Embryotoxic activity of 3C protease of human hepatitis A virus in developing Danio rerio embryos

The 3C protease is a key factor in picornavirus-induced pathologies with a comprehensive action on cell targets. However, the effects induced by the enzyme have not been described at the organismic level. Here, the model of developing Danio rerio embryos was used to analyze possible toxic effects of the 3C protease of human hepatitis A virus (3Cpro) at the whole-body level. The transient 3Cpro expression had a notable lethal effect and induced a number of specific abnormalities in Danio rerio embryos within 24 h. These effects are due to the proteolytic activity of the enzyme. At the same time, the 3Cpro variant with reduced catalytic activity (3Cmut) increased the incidence of embryonic abnormalities; however, this effect was smaller compared to the native enzyme form. While the expression of 3Cmut increased the overall rate of abnormalities, no predominance of specific ones was observed. The data obtained point to a presence significant impact of picornavirus 3Cprotease at the whole-organism level and make contribution to the study of the infectious process caused by human hepatitis A virus.

Pactacin is a novel digestive enzyme in teleosts

Generally, animals extract nutrients from food by degradation using digestive enzymes. Trypsin and chymotrypsin, one of the major digestive enzymes in vertebrates, are pancreatic proenzymes secreted into the intestines. In this investigation, we report the identification of a digestive teleost enzyme, a pancreatic astacin that we termed pactacin. Pactacin, which belongs to the astacin metalloprotease family, emerged during the evolution of teleosts through gene duplication of astacin family enzymes containing six cysteine residues (C6astacin, or C6AST). In this study, we first cloned C6AST genes from pot-bellied seahorse (Hippocampus abdominalis) and analyzed their phylogenetic relationships using over 100 C6AST genes. Nearly all these genes belong to one of three clades: pactacin, nephrosin, and patristacin. Genes of the pactacin clade were further divided into three subclades. To compare the localization and functions of the three pactacin subclades, we studied pactacin enzymes in pot-bellied seahorse and medaka (Oryzias latipes). In situ hybridization revealed that genes of all three subclades were commonly expressed in the pancreas. Western blot analysis indicated storage of pactacin pro-enzyme form in the pancreas, and conversion to the active forms in the intestine. Finally, we partially purified the pactacin from digestive fluid, and found that pactacin is novel digestive enzyme that is specific in teleosts.

Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins

Puromycin and the Streptomyces alboniger-derived puromycin N-acetyltransferase (PAC) enzyme form a commonly used system for selecting stably transfected cultured cells. The crystal structure of PAC has been solved using X-ray crystallography, revealing it to be a member of the GCN5-related N-acetyltransferase (GNAT) family of acetyltransferases. Based on structures in complex with acetyl-CoA or the reaction products CoA and acetylated puromycin, four classes of mutations in and around the catalytic site were designed and tested for activity. Single-residue mutations were identified that displayed a range of enzymatic activities, from complete ablation to enhanced activity relative to wild-type (WT) PAC. Cell pools of stably transfected HEK293 cells derived using two PAC mutants with attenuated activity, Y30F and A142D, were found to secrete up to three-fold higher levels of a soluble, recombinant target protein than corresponding pools derived with the WT enzyme. A third mutant, Y171F, appeared to stabilise the intracellular turnover of PAC, resulting in an apparent loss of selection stringency. Our results indicate that the structure-guided manipulation of PAC function can be utilised to enhance selection stringency for the derivation of mammalian cell lines secreting elevated levels of recombinant proteins.

Molecular basis for metabolite channeling in a ring opening enzyme of the phenylacetate degradation pathway

Substrate channeling is a mechanism for the internal transfer of hydrophobic, unstable or toxic intermediates from the active site of one enzyme to another. Such transfer has previously been described to be mediated by a hydrophobic tunnel, the use of electrostatic highways or pivoting and by conformational changes. The enzyme PaaZ is used by many bacteria to degrade environmental pollutants. PaaZ is a bifunctional enzyme that catalyzes the ring opening of oxepin-CoA and converts it to 3-oxo-5,6-dehydrosuberyl-CoA. Here we report the structures of PaaZ determined by electron cryomicroscopy with and without bound ligands. The structures reveal that three domain-swapped dimers of the enzyme form a trilobed structure. A combination of small-angle X-ray scattering (SAXS), computational studies, mutagenesis and microbial growth experiments suggests that the key intermediate is transferred from one active site to the other by a mechanism of electrostatic pivoting of the CoA moiety, mediated by a set of conserved positively charged residues.

Structure of a critical metabolic enzyme: S-adenosylmethionine synthetase from Cryptosporidium parvum

S-Adenosyl-L-methionine (AdoMet), the primary methyl donor in most biological methylation reactions, is produced from ATP and methionine in a multistep reaction catalyzed by AdoMet synthetase. The diversity of group-transfer reactions that involve AdoMet places this compound at a key crossroads in amino-acid, nucleic acid and lipid metabolism, and disruption of its synthesis has adverse consequences for all forms of life. The family of AdoMet synthetases is highly conserved, and structures of this enzyme have been determined from organisms ranging from bacteria to humans. Here, the structure of an AdoMet synthetase from the infectious parasite Cryptosporidium parvum has been determined as part of an effort to identify structural differences in this enzyme family that can guide the development of species-selective inhibitors. This enzyme form has a less extensive subunit interface than some previously determined structures, and contains some key structural differences from the human enzyme in an allosteric site, presenting an opportunity for the design of selective inhibitors against the AdoMet synthetase from this organism.

The Effect of Myostatin Gene Polymorphisms onSome Biochemist Traits for Blood in Broiler Chicken

This study is conducted at the Poultry Farm of Animal Production Department of College of Agriculture, University of Al-Qadisiyah during the period 9/4/2015 - 20/5/2015 and in a laboratory of molecular genetic analysis of the College of Agriculture, University of Baghdad and Almusayab Bridge Company. The objective of this study is to identify the genotypes for Myostatin gene (GDF-8), and its relationship with physiological traits of broiler chicken. Three hundreds of Ross 308 chicks at day-old were used. The experiment continues until the sixth week of age. Three types of restriction enzymes (Aci I, Bbv I and Bbs I) are used The results of this study could be summarized as follows. The polymorphisms of the Myostatin gene achieves using Restriction enzyme (Aci) are GG, GA and AA respectively. The genotype of Myostatin gene has no effect on biochemical traits during 21 and 42 day, except which the effect of the genotypes of the Myostatin gene on the serum albumin is highly significant (P<0.01) during 21 day. And also the form genotype of the Myostatin gene with Restriction enzyme form (Bbs I) are CC, CT and TT respectively. The genotype of Myostatin gene has no effect on the blood biochemical during 21 and 42 day. The results show that the genotype of the Myostatin gene with Restriction enzyme form (Bbv I) are AA and GA respectively. The effect of has no effect on the blood biochemical during 21 and 42 day.

Isolation and identification of an extracellular enzyme from Aspergillus Niger with Deoxynivalenol biotransformation capability

In this study, the purification and characterization of an extracellular enzyme form Aspergillus niger was performed. With an optimized protocol, it was conducted a 42.6-fold purification with a yield of 26.2%. The purified lipase had a monomeric molecular weight of 40.5kDa and an isoelectric point of 6.01, and its maximum enzyme activity could be achieved at 40°C and pH 7.5-9.0. The enzyme could be activated by Ca2+, Mg2+ and Fe2+, while its activity could be inhibited by Zn2+ and Cu2+. Additionally, organic compounds exerted an inhibitory effect on the enzyme activity in a descending order of methanol, ethanol, DMSO, EDTA, acetone. Meanwhile, the specificity analysis of the enzyme indicated a preference to tributyrin and vegetable oils as well as long-chain fatty acid methyl esters (C12-C18). Most importantly, this enzyme could successfully transform deoxynivalenol (DON). Using HPLC analysis，it was detected a biotransformation rate of more than 70%.The liquid chromatography-mass spectrometry (LC-MS) analysis showed that the molecular weight of the transformation product was 18.0 larger than that of DON, indicating that DON could be hydrolyzed by the enzyme. Overall, the proposed method here provides a new avenue for reducing the toxicity of DON, which appears to have a wide application outlook for DON biotransformation.

Analysis of the Structure and Function of FOX-4 Cephamycinase

ABSTRACTClass C β-lactamases poorly hydrolyze cephamycins (e.g., cefoxitin, cefotetan, and moxalactam). In the past 2 decades, a new family of plasmid-based AmpC β-lactamases conferring resistance to cefoxitin, the FOX family, has grown to include nine unique members descended from theAeromonas caviaechromosomal AmpC. To understand the basis for the unique cephamycinase activity in the FOX family, we determined the first X-ray crystal structures of FOX-4, apo enzyme and the acyl-enzyme with its namesake compound, cefoxitin, using the Y150F deacylation-deficient variant. Notably, recombinant expression of N-terminally tagged FOX-4 also yielded an inactive adenylylated enzyme form not previously observed in β-lactamases. The posttranslational modification (PTM), which occurs on the active site Ser64, would not seem to provide a selective advantage, yet might present an opportunity for the design of novel antibacterial drugs. Substantial ligand-induced changes in the enzyme are seen in the acyl-enzyme complex, particularly the R2 loop and helix H10 (P289 to N297), with movement of F293 by 10.3 Å. Taken together, this study provides the first picture of this highly proficient class C cephamycinase, uncovers a novel PTM, and suggests a possible cephamycin resistance mechanism involving repositioning of the substrate due to the presence of S153P, N289P, and N346I substitutions in the ligand binding pocket.

Optimal Immobilization ofβ-Galactosidase ontoκ-Carrageenan Gel Beads Using Response Surface Methodology and Its Applications

β-Galactosidase (β-gal) was immobilized by covalent binding on novelκ-carrageenan gel beads activated by two-step method; the gel beads were soaked in polyethyleneimine followed by glutaraldehyde. 22full-factorial central composite experiment designs were employed to optimize the conditions for the maximum enzyme loading efficiency. 11.443 U of enzyme/g gel beads was achieved by soaking 40 units of enzyme with the gel beads for eight hours. Immobilization process increased the pH from 4.5 to 5.5 and operational temperature from 50 to 55°C compared to the free enzyme. The apparentKmafter immobilization was 61.6 mM compared to 22.9 mM for free enzyme. Maximum velocityVmaxwas 131.2 μmol·min−1while it was 177.1 μmol·min−1for free enzyme. The full conversion experiment showed that the immobilized enzyme form is active as that of the free enzyme as both of them reached their maximum 100% relative hydrolysis at 4 h. The reusability test proved the durability of theκ-carrageenan beads loaded withβ-galactosidase for 20 cycles with retention of 60% of the immobilized enzyme activity to be more convenient for industrial uses.

The activity of NADH-, NADPH- and Fd-dependent glutamate synthase in the plastids and cytosol of Pisum arvense L. root cells

Three forms of glutamate synthase (NADH-GOGAT, NADPH-GOGAT and Fd-(ferredoxin) GOGAT) were found in the plastids and cytosol of <em>Pisum arvense </em>root cells. The activities of the enzymes of both fractions decreased with increasing age of the plants, with the exception of plastid NADPH-GOGAT which exhibited markedly stable activity. NADH-GOGAT dominated in the cytosol of root cells of several day-old plants but after 14 days of cultivation, the activities of all of the GOGAT forms equalized. Plastid NADH-GOGAT and Fd-GOGAT showed similar activities in the root cells of 3-5 day-old plants, with Fd-GOGAT becoming the dominant enzyme form after 14 days. The entire activity of NADH-GOGAT and Fd-GOGAT was confined to the plaslid stroma. The plastid membrane fraction contained 37% of the NADPH-GOGAT activity. Isolated plastids synthesized glutamate from 2-ketoglutarate and glutamine, and glucose-6-phosphate and 6-phosophogluconate clearly stimulated this process. It is supposed that the synthesis of glutamate in <em>Pisum arvense </em>root plastids may be dependent on the intensity of the carbohydrates conversion in the pentose phosphate pathway.