Construction of artificial cyclic amide amidohydrolases using molecular imprinting technique

A general molecular imprinting approach is proposed to synthesize artificial enzymes to mimic the family of cyclic amide amidohydrolases which share similar active site and catalytic mechanism. The artificial enzymes were constructed by co-polymerizing 4(5)-vinylimidazole-Co²⁺-methacrylic acid clusters with divinylbenzene micro-spheres in the presence of corresponding substrates. The artificial enzymes mimicked creatininase and hydantoinase by showing specific affinity towards the corresponding substrates in buffer. The artificial hydantoinase also showed specific affinity towards corresponding substrate in organic solvent, and catalyzed the hydrolysis of hydantoin.     

High-level expression and one-step purification of cyclic amidohydrolase family enzymes

The cyclic amidohydrolase family enzymes, including hydantoinase, dihydropyrimidinase, allantoinase and dihydroorotase, are metal-dependent hydrolases and play a crucial role in the metabolism of purine and pyrimidine in prokaryotic and eukaryotic cells. With the increasing demand for the elucidation of enzyme structures and functions, along with industrial applications, the research on the family enzymes has recently been proliferating, but the related enzymes had been purified conventionally by multistep purification procedures. Here, we reported the expression in Escherichia coli cells of maltose-binding protein-fused family enzymes and their one-step purification. The expression levels of the fusion proteins account for 20-35% of the total protein in E. coli, allowing approximately 2-3 mg of the purified proteins by affinity chromatography to be obtained per 0.3 L of bacterial culture. As more promising results, their nascent biochemical properties, after the cleavage of the fusion proteins with Factor Xa, in terms of oligomeric structure, optimal pH, specific activity, and kinetic property, were also conserved as those from the native enzymes. The availability of the family enzymes to fusion strategy shows potential as a convenient procedure to recombinant protein purification and accelerates the structure-function study of the related family enzymes.     

Functional expression and characterization of the two cyclic amidohydrolase enzymes, allantoinase and a novel phenylhydantoinase, from Escherichia coli

A superfamily of cyclic amidohydrolases, including dihydropyrimidinase, allantoinase, hydantoinase, and dihydroorotase, all of which are involved in the metabolism of purine and pyrimidine rings, was recently proposed based on the rigidly conserved structural domains in identical positions of the related enzymes. With these conserved domains, two putative cyclic amidohydrolase genes from Escherichia coli, flanked by related genes, were identified and characterized. From the genome sequence of E. coli, the allB gene and a putative open reading frame, tentatively designated as a hyuA (for hydantoin-utilizing enzyme) gene, were predicted to express hydrolases. In contrast to allB, high-level expression of hyuA in E. coli of a single protein was unsuccessful even under various induction conditions. We expressed HyuA as a maltose binding protein fusion protein and AllB in its native form and then purified each of them by conventional procedures. allB was found to encode a tetrameric allantoinase (453 amino acids) which specifically hydrolyzes the purine metabolite allantoin to allantoic acid. Another open reading frame, hyuA, located near 64.4 min on the physical map and known as a UUG start, coded for D-stereospecific phenylhydantoinase (465 amino acids) which is a homotetramer. As a novel enzyme belonging to a cyclic amidohydrolase superfamily, E. coli phenylhydantoinase exhibited a distinct activity toward the hydantoin derivative with an aromatic side chain at the 5' position but did not readily hydrolyze the simple cyclic ureides. The deduced amino acid sequence of the novel phenylhydantoinase shared a significant homology (>45%) with those of allantoinase and dihydropyrimidinase, but its functional role still remains to be elucidated. Despite the unclear physiological function of HyuA, its presence, along with the allantoin-utilizing AllB, strongly suggested that the cyclic ureides might be utilized as nutrient sources in E. coli.     

Prostaglandins and cyclic AMP stimulate creatine kinase synthesis but not fusion in cultured embryonic chick muscle cells

Cyclic AMP levels have been measured in cultures derived from 12-day-old chick embryonic muscle. A rise in concentration was found after the onset of myoblast fusion. Cells cultured at a medium Ca²⁺ concentration of 0.1 μM did not fuse and exhibited only a small rise in cyclic AMP concentration during culture. Addition of 1.4 mM Ca²⁺ to these cells after 50 h in culture caused rapid, synchronous fusion with a concomitant rise in cyclic AMP levels. Indomethacin, an inhibitor of prostaglandin synthesis, did not inhibit fusion, but inhibited the rise in cyclic AMP concentration. Indomethacin-treated cultures exhibited lower creatine kinase levels, though no change in the ratio of the three isoenzymes was observed. Addition of prostaglandins E₁ and E₂ to indomethacin-treated cultures overcame this inhibition. We propose that prostaglandin synthesis is a consequence of the stimulation of myoblast fusion and that via cyclic AMP it stimulates protein synthesis.     

Characterization of the RND family of multidrug efflux pumps: in silico to in vivo confirmation of four functionally distinct subgroups

We have developed a generalized profile that identifies members of the root-nodulation-cell-division (RND) family of efflux pumps and classifies them into four functional subfamilies. According to Z-score values, efflux pumps can be grouped by their metabolic function, thus making it possible to distinguish pumps involved in antibiotic resistance (group 1) from those involved in metal resistance (group 3). In silico data regarding efflux pumps in group 1 were validated after identification of RND efflux pumps in a number of environmental microbes that were isolated as resistant to ethidium bromide. Analysis of the Pseudomonas putida KT2440 genome identified efflux pumps in all groups. A collection of mutants in efflux pumps and a screening platform consisting of 50 drugs were created to assign a function to the efflux pumps. We validated in silico data regarding efflux pumps in groups 1 and 3 using 9 different mutants. Four mutants belonging to group 2 were found to be more sensitive than the wild-type to oxidative stress-inducing agents such as bipyridyl and methyl viologen. The two remaining mutants belonging to group 4 were found to be more sensitive than the parental to tetracycline and one of them was particularly sensitive to rubidium and chromate. By effectively combining in vivo data with generalized profiles and gene annotation data, this approach allowed the assignment, according to metabolic function, of both known and uncharacterized RND efflux pumps into subgroups, thereby providing important new insight into the functions of proteins within this family.     

Establishment of a new method to quantitatively evaluate hyphal fusion ability in Aspergillus oryzae

Hyphal fusion is involved in the formation of an interconnected colony in filamentous fungi, and it is the first process in sexual/parasexual reproduction. However, it was difficult to evaluate hyphal fusion efficiency due to the low frequency in Aspergillus oryzae in spite of its industrial significance. Here, we established a method to quantitatively evaluate the hyphal fusion ability of A. oryzae with mixed culture of two different auxotrophic strains, where the ratio of heterokaryotic conidia growing without the auxotrophic requirements reflects the hyphal fusion efficiency. By employing this method, it was demonstrated that AoSO and AoFus3 are required for hyphal fusion, and that hyphal fusion efficiency of A. oryzae was increased by depleting nitrogen source, including large amounts of carbon source, and adjusting pH to 7.0.     

Characterization of the functionally related sites in the neural inducing gene noggin

Previously we have shown that blocking bone morphogenetic protein (BMP) receptor signaling by a dominant negative BMP receptor causes neurogenesis in Xenopus animal caps (ACs), whereas the physiological neural inducer noggin acts as a homodimer physically binding to BMP-4 and disrupting its signaling at the ligand level. The present study attempted to elucidate the relationship between the structure and function of noggin. By replacing some cysteine residues with serine residues through a site-directed mutagenesis strategy, we generated three noggin mutants, C145S, C205S, and C(218, 220, 222)S (3CS). Although mRNAs encoded by these mutants were translated as efficiently as wild-type (WT) noggin mRNA, they behaved differently when expressed in vivo. Expression of WT noggin or C205S in Xenopus ACs converted the explants (prospective ectoderm) into neural tissue, indicated by the neural-like morphology and expression of the pan neural marker NCAM in the ACs. In contrast, ACs expressing C145S or 3CS sustained an epidermal fate like the control caps. Similar results were observed in the mesoderm where C205S (but not C145S and 3CS) displayed dorsalizing activity as well as WT noggin. Altogether, our results suggest that Cys145 alone or Cys(218, 220, 222) as a whole in noggin protein is required for the biological activities of noggin, probably participating in the dimerization of noggin with BMP-4 or itself.     

Rapid changes in the activities of the enzymes of cyclic AMP metabolism after addition of A23187 to macrophages

The ionophore A23187 stimulated adenylate cyclase activity in intact macrophages within 1 min. This action was blocked by pretreatment with indomethacin (25 μmol/l) suggesting the involvement of a prostaglandin (PG). PGE₂ (500 nmol/l) also stimulated adenylate cyclase activity in intact cells, but this was not prevented by indomethacin pretreatment. Colchicine (100 μmol/l) potentiated the increases in macrophage cyclic AMP production seen after addition of PGE₂ or A23187. The high affinity form of cyclic AMP phosphodiesterase (PDE) was activated within 1 min of the addition of A23187 to intact macrophages. The data suggest that the increase in macrophage cyclic AMP production after A23187 is a consequence of adenylate cyclase activation and not PDE inhibition. The endogenous production of a prostaglandin probably mediates this effect of A23187, emphasizing the importance of arachidonic acid metabolites in the regulation of macrophage functions.     

Solution and solid state conformational preferences of a family of cyclic disulphide bridged tetrapeptides

A set of cyclic tetrapeptides of the general form cyclo (Boc‐Cys‐Pro‐ X ‐Cys‐OMe) with X being L‐ / D‐Ala , L‐ / D‐Val , and L‐ / D‐Trp was synthesized. These peptides serve as model systems for structure elucidation in solution and feature a variety of structural motifs — namely a β‐turn with intramolecular hydrogen bonding interactions, cis/trans isomerism, and a disulphide bond. In this work, we performed a comprehensive structural analysis focussing on their β‐turn conformational preferences using NMR, VCD, and Raman spectroscopy. Our results provide evidence for a strong influence of a single stereocenter on the structures of the peptides whereas solvent polarity does not significantly affect them. Additionally, the solid state conformational preferences were studied by crystal structure analysis. Overall, a general trend for the conformational preferences of this set of peptides can be concluded from the results of the complementary investigations.     

Efficient production of the industrial biocatalysts hydantoinase and N-carbamyl amino acid amidohydrolase: Novel non-metabolizable inducers

Abstract The biosynthesis of the hydantoin-hydrolysing enzymes hydantoinase and N -carbamyl amino acid amidohydrolase from Agrobacterium sp. IP I-671, a Gram-negative bacterium used as a biocatalyst for the production of enantiomerically pure ( R ) amino acids, was found to be highly inducible by the addition to the cultivation medium of different non-metabolizable thiolated hydantoins or pyrimidines. Among these inducers the hexacyclic pyrimidine thioderivatives were more potent than all the pentacyclic thiohydantoin compounds. Addition of 2,4-thiouracil to the cultures, at a rate of 0.1 g (g cell dry mass)⁻¹, led to no appreciable growth inhibition and yielded a biocatalyst exhibiting a 40-fold higher hydantoinase and a 15-fold higher N -carbamyl amino acid amidohydrolase activity than the corresponding inducer-free cultures.     

Evaluation of pyrimidine- and hydantoin-degrading enzyme activities in aerobic bacteria

Abstract The enzyme activities responsible for the reductive pyrimidine base degradation by aerobic bacteria, which produce hydantoin-degrading enzymes, were investigated. Pseudomonas putida IFO 12996, which is a d-stereospecific hydantoinase producer, has dihydropyrimidinase activity, and Comamonas sp. E222c and Blastobacter sp. A17p-4, which are N-carbamoyl-D-amino acid amidohydrolase producers, have β-ureidopropionase activity. Blastobacter sp. also possesses both d-stereospecific hydantoinase and dihydropyrimidinase activities. Thus, two amide ring-opening activities and/or two N -carbamoyl amino acid-hydrolyzing activities coexist in these bacteria. However, the differences of the induction levels of each enzyme activities for the several pyrimidine- and hydantoin-related compounds suggest that these corresponding amide ring-opening or N -carbamoyl amino acid-hydrolyzing activities are not always catalyzed by the same enzymes.     

Synthesis and biological evaluation of analogues of the marine cyclic depsipeptide obyanamide

On the basis of the total synthesis of obyanamide, 20 analogues of this marine cyclic depsipeptide have been synthesized by (i) preparation of the tripeptide fragments in the western hemisphere using Z/OtBu protocol; (ii) preparation of the dipeptide fragments in the eastern hemisphere using Boc/OMe protocol; and (iii) fragments coupling, removal of protecting groups (Boc and OtBu, in one pot), and macrocyclizaion in the last step. The cytotoxic test showed that three synthetic compounds exhibited moderate activities against HL‐60, KB, LOVO, and A549 cell lines. According to the results, the β‐amino acid residue was found to play a critical role in the biological activities. Additionally, the ester bond along with the Ala(Thz) moiety was also essential for biological activities. However, it seems too early to draw a conclusion that the N‐methylation of Val/Phe can lead to higher or lower cytotoxic activities. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Deduction of the evaluation limit and termination timing of multi-round protein misfolding cyclic amplification from a titration curve

In this study, the efficacy of disinfectants in reducing the partially protease-resistant isoform of prion protein was evaluated by a multi-round protein misfolding cyclic amplification (PMCA) technique. Hamster brains infected with scrapie-derived strain 263K were homogenized, treated under inactivating or mock conditions, and subjected to multi-round PMCA. Four sets of serial 10-fold dilutions of mock-treated samples were analyzed. Although considerable variability was observed in the signal patterns, between the second and sixth rounds the number of the PMCA round correlated in a linear fashion with the mean dilution factor of mock-treated, infected brains, corresponding to a log reduction factor (LRF) of 3.8-7.3 log. No signals were observed in the PMCA products amplified from normal hamster brain homogenates. The mean numbers of rounds at the first appearance of the signal for 1 M and 2 M NaOH-treated samples were 4.33 and 4, respectively. Using the linear regression line as the titration curve, the LRFs of these disinfectants were found to be 6.1 and 5.8 log, respectively; these values were not significantly different. The mean number of rounds for the alkaline cleaner and sodium dodecyl sulfate were 9 and 10.33, respectively, and were outside the range of both the linear regression line and evaluation limit. The disinfectants were considered very effective because their LRFs were ≥7.3 log. These estimations were concordant with previous bioassay-based reports. Thus, the evaluation limit seems to be valuable in some applications of multi-round PMCA, such as disinfectant assessment and process validation.     

Disorders in the system of cyclic nucleotides in atherosclerosis: cyclic AMP and cyclic GMP content and activity of related enzymes in human aorta

Cyclic AMP and cyclic GMP content and activities of cyclic nucleotide metabolic enzymes were determined in intima and media of atherosclerotic and unaffected human aorta obtained shortly after death due to myocardial infarction. Cyclic AMP content in fatty streaks and atherosclerotic plaques was lower by three- and five-fold, respectively, as compared with uninvolved intima. Cyclic GMP level in atherosclerotic lesions was estimated to be three-fold higher than in grossly normal area. Basal activity of adenylate cyclase in fatty streaks and plaques was two- to six-fold lower than in unaffected intima. Besides, the ability of adenylate cyclase to be stimulated by the stable analogue of prostacyclin, carbacyclin, was suppressed in plaques. Guanylate cyclase activity in fatty streaks was 1.5- to three-fold higher than in normal tissue. The thiol-reducing agent, dithiothreitol, decreased the enzyme activity to normal level, suggesting the oxidative nature of guanylate cyclase activation in the lesion zone. There were no significant changes in cyclic AMP phosphodiestease activity in the regions of the atherosclerotic lesion. Cyclic GMP phosphodiesterase activity in atherosclerotic plaques was two-fold lower than in the intima of unaffected areas. We did not find differences in the content of cyclic nucleotides or related enzyme activities in the media of uninvolved areas of human aorta nor in the media underlying atherosclerotic lesions. Our findings suggest that development of human atherosclerotic lesions is accompanied by dramatic changes in the cyclic nucleotide metabolism featuring gradual hormonal receptor uncoupling from adenylate cyclase, activation of guanylate cyclase in fatty streaks and inhibition of cyclic GMP phosphodiesterase in plaques.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ligand binding and activation in a prokaryotic cyclic nucleotide-modulated channel

We designed a technique that directly determines binding of cyclic nucleotides to the prokaryotic cyclic nucleotide modulated ion channel MloK1. The ability to purify large quantities of MloK1 facilitated equilibrium binding assays, which avoided the inherent problem of relatively low affinity binding which hindered the use of eukaryotic channels. We found that MloK1 specifically binds cAMP and cGMP with affinity values in the range of those observed for activity assays for eukaryotic channels. Notably, the concentration of ligand that elicited 50% of maximum response in (86)Rb flux assays (K1/2), also referred to as ligand sensitivity, was smaller than the corresponding value obtained from binding assays (Kd) potentially indicating significant channel activity in partially liganded states. To gain further insight into the mechanism of binding and activation of these channels, we mutated several amino acids in the ligand-binding pocket of MloK1, known from electrophysiological studies of homologous eukaryotic channels to affect ligand selectivity and binding efficacy. The S308V MloK1 mutant (a mutation which decreases cGMP selectivity in eukaryotic channels) decreased both the observed cGMP binding affinity and the sensitivity to cGMP relative to the wild-type (WT) channel, leaving those for cAMP unchanged. Conversely, the A352D MloK1 mutant (a mutation which increases cGMP selectivity in eukaryotic channels) increased both the affinity and the sensitivity for cGMP relative to the WT channel, again leaving those for cAMP unchanged. Mutations at R307 in MloK1, the most conserved residue in the binding pocket of cyclic nucleotide-binding proteins, were not tolerated as these mutants do not form functional channels. Furthermore, for each mutation, changes in binding affinities were mirrored by equivalent changes in ligand sensitivity. These data, together with the evidence that partially liganded channels open significantly, suggested strong coupling between cyclic nucleotide binding and MloK1 channel opening.     

苯污染对大鼠脑组织环核苷酸与相关蛋白水平的影响

为了探索苯污染对人类健康损伤作用的因素,选用SPF级Wistar大鼠为实验对象,分为4组:低剂量组灌胃苯0.19 g·kg-1,中剂量组灌胃苯0.38 g·kg-1,高剂量组灌胃苯0.76 g·kg-1,对照组灌胃菜籽油2 mL·kg-1;用酶联免疫吸附法(ELISA)检测脑组织中环核苷酸和相关蛋白的水平.结果发现,连...     

Characterization and age dependence of the stimulatory effect of VIP on cyclic AMP accumulation in rat Leydig cells

The neuropeptide vasoactive intestinal peptide (VIP) has been shown to stimulate cyclic AMP accumulation in Leydig cells isolated from rat testis. The effect was dependent on time, temperature and cell concentration. At 15° half-maximal and maximal stimulation were observed at about 1 and 100 nM VIP, respectively. The interaction was specific since an order of potencies chicken VIP> rat VIP> secretin>glucagon and no effect of neurotensin and substance P were obtained. The efficiency of VIP was lower in pubertal rats and then increased in young-adult and adult animals. These results together with the known presence of VIP in the testis support the idea that VIP may be involved in the regulation and function of Leydig cells during development.