Structural and dynamics characterization of norovirus protease

Norovirus protease is an essential enzyme for proteolytic maturation of norovirus nonstructural proteins and has been implicated as a potential target for antiviral drug development. Although X‐ray structural studies of the protease give us wealth of structural information including interactions of the protease with its substrate and dimeric overall structure, the role of protein dynamics in the substrate recognition and the biological relevance of the protease dimer remain unclear. Here we determined the solution NMR structure of the 3C‐like protease from Norwalk virus (NV 3CLpro), a prototype strain of norovirus, and analyzed its backbone dynamics and hydrodynamic behavior in solution. ¹⁵N spin relaxation and analytical ultracentrifugation analyses demonstrate that NV 3CLpro is predominantly a monomer in solution. Solution structure of NV 3CLpro shows significant structural variation in C‐terminal domain compared with crystal structures and among lower energy structure ensembles. Also, ¹⁵N spin relaxation and Carr–Purcell–Meiboom–Gill (CPMG)‐based relaxation dispersion analyses reveal the dynamic properties of residues in the C‐terminal domain over a wide range of timescales. In particular, the long loop spanning residues T123–G133 show fast motion (ps‐ns), and the residues in the bII–cII region forming the large hydrophobic pocket (S2 site) undergo conformational exchanges on slower timescales (μs–ms), suggesting their important role in substrate recognition.     

Pleiotropy in the melanocortin system: expression levels of this system are associated with melanogenesis and pigmentation in the tawny owl (Strix aluco)

The adaptive function of melanin‐based coloration is a long‐standing debate. A recent genetic model suggested that pleiotropy could account for covariations between pigmentation, behaviour, morphology, physiology and life history traits. We explored whether the expression levels of genes belonging to the melanocortin system (MC1R, POMC, PC1/3, PC2 and the antagonist ASIP), which have many pleiotropic effects, are associated with melanogenesis (through variation in the expression of the genes MITF, SLC7A11, TYR, TYRP1) and in turn melanin‐based coloration. We considered the tawny owl (Strix aluco) because individuals vary continuously from light to dark reddish, and thus, colour variation is likely to stem from differences in the levels of gene expression. We measured gene expression in feather bases collected in nestlings at the time of melanin production. As expected, the melanocortin system was associated with the expression of melanogenic genes and pigmentation. Offspring of darker reddish fathers expressed PC1/3 to lower levels but tended to express PC2 to higher levels. The convertase enzyme PC1/3 cleaves the POMC prohormone to obtain ACTH, while the convertase enzyme PC2 cleaves ACTH to produce α‐melanin‐stimulating hormone (α‐MSH). ACTH regulates glucocorticoids, hormones that modulate stress responses, while α‐MSH induces eumelanogenesis. We therefore conclude that the melanocortin system, through the convertase enzymes PC1/3 and PC2, may account for part of the interindividual variation in melanin‐based coloration in nestling tawny owls. Pleiotropy may thus account for the covariation between phenotypic traits involved in social interactions (here pigmentation) and life history, morphology, behaviour and physiology.     

Omptin proteins: an expanding family of outer membrane proteases in Gram-negative Enterobacteriaceae (Review)

The Escherichia coli K-12 outer membrane protein OmpT is a prototype of a unique family of bacterial endopeptidases known as the omptins. This family includes OmpT and OmpP of E. coli, SopA of Shigella flexneri, PgtE of Salmonella enterica, and Pla of Yersinia pestis. Despite their sequence similarities, the omptins vary in their reported functions. The OmpT protease is characterized by narrow cleavage specificity defined by the extracellular loops of the β-barrel protruding above the lipid bilayer. It employs a distinct proteolytic mechanism that involves a histidine and an aspartate residue. Most of the omptin proteins have been implicated in bacterial pathogenesis. As a result, the omptins are potential targets for antimicrobial drug and vaccine development. This review summarizes recent developments in omptins structure and function, emphasizes their role in pathogenesis, proposes evolutionary relation among the existing omptins, and offers possible directions for future research.     

Extracellular Production of l-Lysine α-Oxidase in Wheat Bran Culture of a Strain of Trichoderma viride

A mold strain Y244-2 capable of producing l-lysine α-oxidase, a new enzyme catalyzing the α-oxidative deamination of l-lysine, was identified as Trichoderma viride. Among strains belonging to the genus Trichoderma tested, only Trichoderma viride Y244-2 produced the enzyme in wheat bran culture. The maximum enzyme production by the mold grown on wheat bran was observed after 10 and 14 days incubation with and without NaN0₃, respectively. Addition of NaN0₃, NH₄N0₃, adenine, purine nucleosides, l-histidine, glycine or l-glutamine to wheat bran stimulated the production of the enzyme. In the liquid culture, the enzyme was produced extracellulary under the aerobic conditions, although the production was much lower than that in the wheat bran culture.     

Isolation and Characterization of a Cysteine Protease of Freesia Corms

A protease, freesia protease (FP)-A, was purified to electrophoretic homogeneity from regular freesia (Freesia reflacta) corms in harvest time. The M _r of FP-A was estimated to be 24 k by SDS-PAGE. The optimum pH of the enzyme was 8.0 using a casein substrate. These enzymes were strongly inhibited by p-chloromercuribenzoic acid but not by phenylmethane-sulfonylfluoride and EDTA. These results indicate that FP-A belongs to the cysteine proteases. The amino terminal sequence of FP-A was similar to that of papain, and the sequences was regarded to the conservative residues of cysteine protease. From the hydrolysis of peptidyl-pNAs, the specificity of FP-A was found to be broad. It was thought that FP-A was a new protease from freesia corms.     

Enzymatic Assay of d-Xylose Isomerase with d-Sorbitol Dehydrogenase

Previously a cyclic pathway for the partial oxidation of propionyl-CoA to pyruvate has been proposed. Enzymatic evidence for the presence of the key reactions involved in this pathway is described and discussed herein. The condensation of propionyl-CoA with oxaloacetate into methylcitrate is shown to be catalyzed by an enzyme contained in cell-free extracts of Candida lipolytica; the enzyme seems to differ from the usual citrate synthase. Methylcitrate is easily convertible to a mixture of C₇-acids by the action of cell-free extract of the mutant strain. On the other hand, a similar mixture is changed into pyruvate and succinate by the action of cell-free extract of the parent strain. Evidence is given that methylisocitrate, one of the products of the conversion, is mainly cleaved by the action of an additional enzyme other than the usual isocitrate lyase. The accumulation of methylisocitrate in a large amount from odd-carbon n-alkanes by the mutant strain can be safely ascribed to the absence or a low level of this enzyme in the mutant strain.     

Purification and Characterization of a Cysteine Protease from Corms of Freesia,Freesia reflacta

A protease (freesia protease B) has been purified to electrophoretic homogeneity from corms of freesia, Freesia reflacta by five steps of chromatography. Its M_r was estimated to be about 26,000 by SDS–PAGE. The optimum pH of the enzyme was 6.0–7.0 at 30°C using casein as a substrate. The enzyme was strongly inhibited by p-chloromercuribenzoic acid but not by phenylmethanesulphonylfluoride and EDTA. These results indicate that freesia protease B is a cysteine protease. Nine sites of oxidized insulin B-chain were cleaved by freesia protease B in 24 h of hydrolysis. The four cleavage sites among them resembled those of papain. From the digestion of five peptidyl substrates the specificity of freesia protease B was found to be approximately broad, but the preferential cleavage sites were negatively charged residues at positions. Freesia protease B preferred also the large hydrophobic amino acid residues at the P₂ position, in a similar manner to papain. The amino terminal sequence of freesia protease B was identical with those of papain in regard to the conservative residues of cysteine protease.     

Microbial Oxidation of Cephalosporins to Cephalosporin Sulfoxides

The highest inhibition rate of conidial germination of Pyricularia oryzae was shown by extracts of rice plant leaves inoculated by a pathogen after treatment with probenazole, a rice blast controlling agent. Four anti-conidial germination substances were isolated from these extracts. Substances A, C and D inhibited the germination of the conidia at concentrations between 100 and 200 mcg/ml, and substance B caused morphological changes in the germination tubes of the conidia with a little inhibition of germination. These substances were differentiated from momilactone A, B and the degraded or metabolized products of probenazole. Besides anti-conidial germination activity, they showed antimicrobial activities against several kinds of phytopathogenic bacteria of fungi on agar plates by diffusion method.     

Same Advantages of Using Low Speed Centrifugation for the Purification of Rat Liver Mitochondria

Twelve strains of lactose-fermenting yeast isolated from raw milk were evaluated on β-galactosidase producing ability. The enzymes from the four strains (Tolulopsis versatilis M6, Tolulopsis sphaerica J28, Candida pseudotropicalis B57 and A60), selected by high productivity, showed very similar properties and were characterized by a pH optimum of 7.0 or 7.5 and a relatively low optimal temperature of 30°C. The molecular weights were estimated by gel filtration to be 200,000-233,000. The Km values for o-nitrophenyl-β-d-galactopyranoside were 3.45 mm, 2.09 mm, 3.45 mm and 2.82 mm for enzymes from M6, J28, B57 and A60, respectively. All enzymes were activated by Mn²⁺ and inhibited by Mg²⁺, Zn²⁺ and Ca²⁺. The enzymes are sulfhydryl dependent and were completely inhibited by Hg²⁺ and sulfhydryl reagents. The yeasts may be a potential source for the enzyme for industrial use.