Discovery of GS-9451: an acid inhibitor of the hepatitis C virus NS3/4A protease

The discovery of GS-9451 is reported. Modification of the P3 cap and P2 quinoline with a series of solubilizing groups led to the identification of potent HCV NS3 protease inhibitors with greatly improved pharmacokinetic properties in rats, dogs and monkeys.     

A novel dienelactone hydrolase from the thermoacidophilic archaeon Sulfolobus solfataricus P1: Purification,characterization, and expression

Background

Dienelactone hydrolases catalyze the hydrolysis of dienelactone to maleylacetate, which play a key role for the microbial degradation of chloroaromatics via chlorocatechols. Here, a thermostable dienelactone hydrolase from thermoacidophilic archaeon Sulfolobus solfataricus P1 was the first purified and characterized and then expressed in Escherichia coli.

Methods

The enzyme was purified by using several column chromatographys and characterized by determining the enzyme activity using p-nitrophenyl caprylate and dienelactones. In addition, the amino acids related to the catalytic mechanism were examined by site-directed mutagenesis using the identified gene.

Results

The enzyme, approximately 29 kDa monomeric, showed the maximal activity at 74 °C and pH 5.0, respectively. The enzyme displayed remarkable thermostability: it retained approximately 50% of its activity after 50 h of incubation at 90 °C, and showed high stability against denaturing agents, including various detergents, urea, and organic solvents. The enzyme displayed substrate specificities toward trans-dienelactone, not cis-isomer, and also carboxylesterase activity toward p-nitrophenyl esters ranging from butyrate (C₄) to laurate (C₁₂). The k_cat/K_m ratios for trans-dienelactone and p-nitrophenyl caprylate (C₈), the best substrate, were 92.5 and 54.7 s⁻¹ μM⁻¹, respectively.

Conclusions

The enzyme is a typical dienelactone hydrolase belonging to α/β hydrolase family and containing a catalytic triad composed of Cys151, Asp198, and His229 in the active site.

General significance

The enzyme is the first characterized archaeal dienelactone hydrolase.     

Optimization of biphasic growth of Saccharomyces carlsbergensis in fed-batch culture

The optimal glucose feeding policy for the fed-batch culture of Saccharomyces carlsbergensis is presented. The biphasic nature of growth results in a singular feed rate policy that is unique to this organism. When the operating cost is high, the reduction in operating time forces the cells to utilize both glucose and ethanol toward the end of fermentation time and results in a decreasing rate of glucose addition, unlike the normally observed in creasing feed rate. The optimal feeding policy depends heavily on the initial conditions and is highly sensitive to changes in kinetic parameters. A semiempirical scheme for feedback optimization is suggested for the fed-batch yeast culture.     

Purifications and characterizations of a ferredoxin and its related 2-oxoacid:ferredoxin oxidoreductase from the hyperthermophilic archaeon, Sulfolobus solfataricus P1

The coenzyme A-acylating 2-oxoacid:ferredoxin oxidoreductase and ferredoxin (an effective electron acceptor) were purified from the hyperthermophilic archaeon, Sulfolobus solfataricus P1 (DSM1616). The purified ferredoxin is a monomeric protein with an apparent molecular mass of approximately 11 kDa by SDS-PAGE and of 11,180+/-50 Da by MALDI-TOF mass spectrometry. Ferredoxin was identified to be a dicluster, [3Fe-4S][4Fe-4S], type ferredoxin by spectrophotometric and EPR studies, and appeared to be zinc-containing based on the shared homology of its N-terminal sequence with those of known zinc-containing ferredoxins. On the other hand, the purified 2-oxoacid: ferredoxin oxidoreductase was found to be a heterodimeric enzyme consisting of 69 kDa alpha and 34 kDa beta subunits by SDS-PAGE and MALDI-TOF mass spectrometry. The purified enzyme showed a specific activity of 52.6 units/mg for the reduction of cytochrome c with 2-oxoglutarate as substrate at 55 degrees C, pH 7.0. Maximum activity was observed at 70 degrees C and the optimum pH for enzymatic activity was 7.0 -8.0. The enzyme displays broad substrate specificity toward 2-oxoacids, such as pyruvate, 2-oxobutyrate, and 2-oxoglutarate. Among the 2-oxoacids tested (pyruvate, 2-oxobutyrate, and 2-oxoglutarate), 2-oxoglutarate was found to be the best substrate with Km and kcat values of 163 microM and 452 min(-1), respectively. These results provide useful information for structural studies on these two proteins and for studies on the mechanism of electron transfer between the two.     

Purification and characterization of a thermostable chitinase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> Mb-2

Summary A chitinase produced by Bacillus licheniformis MB-2 isolated from Tompaso geothermal springs, Indonesia, was purified and characterized. The extracellular enzyme was isolated by successive hydrophobic interaction, anion exchange, and gel filtration chromatographies. The purified enzyme was a monomer with an apparent molecular weight of 67 kDa. The optimal temperature and pH of the enzyme were 70 °C and 6.0, respectively. It was stable below 60 °C for 2 h and over a broad pH range of 4.0–11.0 for 4 h. The enzyme was resistant to denaturation by urea (1 M), Tween-20 (1%) and Triton-X (1%), but unstable toward organic solvents such as dimethyl sulphoxide, DMSO, (5%) and polyethylene glycol, PEG, (5%) for 30 min. The enzyme hydrolysed colloidal chitin, glycol chitin, chitosan, and glycol chitosan. The first 13 N-terminal amino acids of the enzyme were determined as SGKNYKIIGYYPS, which is identical to those in chitinases from B. licheniformis and B. circulans.     

Secretion of recombinant pediocin PA-1 by Bifidobacterium longum, using the signal sequence for bifidobacterial alpha-amylase

A recombinant DNA, encoding the chimeric protein of the signal sequence for bifidobacterial alpha-amylase mature pediocin PA-1, was introduced into Bifidobacterium longum MG1. Biologically active pediocin PA-1 was successfully secreted from the strain and showed bactericidal activity against Listeria monocytogenes and the same molecular mass as native pediocin PA-1.     

Stereospecificity and kinetic mechanism of human prenylcysteine lyase,an unusual thioether oxidase

Prenylated proteins contain either a 15-carbon farnesyl or a 20-carbon geranylgeranyl isoprenoid covalently attached to cysteine residues at or near their C terminus. The cellular abundance of prenylated proteins, as well as the stability of the thioether bond, poses a metabolic challenge to cells. A lysosomal enzyme termed prenylcysteine lyase has been identified that degrades a variety of prenylcysteines. Prenylcysteine lyase is a FAD-dependent thioether oxidase that produces free cysteine, an isoprenoid aldehyde, and hydrogen peroxide as products of the reaction. Here we report initial studies of the kinetic mechanism and stereospecificity of this unusual enzyme. We utilized product and dead end inhibitors of prenylcysteine lyase to probe the kinetic mechanism of the multistep reaction. The results with these inhibitors, together with those of other experiments, suggest that the reaction catalyzed by prenylcysteine lyase proceeds through a sequential mechanism. The reaction catalyzed by the enzyme is stereospecific, in that the pro-S hydride of the farnesylcysteine is transferred to FAD to initiate the reaction. With (2R,1'S)-[1'-(2)H(1)]farnesylcysteine as a substrate, a primary deuterium isotope effect of 2 was observed on the steady state rate. However, the absence of an isotope effect on an observed pre-steady-state burst of hydrogen peroxide formation implicates a partially rate-determining proton transfer after a relatively fast C-H (C-D) bond cleavage step. Furthermore, no pre-steady-state burst of cysteine was observed. The finding that the rate of cysteine formation was within 2-fold of the steady-state k(cat) value indicates that cysteine production is one of the primary rate-limiting steps in the reaction. These results provide substantial new information on the catalytic mechanism of prenylcysteine lyase.     

A novel subtilisin-like serine protease from<Emphasis Type="Italic"> Thermoanaerobacter yonseiensis</Emphasis> KB-1: its cloning,expression, and biochemical properties

A gene, tayI, encoding a novel subtilisin-like protease, designated thermicin, from the extremely thermophilic bacterium Thermoanaerobacter yonseiensis KB-1 (DSM 13777) was cloned by using a sequence tag containing the consensus sequence of proteases. The gene consisted of 1,239 nucleotides, and the deduced amino acid sequence indicated that it is a preproenzyme with a 311-residue mature protein composed of canonical catalytic residues (Asp29, His64, and Ser252). Thermicin was overproduced in E. coli as a fusion protein with a histidine tag and purified by nickel nitrilotriacetic acid affinity chromatography. Thermicin from E. coli showed maximum proteolytic activity at 92.5 degrees C and pH 9.0, and its half-life was 30 h at 80 degrees C. In order to determine cleavage specificity, thermicin was incubated with insulin beta chain, and the resulting peptides were analyzed by matrix assisted laser desorption/ionization-time of flight mass spectrometry. The carboxyl group side of the Val12, Leu15,17, Gly23, and Pro28 residues was cleaved. Thermicin is well known to hydrolyze Gly- and Pro-rich collagens. The K (m) and k (cat)/ K (m) values of thermicin for the hydrolysis of the synthetic substrate L-Gly-Pro- p-nitroaniline were 54.16 microM and 142.05 (10(5) s(-1) M(-1)), respectively, at 92.5 degrees C and pH 9.0. Amino acid sequence comparison and phylogenetic analysis indicated that this enzyme belongs to a new subgroup with respect to its molecular evolution, when compared with previously characterized subtilisins. This result indicates that thermicin is a novel enzyme different from other thermostable proteases.     

New immobilization method for immunoaffinity biosensors by using thiolated proteins

A new immobilization method for immunoaffinity (IA) biosensors that ensures the high surface density and the stability of the IA layer was developed. For the immobilization of biomolecules, the molecular recognition protein was first thiolated by covalent conjugation of mercaptopropionic acid, and then the thiolated protein was attached on the gold surface of the transducer. In this work, horseradish peroxidase (HRP) and its antibody were used as a model antigen-antibody, and the following properties of the IA layer prepared by thiolated protein were estimated: (i) biological integrity of HRP after the immobilization process by using activity assay, (ii) charge transfer resistance by immobilization, (iii) mass loading by the surface plasmon resonance (SPR) biosensor, (iv) number of binding sites, and (v) feasibility test for the measurement of capacitive change by the antigen-antibody interaction. Based on these parameters, the immobilization method by using thiolated protein was determined to be feasible for application to IA biosensors.     

Mutual regulation between DNA-PKcs and snail1 leads to increased genomic instability and aggressive tumor characteristics

Although the roles of DNA-dependent protein kinase catalytic subunits (DNA-PKcs) in the non-homologous end joining (NHEJ) of DNA repair are well-recognized, the biological mechanisms and regulators by DNA-PKcs besides DNA repair, have not been clearly described. Here, we show that active DNA-PKcs caused by ionizing radiation, phosphorylated Snail1 at serine (Ser) 100, led to increased Snail1 stability. Furthermore, phosphorylated Snail1 at Ser100 reciprocally inhibited the kinase activity of DNA-PKcs, resulting in an inhibition of DNA repair activity. Moreover, Snail1 phosphorylation by DNA-PKcs was involved in genomic instability and aggressive tumor characteristics. Our results describe novel cellular mechanisms that affect genomic instability, sensitivity to DNA-damaging agents, and the migration of tumor cells by reciprocal regulation between DNA-PKcs and Snail1.