Cyclic-imide-hydrolyzing activity of D-hydantoinase from Blastobacter sp. strain A17p-4.

The cyclic-imide-hydrolyzing activity of a prokaryotic cyclic-ureide-hydrolyzing enzyme, D-hydantoinase, was investigated. The enzyme hydrolyzed cyclic imides with bulky substituents such as 2-methylsuccinimide, 2-phenylsuccinimide, phthalimide, and 3,4-pyridine dicarboximide to the corresponding half-amides. However, simple cyclic imides without substituents, which are substrates of imidase (ie.g., succinimide, glutarimide, and sulfur-containing cyclic imides such as 2,4-thiazolidinedione and rhodanine), were not hydrolyzed. The combined catalytic actions of bacterial D-hydantoinase and imidase can cover the function of a single mammalian enzyme, dihydropyrimidinase. Prokaryotic D-hydantoinase also catalyzed the dehyrative cyclization of the half-amide phthalamidic acid to the corresponding cyclic imide, phthalimide. The reversible hydrolysis of cyclic imides shown by prokaryotic D-hydantoinase suggested that, in addition to pyrimidine metabolism, it may also function in cyclic-imide metabolism.     

Cyclic ureide and imide metabolism in microorganisms producing a -hydantoinase useful for -amino acid production

The microbial transformation of -5-monosubstituted hydantoins has been applied to industrial scale production of optically active amino acids. Hydantoinase and N-carbamoyl amino acid amidohydrolase, which are the key enzymes in this transformation, from various microorganisms have been studied extensively. Blastobacter sp. A17p-4, which was isolated for -amino acid production through hydantoin transformation, shows not only diverse cyclic ureide-metabolizing activities including those of -hydantoinase and N-carbamoyl- -amino acid amidohydrolase, but also cyclic imide-metabolizing activities. A recent study revealed the participation of -hydantoinase in the metabolism of cyclic imides and the existence of novel enzymes, imidase and half-amidase, in this bacterium. -hydantoinase functions in the metabolism of bulky cyclic imides, while imidase functions in that of simple cyclic imides in combination with half-amidase, which functions in the hydrolysis of the imidase reaction products, half-amides. Imidase and half-amidase are different from reported cyclic-amide-metabolizing enzymes, and are widely found in bacteria, yeasts and molds.     

Gene Cloning,Expression, and Substrate Specificity of an Imidase from the Strain <Emphasis Type="Italic">Pseudomonas putida</Emphasis> YZ-26

A gene-encoding imidase was isolated from Pseudomonas putdia YZ-26 genomic DNA using a combination of polymerase chain reaction and activity screening the recombinant. Analysis of the nucleotide sequence revealed that an open reading frame (ORF) of 879 bp encoded a protein of 293 amino acids with a calculated molecular weight of 33712.6 kDa. The deduced amino-acid sequence showed 78% identity with the imidase from Alcaligenes eutrophus 112R4 and 80% identity with N-terminal 20 amino-acid imidase from Blastobacter sp. A17p-4. Next, the ORF was subcloned into vector pET32a to form recombinant plasmid pEI. The enzyme was overexpressed in Escherichia coli and purified to homogeneity by Ni²⁺–NTA column, with 75% activity recovery. The subunit molecular mass of the recombinant imidase as estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis was approximately 36 kDa, whereas its functional unit was approximately 141 kDa with four identical subunits determined by size-exclusion chromatography. The purified enzyme showed the highest activity and affinity toward succinimide, and some other substrates, such as dihydrouracil, hydantoin, succinimide, and maleimde, were investigated.     

真养产碱杆菌112R4酰亚胺酶基因的克隆、序列分析及其在大肠杆菌中的表达

筛选到一株具海因水解活力的微生物,经鉴定后命名为真养产碱杆菌112R4。该菌能水解海因、二氢尿嘧啶和琥珀酰亚胺,且对琥珀酰亚胺活力最高,但不水解5单取代海因和5,5’双取代海因,因而被确定为含有酰亚胺酶。真养产碱杆菌112R4能在以琥珀酰亚胺为唯一碳、氮源的培养基上生长,表明该菌中存在琥珀酰亚胺完整的转化途径。从112R4基因组DNA出发,用鸟枪法克隆了一个6kb的与环酰亚胺水解相关的DNA片段;进一步亚克隆得到了带酰亚胺酶基因的2kb的DNA片段,并进行了序列测定。缺失分析确定了一个876bp的ORF为真养产碱杆菌112R4的酰亚胺酶基因,推测编码一个291个氨基酸的多肽,这是第一次报道微生物酰亚胺酶的核酸和蛋白序列。推测的氨基酸序列在蛋白数据库中进行了比较,结果表明,酰亚胺酶与已知的环酰胺酶没有明显的同源性,也不属于氨酰水解酶蛋白超家族,因而被分类为一种新的环酰胺酶。真养产碱杆菌112R4的酰亚胺酶与芽生杆菌A17p4的酰亚胺酶N端的20个氨基酸有较高的同源性,一致性为60%,与多糖脱乙酰酶保守序列也部分同源,一致性为14%。带有酰亚胺酶基因的重组质粒在大肠杆菌中得到表达,在lac启动子控制下,使用1mmol/L IPTG诱导5h,酰亚胺酶活力达到3200U/L,为供体菌真养产碱杆菌112R４的7倍。     

Identification, purification, and characterization of a thermophilic imidase from pig liver

This study investigates thermophilic imidase activity of the liver. We demonstrate that imidase catalyzes the hydrolysis of imides at a temperature substantially higher than that of its native environment. Then, a thermophilic imidase is purified to homogeneity from pig liver, and its thermoproperties are studied. About 2500-fold of purification and 15% yield of imidase activity are obtained after ammonium sulfate precipitation, octyl, DEAE, chelation, and gel filtration chromatography. While avoiding heat treatment for the protein purification, this study also indicates that only one enzyme is responsible for the imidase activity. This homogenous enzyme prefers to catalyze hydrolysis of imides at above 60 degrees C rather than at the body temperature of a pig. Although stable at below 50 degrees C, imidase quickly loses its activity at above 65 degrees C. Thus, the temperature effect on imidase activity is limited mainly by its thermostability. Substrate specificity of imidase is also temperature dependent. Our results demonstrate that the hydrolysis of physiological substrates is the most temperature dependent and that of hydantoins is the least temperature dependent. When increasing the reaction temperature from 25 to 60 degrees C, specific activities increase 50- and 60-fold for dihydrouracil and dihydrothymine, respectively. The temperature effect on the K(m) and V(max) of imidase is substrate dependent.     

真养产碱杆菌112R4的二羧酸单酰胺酰胺水解酶

在一株具有环酰亚胺转化活性的真养产碱杆菌112R4中发现了一种特异性的二羧酸单酰胺酰胺水解酶（半酰胺酶）,它催化环酰亚胺代谢的第二步反应,将二羧酸单酰胺水解为二羧酸和氨。该酶的底物仅限于此代谢途径的第一个酶——酰亚胺酶的产物二羧酸单酰胺,而对其它的酰胺类化合物没有明显水解活性。真养产碱杆菌112R4中的半酰胺酶和酰亚胺酶在表达上具有相关性,环酰亚胺（如琥珀酰亚胺）和二羧酸单酰胺（如琥珀酰胺酸）对它们有正调控作用,游离氨离子显示出负调控作用,琥珀酸则在酶合成和活性两方面均表现出影响作用。对重组大肠杆菌中表达的半酰胺酶粗酶的部分性质进行了研究。钴离子对半酰胺酶的活性表现出促进作用,比活力提高到3.37倍,表明半酰胺酶可能是一种金属结合酶。     

A comparison of amide and ureide nitrogen sources in tissue culture of tree legume Prosopis alba clone B2 V50

The effects of medium nitrogen sources on the recalcitrant nature of Prosopis alba clone B2V50 in tissue culture were compared involving shoot development using axillary bud explants from 2 to 4-year-old greenhouse-grown trees. A significant difference (P<0.05) was found between the amino acids aspartic acid and glutamic acid and their corresponding amide-containing compounds asparagine and glutamine. A comparison between amide and ureide nitrogen sources showed that allantoin, a ureide, was an acceptable replacement for asparagine or glutamine. Allantoin, asparagine, and glutamine could be used as the sole nitrogen sources. Allantoin at a concentration of 20 mM was adopted for use in future research. Although shoots were consistently induced, all explants showed complete shoot-tip necrosis after 12 weeks of in vitro culture.     

The role of metal on imide hydrolysis: metal content and pH profiles of metal ion-replaced mammalian imidase

Imidase catalyzes the hydrolysis of a variety of imides. The removal of metal from imidase eliminates its activity but does not affect its tetrameric and secondary structure. The reactivation of the apoenzyme with transition metal ions Co(2+), Zn(2+), Mn(2+), and Cd(2+) shows that imidase activity is linearly dependent on the amount of metal ions added. Ni(2+) and Cu(2+) are also inserted, one per enzyme subunit, into the apoimidase, but do not restore imidase activity. Enzyme activity with different metal replaced imidase varies significantly. However, the changes of the metal contents do not appear to affect the pK(a)s obtained from the bell-shaped pH profiles of metal reconstituted imidase. The metal-hydroxide mechanism for imidase action is not supported based on the novel findings from this study. It is proposed that metal ion in mammalian imidase functions as a Lewis acid, which stabilizes the developing negative charge of imide substrate in transition state.     

Novel Metabolic Transformation Pathway for Cyclic Imides in Blastobacter sp. Strain A17p-4

The metabolic transformation pathway for cyclic imides in microorganisms was studied in Blastobacter sp. strain A17p-4. This novel pathway involves, in turn, hydrolytic ring opening of a cyclic imide to yield a monoamidated dicarboxylate, hydrolytic deamidation of the monoamidated dicarboxylate to yield a dicarboxylate, and dicarboxylate transformation similar to that in the tricarboxylic acid cycle. The initial step is catalyzed by a novel enzyme, imidase. Imidase and subsequent enzymes involved in this metabolic pathway are induced by some cyclic imides, such as succinimide and glutarimide. Induced cells metabolize various cyclic imides.     

Characterization of zinc-binding properties of a novel imidase from Pseudomonas putida YZ-26

The imidase from Pseudomonas putida YZ-26 consisting of 293-amino acid residues is a novel imidase with four subunits as the holo-enzyme and low molecular weight which is significantly different from known mammalian imidase. This study measured the zinc-binding properties of the imidase using inductively coupled plasma-atomic emission spectrometry and competition assay combined with activity determinations. Results show that each subunit of the imidase binds the zinc ion by 1:1 stoichiometry with apparent binding constant of 9.5 × 10⁸ M⁻¹. The activity of the apo-imidase (20 μM) was recovered with the addition of zinc in the lower concentration (0-20 μM), whereas the enzymatic activity is decreased in the presence of high concentration of zinc (above 100 μM). The site-directed mutagenesis of His₂₄₇, His₈₆ or Cys₇, Cys₁₀₈ in imidase resulted in loss of activity and zinc-binding abilities at different degrees, showing that these residues may critically affect both enzymatic activity and conformation.     

Inhibition of a Putative Dihydropyrimidinase from Pseudomonas aeruginosa PAO1 by Flavonoids and Substrates of Cyclic Amidohydrolases

Dihydropyrimidinase is a member of the cyclic amidohydrolase family, which also includes allantoinase, dihydroorotase, hydantoinase, and imidase. These metalloenzymes possess very similar active sites and may use a similar mechanism for catalysis. However, whether the substrates and inhibitors of other cyclic amidohydrolases can inhibit dihydropyrimidinase remains unclear. This study investigated the inhibition of dihydropyrimidinase by flavonoids and substrates of other cyclic amidohydrolases. Allantoin, dihydroorotate, 5-hydantoin acetic acid, acetohydroxamate, orotic acid, and 3-amino-1,2,4-triazole could slightly inhibit dihydropyrimidinase, and the IC₅₀ values of these compounds were within the millimolar range. The inhibition of dihydropyrimidinase by flavonoids, such as myricetin, quercetin, kaempferol, galangin, dihydromyricetin, and myricitrin, was also investigated. Some of these compounds are known as inhibitors of allantoinase and dihydroorotase. Although the inhibitory effects of these flavonoids on dihydropyrimidinase were substrate-dependent, dihydromyricetin significantly inhibited dihydropyrimidinase with IC₅₀ values of 48 and 40 μM for the substrates dihydrouracil and 5-propyl-hydantoin, respectively. The results from the Lineweaver−Burk plot indicated that dihydromyricetin was a competitive inhibitor. Results from fluorescence quenching analysis indicated that dihydromyricetin could form a stable complex with dihydropyrimidinase with the K_d value of 22.6 μM. A structural study using PatchDock showed that dihydromyricetin was docked in the active site pocket of dihydropyrimidinase, which was consistent with the findings from kinetic and fluorescence studies. This study was the first to demonstrate that naturally occurring product dihydromyricetin inhibited dihydropyrimidinase, even more than the substrate analogs (>3 orders of magnitude). These flavonols, particularly myricetin, may serve as drug leads and dirty drugs (for multiple targets) for designing compounds that target several cyclic amidohydrolases.     

Effects of Saccharomyces cerevisiae supplementation on immune response,hematological parameters,body composition and disease resistance in rainbow trout,Oncorhynchus mykiss (Walbaum, 1792)

Natural substances are now generally preferred over chemical and synthetic compounds for the growth and immune enhancement of aquatic organisms. The aim of this study was to evaluate the effect of Saccharomyces cerevisiae extract and hydrolyzed powder on immunity, hematological parameters and body composition in rainbow trout, Oncorhynchus mykiss. Six hundred rainbow trout (50 ± 5 g mean weight) were acclimated to laboratory conditions and then randomly divided into four groups of triplicate tanks. The first group was fed with a commercial diet (control) without supplementation. The second and third groups were given a diet supplemented with 1% of yeast extract and hydrolyzed powder, respectively. The fourth group was also fed with a basal diet supplemented with 0.5% of both substances. Fish were cultured in 300‐L polyethylene tanks for 60 days; immune and hematological parameters, fillet composition and disease resistance were analyzed at days 0, 30 and 60. Results showed that a combination of Saccharomyces cerevisiae extract and hydrolyzed powder could improve the immunity and alter hematological parameters of the rainbow trout compared to the control. Mortality rates of fish fed yeast extract and hydrolyzed powder were also lower than in fish fed the control diet after challenging with Yersinia ruckeri. There were no significant changes in rainbow trout fillet composition compared to the control. It can be concluded that fish diet supplementation with a mixture of yeast extract hydrolyzed powder is preferable compared to each one used alone.     

Analogs of cyclic AMP and cyclic GMP: general methods of synthesis and the relationship of structure to enzymic activity

The syntheses of a large variety of analogs of adenosine cyclic 3′, 5′-phosphate and guanosine cyclic 3′, 5′-phosphate are reviewed. The ability of these compounds to stimulate cyclic nucleotide-dependent protein kinases and to be hydrolyzed by cyclic nucleotide phosphodiesterases is discussed with an emphasis on the structure-activity relationships.     

A novel cold-adapted imidase from fish Oreochromis niloticus that catalyzes hydrolysis of maleimide

In this paper we report the first comparative study of cold-adapted imidase (EC 3.5.2.2) from the fish (Oreochromis niloticus) liver and its thermophilic counterparts taken from pig liver and Escherichia coli (overexpressed recombinant hydantoinase from Agrobacterium radiobacter NRRL B1). Approximately 6000-fold purification and a 40% yield of fish imidase activity were obtained through ammonium sulfate precipitation, octyl, chelating, DEAE, and hydroxyapatite chromatography. This cold-adapted imidase was characterized by a specific activity 10- to a 100-fold higher than those of its thermophilic counterparts below room temperature (25 degrees C or lower) conditions but less stable at elevated temperatures (40 degrees C or higher). A less organized helical structure (compared to those of pig liver and bacterial imidases) was observed by circular dichroism. Furthermore, maleimide was first identified as a novel substrate of all imidases examined, and confirmed by HPLC and NMR analysis. These results constituted a first study to discover a novel cold-adapted imidase with surprising high activity. These findings might be also helpful for industrial application of imidase.     

Cyclic 3′,5′-AMP phosphodiesterase in Physarum polycephalum: II. Kinetic properties

The effect of several inhibitors of the enzyme cyclic 3′,5′-AMP phosphodiesterase as chemoattractants in Physarum polycephalum was examined. Of the compounds tested, 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Roche 20-1724/001) and 1-ethyl-4-(isopropylidinehydrazino)-1H-pyrazolo-(3,4-b)-pyridine-5-carboxylic acid ethyl ester, hydrochloride (Squibb 20009) were the most potent attractants. 3-Isobutyl-1-methyl xanthine, theophylline, and morin (a flavanoid) were moderate attractants and sometimes gave negative chemotaxis at high concentrations. Cyclic 3′,5′-AMP was an effective, but not potent attractant. A repellent effect following the positive chemotactic action was sometimes observed with cyclic 3′,5′-AMP at concentrations as high as 1 · 10⁻² M. Dibutyryl cyclic AMP appeared to be a somewhat more potent attractant than cyclic 3′,5′-AMP. The 8-thiomethyl and 8-bromoderivatives of cyclic AMP, which are poorly hydrolyzed by the phosphodiesterase, were not attractants in Physarum. Possible participation of cyclic 3′,5′-AMP in the directional movement in P. polycephalum is discussed.     

Distribution of cyclic imide-transforming activity in microorganisms

Cyclic imide-transforming activity was found to be widely distributed in bacteria, yeast and molds. This activity was not correlated with cyclic ureide-transforming activity in bacteria, but there was some correlation in yeast and molds. These two activities are probably catalyzed by different enzymes in bacteria. Besides the well-known cyclic ureide transformation, cyclic imide transformation by microorganisms was common.     

Acetonitrile-based solvents: Their application in thin-layer chromatography of cyclic nucleotides, nucleosides, purine, and pyrimidines

Acetonitrile-based solvent mixtures have been applied to the separation of nucleotides, nucleosides, purines, pyrimidines, and cyclic nucleotides by thin-layer chromatography. The R_f's of over 35 compounds are presented. Development times with some of the systems were as low as 16 min. The use of acetonitrile-containing solvents in adenyl cyclase and cyclic phosphodiesterase assays and in the nucleotides and nucleic acid fields is discussed.     

Relationship between Ureide N and N(2) Fixation, Aboveground N Accumulation, Acetylene Reduction, and Nodule Mass in Greenhouse and Field Studies with Glycine max L. (Merr)

The relationship between ureide N and N₂ fixation was evaluated in greenhouse-grown soybean (Glycine max L. Merr.) and lima bean (Phaseolus lunatus L.) and in field studies with soybean. In the greenhouse, plant N accumulation from N₂ fixation in soybean and lima bean correlated with ureide N. In soybean, N₂ fixation, ureide N, acetylene reduction, and nodule mass were correlated when N₂ fixation was inhibited by applying KNO₃ solutions to the plants. The ureide-N concentrations of different plant tissues and of total plant ureide N varied according to the effectiveness of the strain of Bradyrhizobium japonicum used to inoculate plants. The ureide-N concentrations in the different plant tissues correlated with N₂ fixation. Ureide N determinations in field studies with soybean correlated with N₂ fixation, aboveground N accumulation, nodule weight, and acetylene reduction. N₂ fixation was estimated by ¹⁵N isotope dilution with nine and ten soybean genotypes in 1979 and 1980, respectively, at the V9, R2, and R5 growth stages. In 1981, we investigated the relationship between ureide N, aboveground N accumulation, acetylene reduction, and nodule mass using four soybean genotypes harvested at the V4, V6, R2, R4, R5, and R6 growth stages. Ureide N concentrations of young stem tissues or plants or aboveground ureide N content of the four soybean genotypes varied throughout growth correlating with acetylene reduction, nodule mass, and aboveground N accumulation. The ureide-N concentrations of young stem tissues or plants or aboveground ureide-N content in three soybean genotypes varied across inoculation treatments of 14 and 13 strains of Bradyrhizobium japonicum in 1981 and 1982, respectively, and correlated with nodule mass and acetylene reduction. In the greenhouse, results correlating nodule mass with N₂ fixation and ureide N across strains were variable. Acetylene reduction in soybean across host-strain combinations did not correlate with N₂ fixation and ureide N. N₂ fixation, ureide N, acetylene reduction, and nodule mass correlated across inoculation treatments with strains of Bradyrhizobium spp. varying in effectiveness on lima beans. Our data indicate that ureide-N determinations may be used as an additional method to acetylene reduction in studies of the physiology of N₂ fixation in soybean. Ureide-N measurements also may be useful to rank strains of B. japonicum for effectiveness of N₂ fixation.     

Calcium-dependent cyclic nucleotide phosphodiesterase from brain: comparison of adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate as substrates.

A Ca²⁺-dependent cyclic nucleotide phosphodiesterase has been partially purified from extracts of porcine brain by column chromatography on Sepharose 6 B containing covalently linked protamine residues, ammonium sulfate salt fractionation, and ECTEOLA-cellulose column chromatography. The resultant preparation contained a single form of cyclic nucleotide phosphodiesterase activity by the criteria of isoelectric focusing, gel filtration chromatography on Sephadex G-200, and electrophoretic migration on polyacrylamide gels. When fully activated by the addition of Ca²⁺ and microgram quantities of a purified Ca²⁺-binding protein (CDR), the phosphodiesterase hydrolyzed both adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP), with apparent K_m values of 180 and 8 μm, respectively. Approximately 15% of the total enzymic activity was present in the absence of added CDR and Ca²⁺. This activity exhibited apparent K_m values for the two nucleotides identical to those observed for the maximally activated enzyme. Competitive substrate kinetics and heat destabilization studies demonstrated that both cyclic nucleotides were hydrolyzed by the same phosphodiesterase. The purified enzyme was identical to a Ca²⁺-dependent phosphodiesterase present in crude extract by the criteria of gel filtration chromatography, polyacrylamide-gel electrophoresis, and kinetic behavior.Apparent K_m values of the Ca²⁺-dependent phosphodiesterase for cyclic AMP and cyclic GMP were lowered more than 20-fold as CDR quantities in the assay were increased to microgram amounts, whereas the respective maximal velocities remained constant. The apparent K_m for Mg²⁺ was lowered more than 50-fold as CDR was increased to microgram amounts. Half-maximal activation of the phosphodiesterase occurred with lower amounts of CDR as a function of either increasing degrees of substrate saturation or increasing concentrations of Mg²⁺. At low cyclic nucleotide substrate concentrations i.e., 2.5 μm, cyclic GMP was hydrolyzed at a fourfold greater velocity than cyclic AMP. At high substrate concentrations (millimolar range) cyclic AMP was hydrolyzed at a threefold greater rate than cyclic GMP.