Covalently anchoring of a ligand/metal via polar amino acid side chain(s) is often observed in metalloenzyme, while the substitutability of metal-binding sites remains elusive. In this study, we utilized a zinc-dependent alcohol dehydrogenase from Thermoanaerobacter brockii (TbSADH) as a model enzyme, analyzed the sequence conservation of the three residues Cys37, His59, and Asp150 that bind the zinc ion, and constructed the mutant library. After experimental validation, three out of 224 clones, which showed comparative conversion and ee values as the wild-type enzyme in the asymmetric reduction of the model substrate tetrahydrofuran-3-one, were screened out. The results reveal that the metal-binding sites in TbSADH are substitutable without tradeoff in activity and stereoselectivity, which lay a foundation for designing ADH-catalyzed new reactions via metal ion replacement.
Alcohol Dehydrogenase/metabolism* ; Catalytic Domain ; Ligands ; Protein Domains ; Zinc/metabolism*

Telomerase reverse transcriptase (TERT) is an enzymatic ribonucleoprotein that prolongs the replicative life span of cells by maintaining protective structures at the ends of eukaryotic chromosomes. Telomerase activity is highly up-regulated in 85-90% of human cancers, and is predominately regulated by hTERT expression. In contrast, most normal somatic tissues in humans express low or undetectable levels of telomerase activity. This expression profile identifies TERT as a potential anticancer target. By using an RNA mapping strategy based on a trans-splicing ribozyme library, we identified the regions of mouse TERT (mTERT) RNA that were accessible to ribozymes. We found that particularly accessible sites were present downstream of the AUG start codon. This mTERTspecific ribozyme will be useful for validation of the RNA replacement as cancer gene therapy approach in mouse model with syngeneic tumors.
Animals ; Catalytic Domain* ; Codon, Initiator ; Genes, Neoplasm ; Genetic Therapy ; Humans ; Mice* ; Ribonucleoproteins ; RNA* ; RNA, Catalytic ; Telomerase* ; Trans-Splicing

The methylglyoxal (MGO) trapping constituents from onion (Allium cepa L.) peels were investigated using pre-column incubation of MGO and crude extract followed by HPLC analysis. The peak areas of MGO trapping compounds decreased, and their chemical structures were identified by HPLC-ESI/MS. Among major constituents in outer scale of onion, 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone (2) was more effective MGO scavenger than quercetin (6) and its 4′-glucoside, spiraeoside (3). After 1 h incubation, compound 2 trapped over 90% MGO at a concentration of 0.5 mM under physiological conditions, but compounds 3 and 6 scavenged 45%, 16% MGO, respectively. HPLC-ESI/MS showed that compound 2 trapped two molecules of MGO to form a di-MGO adduct and compounds 3 and 6 captured one molecule of MGO to form mono-MGO adducts, and the positions 6 and 8 of the A ring of flavonoids were major active sites for trapping MGO.
Catalytic Domain ; Chromatography, High Pressure Liquid ; Flavonoids ; Methods* ; Onions* ; Pyruvaldehyde* ; Quercetin

BACKGROUNDTo express in vitro the bovine enterokinase catalytic subunit (EKL) protein, which could be used in the future for the cleavage and purification of fusion proteins.

METHODSBovine enterokinase catalytic subunit cDNA was obtained by RT-PCR from duodenal mucosa of a bovine obtained at wholesale market, and then cloned into a pUCmT cloning vector and sequenced. The desired gene fragment was inserted into a pET39b expression plasmid and the recombinant vector pET39b-EKL was transformed into E. coli BL21 (DE3). Protein expression was induced using IPTG. The recombinant DsbA-EKL was purified with His.Tag affinity chromatography, and it bioactivity was analyzed.

RESULTSCompared with the sequence deposited in GenBank, the sequence of the EKL gene cloned in the present study is correct. It was also confirmed that the nucleotide sequence of expression plasmid pET39b-EKL was correct at the conjunction site between the recombinant DNA 5' terminal multi-cloning site and the recombinant fragment. SDS-PAGE analysis indicated that the target product was about 65 kDa and represented 28% of total cell protein. Purified recombinant protein was obtained by metal chelating chromatography using Ni-IDA resin. After desalting and changing the buffer, the crude kinase was incubated at 21 degrees C overnight and shown to have a high autocatalytic cleavage activity.

CONCLUSIONSThe EKL gene from Chinese bovine has been cloned successfully and expressed. This investigation has layed the foundation for future enterokinase activity research and for further large-scale application of expression products.

Animals ; Catalytic Domain ; genetics ; Cattle ; Cloning, Organism ; DNA, Complementary ; Enteropeptidase ; analysis ; genetics ; Recombinant Proteins

Genistein is a high specific and noncompetitive inhibitor of epidermal growth factor receptor tyramine kinase domain (EGFR-TK). In the paper, a molecular docking between genistein and EGFR-TK was studied to explore the mechanism of their interaction and antitumor mechanism of genistein by AUTODOCK 3.05 program. The results indicated that genistein located in the active cavity of EGFR-TK by high affinity (deltaG = -31.2 kJ/mol), and genistein inhibited EGFR-TK by interfering with forming of Lys721/Glu738 ion pair. The inhibition belonged to noncompetitive interaction, in which hydrophobic force and hydrogen bond played key roles.
Catalytic Domain ; Genistein ; metabolism ; pharmacology ; Models, Molecular ; Receptor, Epidermal Growth Factor ; antagonists & inhibitors ; metabolism

The chalcone synthase (CHS) superfamily of the type III polyketide synthases (PKSs) generates backbones of a variety of plant secondary metabolites. Benzalacetone synthase (BAS) catalyzes a condensation reaction of decarboxylation between the substrates of 4-coumaric coenzyme A and malonyl coenzyme A to generate benzylidene acetone, whose derivatives are series of compounds with various biological activities. A BAS gene Pcpks2 and a bifunctional CHS/BAS PcPKSI were isolated from medicinal plant P. cuspidatum. Crystallographic and structure-based mutagenesis studies indicate that the functional diversity of the CHS-superfamily enzymes is principally derived from small modifications of the active site architecture. In order to obtain an understanding of the biosynthesis of polyketides in P. cuspidatum, which has been poorly described, as well as of its activation mechanism, PcPKS2 was overexpressed in Escherichia coli as a C-terminally poly-His-tagged fusion protein, purified to homogeneity and crystallized, which is helpful for the clarification of the catalytic mechanism of the enzyme and lays the foundation for its genetic engineering manipulation.
Butanones ; Catalytic Domain ; Crystallization ; Fallopia japonica ; enzymology ; Polyketide Synthases ; genetics ; metabolism

Hydrogenases are enzymes that catalyse the oxidation of hydrogen and the reduction of protons. It plays an important role in the process of biohydrogen production. According to the metal atoms within hydrogenase, it can be classified as NiFe-hydrogenase, Fe-hydrogenase and metal-free hydrogenase. The overwhelming majority of hydrogenases are metalloenzymes. The metal atoms are involved in the forming of active site and [Fe-S] clusters. The active site directly catalyzes the reduction of protons and the oxidation of hydrogen. The [Fe-S] clusters are involved in the transport of electrons between the H2-activating site and the redox partners of hydrogenase. Presently, the crystal structures of NiFe-hydrogenase and Fe-hydrogenase from a few kinds of microorganism have been revealed. The metal-free hydrogenase, characterized by the absence of [Fe-S] cluster and the presence of an iron-containing cofactor, shows a great diversity comparing with those of NiFe-hydrogenases and Fe-hydrogenases. Recent progress have also indicated the mechanisms of activation.
Catalysis ; Catalytic Domain ; Hydrogenase ; metabolism ; Iron-Sulfur Proteins ; metabolism ; Oxidation-Reduction

Nine cases of primary localized cutaneous amyloidosis were studied by immunoperoxidase technique (ABC method) employing anti-keratin antibodies. All specimens were examined using consecutive paraffin sections to confirm the correspondence between amyloid existing area and reactive sites. Anti-keratin antibody 34pE which recognize 68, 58, 56.5, 56kd keratin peptides reacted with amyloid deposits in both lichen amyloidosus and macular amyloidosis. However, anti-keratin antibodies 34pB4 and 35pH did not react with amyloids. In general, Dylon staining positive material, keratin reacted with 34pE and amyloid P showed similar distribution in serial sections, but did not show the same one. Several keratin bodies reacted with 34pE, which were not stained with Dylon staining or antiamyloid P were found in the dermis of one specimen. These results suggest that immunohistochemical staining with antikeratin antibody 34pE using formalin-fixed, paraffin-embedded sections appeared to be a useful method in studying the histogenesis of primary localized cutaneous arnyloidosis.
Amyloid* ; Amyloidosis* ; Antibodies* ; Catalytic Domain ; Dermis ; Immunoenzyme Techniques ; Lichens ; Paraffin ; Peptides ; Plaque, Amyloid*

Fatty acid synthase (FASN, EC 2.3.1.85), is a multi-enzyme dimer complex that plays a critical role in lipogenesis. This lipogenic enzyme has gained importance beyond its physiological role due to its implications in several clinical conditions-cancers, obesity, and diabetes. This has made FASN an attractive pharmacological target. Here, we have attempted to predict the theoretical models for the human enoyl reductase (ER) and beta-ketoacyl reductase (KR) domains based on the porcine FASN crystal structure, which was the structurally closest template available at the time of this study. Comparative modeling methods were used for studying the structure-function relationships. Different validation studies revealed the predicted structures to be highly plausible. The respective substrates of ER and KR domains-namely, trans-butenoyl and beta-ketobutyryl-were computationally docked into active sites using Glide in order to understand the probable binding mode. The molecular dynamics simulations of the apo and holo states of ER and KR showed stable backbone root mean square deviation trajectories with minimal deviation. Ramachandran plot analysis showed 96.0% of residues in the most favorable region for ER and 90.3% for the KR domain, respectively. Thus, the predicted models yielded significant insights into the substrate binding modes of the ER and KR catalytic domains and will aid in identifying novel chemical inhibitors of human FASN that target these domains.
Catalytic Domain* ; Humans ; Lipogenesis ; Models, Theoretical ; Molecular Dynamics Simulation* ; Obesity ; Oxidoreductases*

An RNase P ribozyme library has been developed as a tool for functional genomics studies. Each clone of this library contains a random 18-mer and the sequence of M1 RNA, the catalytic subunit of RNase P. Repression of target gene expression is thus achieved by the complementary binding of mRNA to the random guide sequence and the successive target cleavage via M1 RNA. Cellular expression of the ribozyme expression was confirmed, and EGFP mRNA was used as a model to demonstrate that the RNase P ribozyme expression system can inhibit the target gene expression. The constructed RNase P ribozyme library has a complexity of 1.4x10(7). This novel library system should become a useful in functional genomics, to identify novel gene functions in mammalian cells.
Catalytic Domain ; Clone Cells ; Gene Expression ; Genomics* ; Repression, Psychology ; Ribonuclease P* ; Ribonucleases* ; RNA ; RNA, Messenger