Functional expression of nitrile hydratase in Escherichia coli: requirement of a nitrile hydratase activator and post-translational modification of a ligand cysteine

The nitrile hydratase (NHase) from Rhodococcus sp. N-771 is a photoreactive enzyme that is inactivated on nitrosylation of the non-heme iron center and activated on photo-dissociation of nitric oxide (NO). The nitrile hydratase operon consists of six genes encoding NHase regulator 2, NHase regulator 1, amidase, NHase alpha subunit, NHase beta subunit and NHase activator. We overproduced the NHase in Escherichia coli using a T7 expression system. The NHase was functionally expressed in E. coli only when the NHase activator encoded downstream of the beta subunit gene was co-expressed and the transformant was grown at 30 degrees C or less. A ligand cysteine, alphaCys112, of the recombinant NHase was also post-translationally modified to a cysteine-sulfinic acid similar to for the native NHase. Although another modification of alphaCys114 could not be identified because of the instability under acidic conditions, the recombinant NHase could be reversibly inactivated by nitric oxide.     

Surface modification of polyacrylonitrile with nitrile hydratase and amidase from Agrobacterium tumefaciens

A new strain of Agrobacterium tumefaciens (BST05) was found to grow on polyacrylonitrile (PAN; ¹³C labelled) converting the polymer to polyacrylic acid as shown by solid state NMR. When cultivated in a medium containing acetonitrile the bacterium produced nitrile hydratase and amidase activity. Activity recovery after lyophilisation and enzyme stability was significantly enhanced in the presence of 5% sorbitol leading to half life times of 12, 72 and 154 days at 25°C, 4°C and -20°C. The enzymes were able to convert 1.1% of the nitrile groups of PAN-powder to the corresponding acids. PAN fabrics were mainly converted to the amides as shown by an 80% increase of the O/C ratio in ESCA analysis. These data were confirmed by cationic dyeing and FTIR-ATR analysis.     

Surface modification of polyacrylonitrile with nitrile hydratase and amidase from Agrobacterium tumefaciens

A new strain of Agrobacterium tumefaciens (BST05) was found to grow on polyacrylonitrile (PAN; ¹³C labelled) converting the polymer to polyacrylic acid as shown by solid state NMR. When cultivated in a medium containing acetonitrile the bacterium produced nitrile hydratase and amidase activity. Activity recovery after lyophilisation and enzyme stability was significantly enhanced in the presence of 5% sorbitol leading to half life times of 12, 72 and 154 days at 25°C, 4°C and –20°C. The enzymes were able to convert 1.1% of the nitrile groups of PAN-powder to the corresponding acids. PAN fabrics were mainly converted to the amides as shown by an 80% increase of the O/C ratio in ESCA analysis. These data were confirmed by cationic dyeing and FTIR-ATR analysis.     

Mutational and structural analysis of cobalt-containing nitrile hydratase on substrate and metal binding.

Mutants of a cobalt-containing nitrile hydratase (NHase, EC 4.2.1.84) from Pseudonocardia thermophila JCM 3095 involved in substrate binding, catalysis and formation of the active center were constructed, and their characteristics and crystal structures were investigated. As expected from the structure of the substrate binding pocket, the wild-type enzyme showed significantly lower K(m) and K(i) values for aromatic substrates and inhibitors, respectively, than aliphatic ones. In the crystal structure of a complex with an inhibitor (n-butyric acid) the hydroxyl group of betaTyr68 formed hydrogen bonds with both n-butyric acid and alphaSer112, which is located in the active center. The betaY68F mutant showed an elevated K(m) value and a significantly decreased k(cat) value. The apoenzyme, which contains no detectable cobalt atom, was prepared from Escherichia coli cells grown in medium without cobalt ions. It showed no detectable activity. A disulfide bond between alphaCys108 and alphaCys113 was formed in the apoenzyme structure. In the highly conserved sequence motif in the cysteine cluster region, two positions are exclusively conserved in cobalt-containing or iron-containing nitrile hydratases. Two mutants (alphaT109S and alphaY114T) were constructed, each residue being replaced with an iron-containing one. The alphaT109S mutant showed similar characteristics to the wild-type enzyme. However, the alphaY114T mutant showed a very low cobalt content and catalytic activity compared with the wild-type enzyme, and oxidative modifications of alphaCys111 and alphaCys113 residues were not observed. The alphaTyr114 residue may be involved in the interaction with the nitrile hydratase activator protein of P. thermophila.     

A gene cluster responsible for alkylaldoxime metabolism coexisting with nitrile hydratase and amidase in Rhodococcus globerulus A-4

An enzyme "alkylaldoxime dehydratase (OxdRG)" was purified and characterized from Rhodococcus globerulus A-4, in which nitrile hydratase (NHase) and amidase coexisted with the enzyme. The enzyme contains heme b as a prosthetic group, requires reducing reagents for the reaction, and is most active at a neutral pH and at around 30 degrees C, similar to the phenylacetaldoxime dehydratase from Bacillus sp. OxB-1 (OxdB). However, some differences were seen in subunit structure, substrate specificity, and effects of activators and inhibitors. The corresponding gene, oxd, encoding a 1059-base pair ORF consisting of 353 codons, was cloned, sequenced, and overexpressed in Escherichia coli. The predicted polypeptide showed 30.3% identity to OxdB. The gene is mapped just upstream of the gene cluster encoding the enzymes involved in the metabolism of aliphatic nitriles, i.e., NHase and amidase, and their regulatory and activator proteins. We report here the existence of an aldoxime dehydratase genetically linked with NHase and amidase, and responsible for the metabolism of alkylaldoxime in R. globerulus.     

Expression control of nitrile hydratase and amidase genes in Rhodococcus erythropolis and substrate specificities of the enzymes

Bacterial amidases and nitrile hydratases can be used for the synthesis of various intermediates and products in the chemical and pharmaceutical industries and for the bioremediation of toxic pollutants. The aim of this study was to analyze the expression of the amidase and nitrile hydratase genes of Rhodococcus erythropolis and test the stereospecific nitrile hydratase and amidase activities on chiral cyanohydrins. The nucleotide sequences of the gene clusters containing the oxd (aldoxime dehydratase), ami (amidase), nha1, nha2 (subunits of the nitrile hydratase), nhr1, nhr2, nhr3 and nhr4 (putative regulatory proteins) genes of two R. erythropolis strains, A4 and CCM2595, were determined. All genes of both of the clusters are transcribed in the same direction. RT-PCR analysis, primer extension and promoter fusions with the gfp reporter gene showed that the ami, nha1 and nha2 genes of R. erythropolis A4 form an operon transcribed from the Pami promoter and an internal Pnha promoter. The activity of Pami was found to be weakly induced when the cells grew in the presence of acetonitrile, whereas the Pnha promoter was moderately induced by both the acetonitrile or acetamide used instead of the inorganic nitrogen source. However, R. erythropolis A4 cells showed no increase in amidase and nitrile hydratase activities in the presence of acetamide or acetonitrile in the medium. R. erythropolis A4 nitrile hydratase and amidase were found to be effective at hydrolysing cyanohydrins and 2-hydroxyamides, respectively.     

Mouse alcohol dehydrogenase 4: kinetic mechanism, substrate specificity and simulation of effects of ethanol on retinoid metabolism

Mouse ADH4 (purified, recombinant) has a low catalytic efficiency for ethanol and acetaldehyde, but very high activity with longer chain alcohols and aldehydes, at pH 7.3 and temperature 37 degrees C. The observed turnover numbers and catalytic efficiencies for the oxidation of all-trans-retinol and the reduction of all-trans-retinal and 9-cis-retinal are low relative to other substrates; 9-cis-retinal is more reactive than all-trans-retinal. The reduction of all-trans- or 9-cis-retinals coupled to the oxidation of ethanol by NAD(+) is as efficient as the reduction with NADH. However, the Michaelis constant for ethanol is about 100 mM, which indicates that the activity would be lower at physiologically relevant concentrations of ethanol. Simulations of the oxidation of retinol to retinoic acid with mouse ADH4 and human aldehyde dehydrogenase (ALDH1), using rate constants estimated for all steps in the mechanism, suggest that ethanol (50 mM) would modestly decrease production of retinoic acid. However, if the K(m) for ethanol were smaller, as for human ADH4, the rate of retinol oxidation and formation of retinoic acid would be significantly decreased during metabolism of 50 mM ethanol. These studies begin to describe quantitatively the roles of enzymes involved in the metabolism of alcohols and carbonyl compounds.     

Mouse alcohol dehydrogenase 4: kinetic mechanism,substrate specificity and simulation of effects of ethanol on retinoid metabolism

Mouse ADH4 (purified, recombinant) has a low catalytic efficiency for ethanol and acetaldehyde, but very high activity with longer chain alcohols and aldehydes, at pH 7.3 and temperature 37°C. The observed turnover numbers and catalytic efficiencies for the oxidation of all-trans-retinol and the reduction of all-trans-retinal and 9-cis-retinal are low relative to other substrates; 9-cis-retinal is more reactive than all-trans-retinal. The reduction of all-trans- or 9-cis-retinals coupled to the oxidation of ethanol by NAD⁺ is as efficient as the reduction with NADH. However, the Michaelis constant for ethanol is about 100 mM, which indicates that the activity would be lower at physiologically relevant concentrations of ethanol. Simulations of the oxidation of retinol to retinoic acid with mouse ADH4 and human aldehyde dehydrogenase (ALDH1), using rate constants estimated for all steps in the mechanism, suggest that ethanol (50 mM) would modestly decrease production of retinoic acid. However, if the K_m for ethanol were smaller, as for human ADH4, the rate of retinol oxidation and formation of retinoic acid would be significantly decreased during metabolism of 50 mM ethanol. These studies begin to describe quantitatively the roles of enzymes involved in the metabolism of alcohols and carbonyl compounds.     

野生植物南山王粗提物对菜青虫的生物活性和光活化作用

野生植物南山王 (Celastrussp .)的根茎叶用甲醇、氯仿和石油醚提取 ,并制备干粉。浓度为 0l. 0 1g mL的粗提物对菜青虫PierisrapaeL .的非选择性拒食率达到 61 . 73 %～ 93 . 5 5 % ;用浸渍法测得根的粗提物对菜青虫的光活化比达到 2 5 . 5～ 3 4. 1。南山王根的氯仿提取物活性最高。     

Purification and characterization of the enantioselective nitrile hydratase from Rhodococcus equi A4

The nitrile hydratase from Rhodococcus equi A4 consisted of two kinds of subunits which slightly differed in molecular weight (both approximately 25 kDa) and showed a significant similarity in the N-terminal amino acid sequences to those of the nitrile hydratase from Rhodococcus sp. N-774. The enzyme preferentially hydrated the S-isomers of racemic 2-(2-, 4-methoxyphenyl)propionitrile, 2-(4-chlorophenyl)propionitrile and 2-(6-methoxynaphthyl)propionitrile (naproxennitrile) with E-values of 5-15. The enzyme functioned in the presence of 5-98% (v/v) of different hydrocarbons, alcohols or diisopropyl ether. The addition of 5% (v/v) of n-hexane, n-heptane, isooctane, n-hexadecane, pristane and methanol increased the E-value for the enzymatic hydration of 2-(6-methoxynaphthyl)propionitrile.     

2-羟基-4-甲氧基苯甲醛缩苯胺对菜青虫酚氧化酶的抑制作用

为筛选对十字花科蔬菜害虫菜青虫Pieris rapae（L.）酚氧化酶具有高抑制活性的化合物,为寻找新型害虫控制剂提供线索,采用酶标仪微量法以室内合成、筛选的高活性化合物2-羟基-4-甲氧基苯甲醛缩苯胺为抑制剂,研究了其对菜青虫酚氧化酶的抑制活性及抑制类型。结果表明,供试化合物对菜青虫酚氧化酶的抑制中浓度（IC₅₀）为0.116 mmol/L;该化合物为典型的可逆非竞争型抑制剂,抑制常数（K_i）为1.96 mmol/L。该化合物直接对靶标酚氧化酶产生作用,而不是通过影响酶结构内的铜离子来产生作用的。     

Larval gut phosphatases of Pieris rapae: Isolation and partial characterization

Two isozymes of alkaline phosphatase are present in the larval gut of the imported cabbage butterfly, Pieris rapae. These have been isolated by means of ammonium sulfate fractionation, column chromatography, and preparative electrophoresis. The affinity of each form for each of eighteen substrates was determined. Both forms exhibit high affinity for glucose-6-phosphate. The faster migrating form, which is specific to the larval gut, does not hydrolyze α-glycerophosphate or 6-phosphogluconate but has a high affinity for ribose derivatives, including the nucleotides. The slower migrating isozyme, which is found in many tissues of the larva and the adult, readily hydrolyzes α-glycerophosphate, 6-phosphogluconate and the phosphoamino acids. Ribose derivatives, with the exception of ADP, are poor or unsuitable substrates for this form. It is suggested that the additional capacity for active transport and rapid nucleotide metabolism represented by the larval gut-specific form may be of great advantage in carrying out the diverse and often unique functions of this tissue.     

Axonal pathfinding in developing labial palps of the butterflies,Pieris rapae and Pieris brassicae

Summary Electron microscopy was used to study the developmental changes in the pupal labial palp of Pieris rapae L. and P. brassicae L. prior to axogenesis of the peripheral receptor organs. Two cells emigrate from the anlage of the labial palp-pit organ (LPPO). They develop growth cones and filopodia, which are directed distally. The filopodia insert into stem cells of the LPPO. The perikarya of the cells proceed into the hemolymph space of the palp such that, eventually, these cells bridge the gap between the LPPO and the apical scolopidial organ (ASO) of the labial palp. This bridge is established at about 11% of total pupal development. Only 6 h later, at 15% of pupal development, the cells are no longer visible. We suggest that these cells are luminal neurons and name them pit-organ luminal (POL) cells. These POL cells may act as primary guiding structures for the axons of the sensory cells of the LPPO, which are assumed to orientate along this structure once they reach the ASO.Supported by the Deutsche Forschungsgemeinschaft (H.A.: SFB4/G1).     

Parental effects in Pieris rapae in response to variation in food quality: adaptive plasticity across generations?

1. Herbivores using seasonal resources must cope with variation in the quality of their host plants. The effects of variation in protein concentration of artificial diet and glucosinolate concentration in canola, Brassica napus, on Pieris rapae parental and progeny growth were investigated. 2. The hypothesis that parents respond to variation in food quality by altering the phenotype of their progeny to enhance progeny fitness was tested. Consistent with previous studies, P. rapae was not affected strongly by variation in the protein concentration of artificial diet and had equal mass on completing development. 3. The mass of individual eggs of P. rapae progeny was correlated negatively with the amount of protein in the diet on which parents fed. Moreover, mothers reared in extreme conditions (high and low protein) produced progeny that grew best under those conditions. These potentially adaptive parental effects were detected early in progeny growth but not later in their development. 4. Early larval growth of P. rapae was affected negatively by increasing glucosinolates in B. napus plants, although no effects of glucosinolates were detected later in growth or on the progeny's phenotype. 5. Thus, evidence is presented that variation in food quality (protein concentration) has major consequences for the progeny of P. rapae. Given the multivoltine life history of P. rapae and the seasonal differences in food quality it encounters, such parental effects may be adaptive.     

Host plant specificity in several species of generalist mite predators

1. Species in the genus Neoseiulus are considered to be generalist predators, with some species used in biological control programmes against phytophagous mites and insects. 2. A general survey of Neoseiulus species in inland Australia indicated that different species are associated with particular tree species. This pattern of host plant use was investigated for four Neoseiulus species (N. buxeus, N. cappari, N. brigarinus, N. eremitus) by means of a sampling programme through time and across space. 3. Each species of Neoseiulus was collected entirely or mostly from one species of tree; little or no overlap was detected despite the tree species growing in well‐mixed stands. Host plant specificity thus appears to be strong in this genus. 4. Species in two other genera (Pholaseius and Australiseiulus), also considered to be predatory, showed a similar association with particular tree species. 5. The implications for the use of these predators in biological control are considerable. In particular, phytoseiid species with specific needs in terms of host plants may not be suitable for use as general purpose predators. Meeting the needs of phytoseiids through the modification of host plant attributes may be a step towards enhancing their efficacy as biological control agents.     

Carbohydrate substrate specificity of bacterial and plant pyrophosphate-dependent phosphofructokinases

Pyrophosphate-dependent phosphofructokinase from the facultative anaerobic bacterium Propionibacterium freudenreichii and from the mung bean Phaseolus aureus has been purified to homogeneity. Potential utilization of carbohydrate substrate analogues for each enzyme was initially screened by using Fourier transform 31P NMR at pH 8 and 25 degrees C and monitoring the appearance of the phosphate resonance in the direction of D-fructose 6-phosphate phosphorylation (forward reaction direction) and, with the bisphosphate analogues, the appearance of the pyrophosphate resonance in the direction of phosphate phosphorylation (reverse reaction direction). Both enzymes are strict in their requirements for the sugar phosphate substrate, with only D-fructose 6-phosphate, D-sedoheptulose 7-phosphate, and 2,5-anhydro-D-mannitol 6-phosphate, or their respective bisphosphates in the reverse reaction direction, utilized as substrates at detectable levels. The dissociation constants for D-psicose 6-phosphate, D-tagatose 6-phosphate, and L-sorbose 6-phosphate are an order of magnitude larger than that for D-fructose 6-phosphate, indicating a stringent steric requirement for the D-threo (trans) configuration at the two nonanomeric furan ring hydroxyl groups. These results strongly suggest that the anomeric, epimeric, and tautomeric form of the sugar phosphate substrates favored by both enzymes is the beta-D-fructofuranose form. Dissociation constants for nonsubstrate analogues were used to provide information on the nature of the active site. Competitive inhibition patterns vs. fructose 1,6-bisphosphate were obtained for a series of 1,n-alkanediol bisphosphates (where n = 2-9).(ABSTRACT TRUNCATED AT 250 WORDS)     

Exofacial membrane composition and lipid metabolism regulates plasma membrane P4-ATPase substrate specificity

The plasma membrane of a cell is characterized by an asymmetric distribution of lipid species across the exofacial and cytofacial aspects of the bilayer. Regulation of membrane asymmetry is a fundamental characteristic of membrane biology and is crucial for signal transduction, vesicle transport, and cell division. The type IV family of P-ATPases, or P4-ATPases, establishes membrane asymmetry by selection and transfer of a subset of membrane lipids from the lumenal or exofacial leaflet to the cytofacial aspect of the bilayer. It is unclear how P4-ATPases sort through the spectrum of membrane lipids to identify their desired substrate(s) and how the membrane environment modulates this activity. Therefore, we tested how the yeast plasma membrane P4-ATPase, Dnf2, responds to changes in membrane composition induced by perturbation of endogenous lipid biosynthetic pathways or exogenous application of lipid. The primary substrates of Dnf2 are glucosylceramide (GlcCer) and phosphatidylcholine (PC, or their lyso-lipid derivatives), and we find that these substrates compete with each other for transport. Acutely inhibiting sphingolipid synthesis using myriocin attenuates transport of exogenously applied GlcCer without perturbing PC transport. Deletion of genes controlling later steps of glycosphingolipid production also perturb GlcCer transport to a greater extent than PC transport. In contrast, perturbation of ergosterol biosynthesis reduces PC and GlcCer transport equivalently. Surprisingly, application of lipids that are poor transport substrates differentially affects PC and GlcCer transport by Dnf2, thus altering substrate preference. Our data indicate that Dnf2 exhibits exquisite sensitivity to the membrane composition, thus providing feedback onto the function of the P4-ATPases.