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

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

BACKGROUND: Human telomerase is a ribonucleoprotein polymerase, which synthesizes telomeric repeat sequences, and human telomerase reverse transcriptase (hTERT) has been identified as the catalytic subunit, as well as the rate-limiting component, of telomerase. In this study, we attempted to identify the modulators of telomerase, and to determine the molecular mechanisms underlying cisplatin-induced apoptosis. METHODS: To determine the role of telomerase in cisplatin-induced apoptosis, we measured telomerase activity and analyzed apoptosis using PI and trypan blue staining. Also, we inhibited the caspase activations using Z-VAD-fmk to analyze the effects on expression of hTERT protein. Finally, we induced the transient co-expression of the Bcl-2 and Bak genes in HEK293 cells, and then, the telomerase activity and expression of hTERT were evaluated. RESULTS: In the Bcl-2-overexpressing HeLa cells, telomerase activity was more enhanced, and cell death was reduced to 40-50% that of the mock controls. This finding suggests that Bcl-2-induced telomerase activity exerts an antiapoptotic effect in cisplatin-induced death. As caspase activation was inhibited via Z-VAD-fmk, the hTERT protein was recovered in the mock controls, but not in the Bcl-2-overexpressing cells. This suggests that the expression of hTERT can be regulated by caspases, but Bcl-2 was located within the upstream pathway. Moreover, when the Bcl-2 and Bak genes were co-transfected into the HEK293, both telomerase activity and hTERT protein were prominently reduced. CONCLUSIONS: Bcl-2-induced telomerase activity inhibits cisplatin-induced apoptosis in HeLa cells, and can be regulated via both caspases and the interaction of Bcl-2 and Bak.
Apoptosis ; Caspases* ; Catalytic Domain ; Cell Death* ; Cisplatin ; HEK293 Cells ; HeLa Cells ; Humans* ; Ribonucleoproteins ; Telomerase* ; Trypan Blue

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

OBJECTIVETo investigate the variety of proliferating ability of umbilical endothelia (UE) transfected by plasmid pBABE-HYGR-hTERT.

METHODSUE was identified from two aspects: morphology and CD34 labeling technique. The plasmid was obtained and identified by alkali splitting and gel electrophoresis. Liposomes were used to transfect UE. RT-PCR based telomeric repeat amplification protocol (TRAP) assay was used to measure the telomerase activity of endothelia.

RESULTSUE arranged as "cobblestone" and were positive of CD34 labeling. Endothelia transfected by pBABE-HYGR-hTERT(HC) had an raised absorbance of 0.889. The shape of growth curve of HC was similar to UE. But the absorbance of MTT test and the amount of HC were prior to UE at every measuring time and the amount of HC increased four times within 8 days (P < 0.05).

CONCLUSIONThe transfection of pBABE-HYGRO-hTERT had greatly improved the proliferating abilities and activated the telomerase of UE.

Catalytic Domain ; genetics ; Cells, Cultured ; Endothelium, Vascular ; cytology ; Humans ; Telomerase ; genetics ; Transfection ; Umbilical Veins ; cytology

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

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

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

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*