metabolism ; Periplasmic Binding Proteins ; Proto-Oncogene Proteins ; RNA Splicing ; Transcription Factors ; metabolism ; Two-Hybrid System Techniques

As thermostable enzymes and organisms are much more needed, researches on heat shock proteins(HSPs) of hyperthermophilic archaea have drawn more concerns. HSPs from hyperthermophilic archaea are concise only with HSP60, sHSP, prefoldin and AAA+proteins, but without HSP100s, HSP90s, HSP70 (DnaK), HSP40 (DnaJ) and GrpE which are common in mesophilic or thermophilic archaea. Accordingly, studies on the structure, function and operation mechanism of these four groups are much more important and meaningful. This review focuses on the recent progress in the researchs on the structure, function, operation mechanism and cooperation of the HSPs from hyperthermophilic archaea. The problems and obfuscations in these HSPs are analyzed, and farther research direction and key points are put out.
Archaea ; classification ; metabolism ; Archaeal Proteins ; metabolism ; Chaperonin 60 ; metabolism ; Heat-Shock Proteins ; genetics ; metabolism ; Molecular Chaperones ; metabolism

Cyclodextrin glucanotransferase, the essential enzyme for the production of cyclodextrins, has become the focus of scientific research nowadays. Although many related enzyme properties are well known, the crucial factors in product specificity determination remain to be answered. Here, the recent research progresses of cyclodextrin glucanotransferase, especially those about the evolution of product specificity, were reviewed, and the scientific problems were discussed.
Archaeal Proteins ; genetics ; metabolism ; Bacillus ; enzymology ; genetics ; Bacterial Proteins ; genetics ; metabolism ; Biocatalysis ; Cyclodextrins ; metabolism ; Evolution, Molecular ; Glucosyltransferases ; classification ; genetics ; metabolism ; Mutation ; Thermoanaerobacterium ; enzymology ; genetics ; Thermococcus

One hundred and forty-eight strains of halophilic archaea were isolated from 40 samples of soil, lake water, and silt. To study and analyze the species and bacteriorhodopsin(BR) protein resource, partial DNA fragments encoding BR protein from helix C to helix G andl6S rRNA encoding genes from 6 strains of halophilic archaea were amplified by polymerase chain(PCR) , and their DNA sequences were determined. The results indicate that the reduced amino acid sequences of BR protein from helix C to helix G of ABDH11 is obviously different from those of other existing proteins. The results of homology analysis on BR gene andl6S rRNA and phylogenetic analysis on 16S rRNA show that strains ABDH10 and ABDH40 are novel members of genus Natronorubrum and Natrinema, respectively; the sequence of ABDH40 was obtained from GenBank and the number of sequence is AY989910. The protein from helix C to helix G of ABDH11 is significantly different from that of other strains.
Amino Acid Sequence ; Bacteriorhodopsins ; genetics ; China ; DNA, Archaeal ; chemistry ; genetics ; Fresh Water ; microbiology ; Halobacteriaceae ; classification ; genetics ; isolation & purification ; Molecular Sequence Data ; Phylogeny ; RNA, Ribosomal, 16S ; genetics ; Sequence Analysis, DNA ; Sequence Homology, Amino Acid

Animals ; Antibodies, Bacterial ; blood ; Bacterial Proteins ; genetics ; immunology ; Brucella Vaccine ; immunology ; Brucella abortus ; immunology ; Female ; Immunization ; Interferon-gamma ; biosynthesis ; Lymphocyte Activation ; Mice ; Mice, Inbred BALB C ; Periplasmic Binding Proteins ; genetics ; immunology ; Ribosomal Proteins ; genetics ; immunology ; Vaccines, DNA ; immunology

A novel member of extremely halophilic archaea, strain AJ2, was isolated from Ayakekum Lake located in Altun Mountain National Nature Reserve of Xinjiang Uygur Autonomous Region in China. The strain AJ2 requires at least 10% (w/v) NaCl and grows 10% to 30% (optimum at 20%). Phylogenetic analysis based on 16S rDNA sequence comparison revealed that strain AJ2 clustered to three Natrinema species with less than 97.7% sequence similarities, suggesting AJ2 is a novel member of Natrinema. A bacteriorhodopsin-encoding (bop) gene was subsequently detected in the AJ2 genome using the polymerase chain reaction technique. The cloning and sequencing of a 401 base pairs fragment indicated the deduced amino acid sequence of bop from AJ2 is different from that reported for bacteriorhodopsins. This is the first reported detection of a bop gene in Natrinema.
Bacteriorhodopsins ; genetics ; metabolism ; Cell Proliferation ; Gene Expression Profiling ; Genome, Archaeal ; Halobacteriaceae ; classification ; isolation & purification ; physiology ; Phylogeny ; Sequence Analysis, DNA ; Species Specificity