SEARCH · PubMed Health

Results for “AMIDOHYDROLASES”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 55 records · Page 3Linked to original sources

PubMed1995

Two novel classes of neuroactive fatty acid amides are substrates for mouse neuroblastoma 'anandamide amidohydrolase'.

The endogenous cannabimimetic substance, anandamide (N-arachidonoyl-ethanolamine) and the recently isolated sleep-inducing factor, oleoyl-amide (cis-9,10-octadecenoamide), belong to two neuroactive fatty acid amide classes whose action in mammals has been shown to be controlled by enzymatic amide bond hydrolysis. Here we report the partial characterisation and purification of 'anandamide amidohydrolase' from membrane fractions of N18 neuroblastoma cells, and provide evidence for a further and previously unsuspected role of this enzyme. An enzymatic activity catalysing the hydrolysis of [14C]anandamide was found in both microsomal and 10,000 x g pellet fractions. The latter fractions, which displayed the highest Vmax for anandamide, were used for further characterisation of the enzyme, and were found to catalyse the hydrolysis also of [14C]oleoyl-amide, with an apparent Km of 9.0 +/- 2.2 microM. [14C]anandamide- and [14C]oleoyl-amide-hydrolysing activities: (i) exhibited identical pH- and temperature-dependency profiles; (ii) were inhibited by alkylating agents; (iii) were competitively inhibited by the phospholipase A2 inhibitor arachidonyl-trifluoromethyl-ketone with the same IC50 (3 microM); (iv) were competitively inhibited by both anandamide (or other polyunsaturated fatty acid-ethanolamides) and oleoyl-amide. Proteins solubilised from 10,000 x g pellets were directly analysed by isoelectric focusing, yielding purified fractions capable of catalysing the hydrolysis of both [14C]anandamide and [14C]oleoyl-amide. These data suggest that 'anandamide amidohydrolase' enzymes, such as that characterised in this study, may be used by neuronal cells also to hydrolyse the novel sleep-inducing factor oleoyl-amide.

Amides↗

PubMed2004

A stable three-enzyme creatinine biosensor. 3. Immobilization of creatinine amidohydrolase and sensor development.

We describe the development of an extended use amperometric three-enzyme creatinine biosensor and the successful chemical modification and immobilization of the enzyme creatinine amidohydrolase using polyurethane prepolymers. Creatinine amidohydrolase is significantly stabilized by immobilization in polyurethane polymers. The half-life increases from six to more than 80 days in buffer at 37 degrees C. The effect of silver ions leached from amperometric reference electrodes on enzyme and sensor performance is discussed. The use of cellulose acetate cover membranes to prevent silver from reaching the enzyme is investigated. Sensors prepared with cover membranes have half-lives almost an order of magnitude greater than those prepared with no cover membrane over the silver electrode. The complete biosensor has been constructed on a clinical blood analyzer platform and is stable for many days.

Amidohydrolases↗

PubMed2005

Anandamide metabolism by Tetrahymena pyriformis in vitro. Characterization and identification of a 66 kDa fatty acid amidohydrolase.

Fatty acid amidohydrolase, a membrane-bound enzyme found in a variety of mammalian cells, is responsible for the catabolism of neuromodulatory fatty acid amides, including anandamide. In an earlier study we reported that Tetrahymena pyriformis was able to secrete a FAAH-like activity in starvation medium (Karava V., Fasia L., Siafaka-Kapadai A., FEBS Lett. 508 (2001) 327-331). In this study the endocannabinoid anandamide, was found to be metabolized by T. pyriformis homogenate by the action of a FAAH-like enzyme, in a time- and concentration-dependent manner. The main metabolic products of [3H]anandamide hydrolysis were [3H]arachidonic acid and ethanolamine. Amidohydrolase activity was maximal at pH 9-10, it was inhibited by phenylmethylsulfonyl fluoride and arachidonyltrifluoromethyl ketone and was Ca2+ and Mg(2+)-independent. Kinetic experiments demonstrated that the enzyme had an apparent K(m) of 2.5 microM and V(max) of 20.6 nmol/min mg. Subcellular fractionation of T. pyriformis homogenate showed that the activity was present in every subcellular fraction with highest specific activity in the microsomal as well as in non-microsomal membrane fraction. Immunoblot analysis of selected subcellular fractions, using an anti-FAAH polyclonal antibody, revealed the presence of an immunoreactive protein with a molecular mass approximately 66 kDa similar to the molecular mass of the mammalian enzyme. In conclusion, this study demonstrates that a FAAH similar to the mammalian enzyme is present in a unicellular eukaryote, indicating the importance of FAAH activity throughout evolution. It also supports the notion that Tetrahymena species may be a suitable model for metabolic studies on endocannabinoids, as well as for the study of drugs targeted towards FAAH.

Amidohydrolases↗

PubMed2005

Shared promiscuous activities and evolutionary features in various members of the amidohydrolase superfamily.

The amidohydrolase superfamily comprises hundreds of hydrolytic enzymes of the (beta/alpha)8 barrel fold with mono- or binuclear active-site metal centers, and a diverse spectrum of substrates and reactions. Promiscuous activities, or cross-reactivities, between different members of the same superfamily may provide important hints regarding evolutionary and mechanistic relationships. We examined three members: dihydroorotase (DHO), phosphotriesterase (PTE), and PTE-homology protein (PHP). Of particular interest are PTE, which is thought to have evolved within the last several decades, and PHP, an amidohydrolase superfamily member of unknown function, and the closest known homologue of PTE. We found a diverse and partially overlapping pattern of promiscuous activities in these enzymes, including a significant lactonase activity in PTE, esterase activities in both PTE and PHP, and a weak PTE activity in DHO. Directed evolution was applied to improve the promiscuous esterase activities of PTE and PHP. Remarkably, the most recurrent mutation increasing esterase activity in PTE, or PHP, maps to the same location in their superposed 3D structures. The evolved variants also exhibit newly acquired promiscuous activities that were not selected for, including very weak, yet measurable, paraoxonase activity in PHP. Our results illustrate the mechanistic, structural, and evolutionary links between these enzymes, and highlight the importance of studying laboratory evolution intermediates that might resemble node intermediates along the evolutionary pathways leading to the divergence of enzyme superfamilies.

Amidohydrolases↗

PubMed2006

Alpha-amino-beta-carboxymuconic-epsilon-semialdehyde decarboxylase (ACMSD) is a new member of the amidohydrolase superfamily.

The enzymatic activity of Pseudomonas fluorescens alpha-amino-beta-carboxymuconic-epsilon-semialdehyde decarboxylase (ACMSD) is critically dependent on a transition metal ion [Li, T., Walker, A. L., Iwaki, H., Hasegawa, Y., and Liu, A. (2005) J. Am. Chem. Soc. 127, 12282-12290]. Sequence analysis in this study further suggests that ACMSD belongs to the amidohydrolase superfamily, whose structurally characterized members comprise a catalytically essential metal cofactor. To identify ACMSD's metal ligands and assess their functions in catalysis, a site-directed mutagenesis analysis was conducted. Alteration of His-9, His-177, and Asp-294 resulted in a dramatic loss of enzyme activity, substantial reduction of the metal-binding ability, and an altered metallocenter electronic structure. Thus, these residues are confirmed to be the endogenous metal ligands. His-11 is implicated in metal binding because of the strictly conserved HxH motif with His-9. Mutations at the 228 site yielded nearly inactive enzyme variants H228A and H228E. The two His-228 mutant proteins, however, exhibited full metal-binding ability and a metal center similar to that of the wild-type enzyme as shown by EPR spectroscopy. Kinetic analysis on the mutants indicates that His-228 is a critical catalytic residue along with the metal cofactor. Since the identified metal ligands and His-228 are present in all known ACMSD sequences, it is likely that ACMSD proteins from other organisms contain the same cofactor and share similar catalytic mechanisms. ACMSD is therefore the first characterized member in the amidohydrolase superfamily that represents a C-C breaking activity.

Amidohydrolases↗

PubMed2006

Uronate isomerase: a nonhydrolytic member of the amidohydrolase superfamily with an ambivalent requirement for a divalent metal ion.

Uronate isomerase, a member of the amidohydrolase superfamily, catalyzes the isomerization of D-glucuronate and D-fructuronate. During the interconversion of substrate and product the hydrogen at C2 of D-glucuronate is transferred to the pro-R position at C1 of the product, D-fructuronate. The exchange of the transferred hydrogen with solvent deuterium occurs at a rate that is 4 orders of magnitude slower than the interconversion of substrate and product. The enzyme catalyzes the elimination of fluoride from 3-deoxy-3-fluoro-D-glucuronate. These results have been interpreted to suggest a chemical reaction mechanism in which an active site base abstracts the proton from C2 of D-glucuronate to form a cis-enediol intermediate. The conjugate acid then transfers this proton to C1 of the cis-enediol intermediate to form D-fructuronate. The loss of fluoride from 3-deoxy-3-fluoro-D-glucuronate is consistent with a stabilized carbanion at C2 of the substrate during substrate turnover. The slow exchange of the transferred hydrogen with solvent water is consistent with a shielded conjugate acid after abstraction of the proton from either D-glucuronate or D-fructuronate during the isomerization reaction. This conclusion is supported by the competitive inhibition of the enzymatic reaction by D-arabinaric acid and the monohydroxamate derivative with Ki values of 13 and 670 nM, respectively. There is no evidence to support a hydride transfer mechanism for uronate isomerase. The wild type enzyme was found to contain 1 equiv of zinc per subunit. The divalent cation could be removed by dialysis against the metal chelator, dipicolinate. However, the apoenzyme has the same catalytic activity as the Zn-substituted enzyme and thus the divalent metal ion is not required for enzymatic activity. This is the only documented example of a member in the amidohydrolase superfamily that does not require one or two divalent cations for enzymatic activity.

Aldose-Ketose Isomerases↗

PubMed2002

Identification and characterization of a new gene from Variovorax paradoxus Iso1 encoding N-acyl-D-amino acid amidohydrolase responsible for D-amino acid production.

An N-acyl-d-amino acid amidohydrolase (N-D-AAase) was identified in cell extracts of a strain, Iso1, isolated from an environment containing N-acetyl-d-methionine. The bacterium was classified as Variovorax paradoxus by phylogenetic analysis. The gene was cloned and sequenced. The gene consisted of a 1467-bp ORF encoding a polypeptide of 488 amino acids. The V. paradoxusN-D-AAase showed significant amino acid similarity to the N-acyl-d-amino acid amidohydrolases of the two eubacteria Alcaligenes xylosoxydans A-6 (44-56% identity), Alcaligenes facelis DA1 (54% identity) and the hyperthermophilic archaeon Pyrococcus abyssi (42% identity). After over-expression of the N-D-AAase protein in Escherichia coli, the enzyme was purified by multistep chromatography. The native molecular mass was 52.8 kDa, which agreed with the predicted molecular mass of 52 798 Da and the enzyme appeared to be a monomer protein by gel-filtration chromatography. A homogenous protein with a specific activity of 516 U.mg-1 was finally obtained. After peptide sequencing by LC/MS/MS, the results were in agreement with the deduced amino acid sequence of the N-D-AAase. The pI of the enzyme was 5.12 and it had an optimal pH and temperature of 7.5 and 50 degrees C, respectively. After 30 min heat treatment at 45 degrees C, between pH 6 and pH 8, 80% activity remained. The N-D-AAase had higher hydrolysing activity against N-acetyl-d-amino acid derivates containing d-methionine, d-leucine and d-alanine and against N-chloroacetyl-d-phenylalanine. Importantly, the enzyme does not act on the N-acetyl-l-amino acid derivatives. The enzyme was inhibited by chelating agents and certain metal ions, but was activated by 1 mm of Co2+ and Mg2+. Thus, the N-D-AAase from V. paradoxus can be considered a chiral specific and metal-dependent enzyme.

Amidohydrolases↗

PubMed2004

Expression cloning and demonstration of Enterococcus faecalis lipoamidase (pyruvate dehydrogenase inactivase) as a Ser-Ser-Lys triad amidohydrolase.

Enterococcus faecalis lipoamidase was discovered almost 50 years ago (Reed, L. J., Koike, M., Levitch, M. E., and Leach, F. R. (1958) J. Biol. Chem. 232, 143-158) as an enzyme activity that cleaved lipoic acid from small lipoylated molecules and from pyruvate dehydrogenase thereby inactivating the enzyme. Although the partially purified enzyme was a key reagent in proving the crucial role of protein-bound lipoic acid in the reaction mechanism of the 2-oxoacid dehydrogenases, the identity of the lipoamidase protein and the encoding gene remained unknown. We report isolation of the lipoamidase gene by screening an expression library made in an unusual cosmid vector in which the copy number of the vector is readily varied from 1-2 to 40-80 in an appropriate Escherichia coli host. Although designed for manipulation of large genome segments, the vector was also ideally suited to isolation of the gene encoding the extremely toxic lipoamidase. The gene encoding lipoamidase was isolated by screening for expression in E. coli and proved to encode an unexpectedly large protein (80 kDa) that contained the sequence signature of the Ser-Ser-Lys triad amidohydrolase family. The hexa-histidine-tagged protein was expressed in E. coli and purified to near-homogeneity. The purified enzyme was found to cleave both small molecule lipoylated and biotinylated substrates as well as lipoic acid from two 2-oxoacid dehydrogenases and an isolated lipoylated lipoyl domain derived from the pyruvate dehydrogenase E2 subunit. Lipoamidase-mediated inactivation of the 2-oxoacid dehydrogenases was observed both in vivo and in vitro. Mutagenesis studies showed that the residues of the Ser-Ser-Lys triad were required for activity on both small molecule and protein substrates and confirmed that lipoamidase is a member of the Ser-Ser-Lys triad amidohydrolase family.

Amidohydrolases↗

PubMed2006

The crystal structures of dihydropyrimidinases reaffirm the close relationship between cyclic amidohydrolases and explain their substrate specificity.

In eukaryotes, dihydropyrimidinase catalyzes the second step of the reductive pyrimidine degradation, the reversible hydrolytic ring opening of dihydropyrimidines. Here we describe the three-dimensional structures of dihydropyrimidinase from two eukaryotes, the yeast Saccharomyces kluyveri and the slime mold Dictyostelium discoideum, determined and refined to 2.4 and 2.05 angstroms, respectively. Both enzymes have a (beta/alpha)8-barrel structural core embedding the catalytic di-zinc center, which is accompanied by a smaller beta-sandwich domain. Despite loop-forming insertions in the sequence of the yeast enzyme, the overall structures and architectures of the active sites of the dihydropyrimidinases are strikingly similar to each other, as well as to those of hydantoinases, dihydroorotases, and other members of the amidohydrolase superfamily of enzymes. However, formation of the physiologically relevant tetramer shows subtle but nonetheless significant differences. The extension of one of the sheets of the beta-sandwich domain across a subunit-subunit interface in yeast dihydropyrimidinase underlines its closer evolutionary relationship to hydantoinases, whereas the slime mold enzyme shows higher similarity to the noncatalytic collapsin-response mediator proteins involved in neuron development. Catalysis is expected to follow a dihydroorotase-like mechanism but in the opposite direction and with a different substrate. Complexes with dihydrouracil and N-carbamyl-beta-alanine obtained for the yeast dihydropyrimidinase reveal the mode of substrate and product binding and allow conclusions about what determines substrate specificity, stereoselectivity, and the reaction direction among cyclic amidohydrolases.

Amidohydrolases↗

PubMed2003

Inhibition of fatty acid amidohydrolase, the enzyme responsible for the metabolism of the endocannabinoid anandamide, by analogues of arachidonoyl-serotonin.

Arachidonoyl-serotonin inhibits in a mixed-type manner the metabolism of the endocannabinoid anandamide by the enzyme fatty acid amidohydrolase. In the present study, compounds related to arachidonoyl-serotonin have been synthesised and investigated for their ability to inhibit anandamide hydrolysis by this enzyme in rat brain homogenates. Removal of the 5-hydroxy from the serotonin head group of arachidonoyl-serotonin produced a compound (N-arachidonoyltryptamine) that was a 2.3-fold weaker inhibitor of anandamide hydrolysis, but which also produced its inhibition by a mixed-type manner (Ki(slope) 1.3 microM; Ki(intercept) 44 microM). Replacement of the amide linkage in this compound by an ester group further reduced the potency. In contrast, replacement of the arachidonoyl side chain by a linolenoyl side chain did not affect the observed potency. N-(Fur-3-ylmethyl) arachidonamide (UCM707), N-(fur-3-ylmethyl)linolenamide and N-(fur-3-ylmethyl)oleamide inhibited anandamide hydrolysis with pI50 values of 4.53, 5.36 and 5.25, respectively. The linolenamide derivative was also found to be a mixed-type inhibitor. It is concluded that the 5-hydroxy group of arachidonoyl-serotonin contributes to, but is not essential for, inhibitory potency at fatty acid amidohydrolase.

Amidohydrolases↗

PubMed1975

Characterizations of acylagmatine amidohydrolase and carboxypeptidase from Fusarium anguioides.

Previously an enzyme, named acylagmatine amidohydrolase, hydrolyzing bleomycin B2 to bleomycinic acid and agmatine was found in the mycelia of Fusarium anguioides Sherbakoff. In this work the enzyme was purified further, but not completely. The crude enzyme preparation hydrolyzed various acylagmatines and also peptidyl arginine, but the latter activity could be separated from acylagmatine amidohydrolase activity by gel filtration on Sephadex G-100. The enzyme was inhibited by PCMB and its molecular weight was estimated as 65,000 by gel filtration. It showed substrate specificity with respect to the alkyl-chain length of the amine moiety. The other hydrolase fraction with activity toward Bz-Gly-Arg was found to be of a sort of carboxypeptidase, which preferentially hydrolyzed peptides with arginine or lysine at the carboxyl terminus, including bradykinin, but liberated neutral amino acids as well from the terminus when the penultimate residue of the substrates was phenylalanine. With Bz-Gly-Arg as substrate Fusarium carboxypeptidase was sensitive to chelating agents but not to diisopropyfluorophosphate, and its molecular weight was estimated to be 145,000.

Agmatine↗

PubMed2002

N-acylethanolamines are metabolized by lipoxygenase and amidohydrolase in competing pathways during cottonseed imbibition.

Saturated and unsaturated N-acylethanolamines (NAEs) occur in desiccated seeds primarily as 16C and 18C species with N-palmitoylethanolamine and N-linoleoylethanolamine (NAE 18:2) being most abundant. Here, we examined the metabolic fate of NAEs in vitro and in vivo in imbibed cotton (Gossypium hirsutum) seeds. When synthetic [1-(14)C]N-palmitoylethanolamine was used as a substrate, free fatty acids (FFA) were produced by extracts of imbibed cottonseeds. When synthetic [1-(14)C]NAE 18:2 was used as a substrate, FFA and an additional lipid product(s) were formed. On the basis of polarity, we presumed that the unidentified lipid was a product of the lipoxygenase (LOX) pathway and that inclusion of the characteristic LOX inhibitors nordihydroguaiaretic acid and eicosatetraynoic acid reduced its formation in vitro and in vivo. The conversion of NAE 18:2 in imbibed cottonseed extracts to 12-oxo-13-hydroxy-N-(9Z)-octadecanoylethanolamine was confirmed by gas chromatography-mass spectrometry, indicating the presence of 13-LOX and 13-allene oxide synthase, which metabolized NAE 18:2. Cell fractionation studies showed that the NAE amidohydrolase, responsible for FFA production, was associated mostly with microsomes, whereas LOX, responsible for NAE 18:2-oxylipin production, was distributed in cytosol-enriched fractions and microsomes. The highest activity toward NAE by amidohydrolase was observed 4 to 8 h after imbibition and by LOX 8 h after imbibition. Our results collectively indicate that two pathways exist for NAE metabolism during seed imbibition: one to hydrolyze NAEs in a manner similar to the inactivation of endocannabinoid mediators in animal systems and the other to form novel NAE-derived oxylipins. The rapid depletion of NAEs by these pathways continues to point to a role for NAE metabolites in seed germination.

5,8,11,14-Eicosatetraynoic Acid↗

PubMed1989

Penicillin amidohydrolases in fungal autolysis.

The production of penicillin G and penicillin V amidohydrolases or acylases (E.C.3.5.1.11) was studied during the autolysis of filamentous fungi in a mineral medium, and in the same medium with phenoxyacetic acid as inducer. In all the studied fungi, enzymes showing penicillin G and penicillin V amidohydrolase activities were found. Generally, an increase of these activities during fungal autolysis was observed. The presence of phenoxyacetic acid in the medium did not increase these activities. The activities found in the culture fluids were generally higher than that found in the mycelial extracts. Under these conditions, beta-lactamases (penicillinases) were not found. The fungi Alternaria alternata, Fusarium culmorum, Penicillium oxalicum, and the species Penicillium 222 were chosen to study penicillin G and penicillin V acylases. The enzymes were precipitated with tannic acid from the culture fluid of their autolyzed cultures. Some kinetic constants of these activities were determined.

Amidohydrolases↗

PubMed1989

Purification and properties of 5,6-dihydropyrimidine amidohydrolase from calf liver.

5,6-Dihydropyrimidine amidohydrolase was isolated from an acetone powder of calf liver and purified to homogeneity. Purification made use of heat treatment, ammonium sulfate fractionation and chromatography on Chelating Sepharose and DEAE-Sepharose with 44% recovery of total activity. The native enzyme has a molecular mass of 217 kDa consisting of four subunits with a molecular mass of 54 kDa each. The amidohydrolase is a metalloenzyme containing one zinc atom/subunit. The enzyme can slowly be inactivated by chelating agents. The kinetic parameters for substrates, 5,6-dihydrouracil, 5,6-dihydrothymine and glutarimide were determined. From log Vmax/KM data, a pKa of 7.6 could be calculated suggesting the formation of a zinc-bound hydroxyl ion which carries out the nucleophilic attack on the C-4 of dihydrouracil.

Amidohydrolases↗

PubMed1993

N-carbamoyl-D-amino acid amidohydrolase from Comamonas sp. E222c purification and characterization.

N-Carbamoyl-D-amino acid amidohydrolase was purified 119-fold, with 36% overall recovery from a cell-free extract of Comamonas sp. E222c. The purified enzyme was homogeneous as judged by SDS/PAGE. The relative molecular mass of the native enzyme was 120,000 and that of the subunit was 40,000. The purified enzyme hydrolyzed various N-carbamoyl-D-amino acids to D-amino acids, ammonia and carbon dioxide. N-Carbamoyl-D-amino acids having hydrophobic groups served as good substrates for the enzyme. The Km and Vmax values for N-carbamoyl-D-phenylalanine were 19.7 mM and 13.1 units/mg, respectively, and those for N-carbamoyl-D-p-hydroxyphenylglycine were 13.1 mM and 0.56 units/mg, respectively. The enzyme strictly recognized the configuration of the substrate and only the D-enantiomer of the N-carbamoyl amino acid was hydrolyzed. The enzyme activity was not significantly affected by N-carbamoyl-L-amino acids and ammonia. The enzyme was sensitive to thiol reagents and did not require metal ions for its activity. The enzyme did not hydrolyze N-carbamoyl-beta-alanine or N-carbamoyl-DL-aspartate suggesting that the enzyme is different from the N-carbamoylamide-hydrolyzing enzymes involved in the pyrimidine degradation pathway. The enzyme did not hydrolyze allantoin and allantoic acid, which are intermediates in purine degradation, N-carbamoylsarcosine and citrulline, suggesting that it is a novel N-carbamoylamide amidohydrolase.

Amidohydrolases↗

PubMed1993

Purification and biochemical characterization of the hydantoin hydrolyzing enzyme from Agrobacterium species. A hydantoinase with no 5,6-dihydropyrimidine amidohydrolase activity.

A soluble hydantoinase (5,6-dihydropyrimidine amidohydrolase) was purified to homogeneity from a newly isolated Agrobacterium species. This hydrolase consists of about 578 aminoacyl residues and is a slightly acidic protein with an isoelectric point of 6.5. The first 22 N-terminal amino acid residues were determined by Edman degradation. Determination of the relative molecular mass of the protein by gel-filtration chromatography gave an apparent value of 250,000. The subunit M(r) was 62,000, as estimated by analytical SDS/PAGE and 66,500, as estimated by denaturing gel-filtration chromatography. The pure hydantoinase exhibits the following hydrodynamic properties: a sedimentation coefficient of 8.8 S as determined by sedimentation velocity experiments; a Stokes radius of 6.8 nm; a diffusion coefficient of 31.5 microns2.s-1 as determined by analytical gel-filtration chromatography. From these experimental data, the following physical constants could be calculated: a theoretical M(r) of 265,000, a frictional ratio, f/fo, of 1.59, a maximal axial ratio, a/b, of 3.1; a Perrin shape factor, F, of 1.37. As shown by different Km values, the preferred substrates of this hydrolase were 5-monosubstituted hydantoins bearing aromatic substituents. 5,5-Dimethylhydantoin and different thio analogs of the 5-p-hydroxyphenylhydantoin molecule are competitive inhibitors of this hydrolase. The classification of this microbial hydantoinase, which exhibits no hydrolytic activity with all the dihydropyrimidines tested, under the systematic name of 5,6-dihydropyrimidine amidohydrolase, and its putative metabolic role are further discussed.

Amidohydrolases↗

PubMed1993

The isopenicillin-N acyltransferase of Penicillium chrysogenum has isopenicillin-N amidohydrolase, 6-aminopenicillanic acid acyltransferase and penicillin amidase activities, all of which are encoded by the single penDE gene.

The isopenicillin-N acyltransferase of Penicillium chrysogenum catalyzes the conversion of the biosynthetic intermediate isopenicillin N to the hydrophobic penicillins. The isopenicillin-N acyltransferase copurified with the acyl-CoA:6-aminopenicillanic acid (6-APA) acyltransferase activity which transfers an acyl residue from acyl-CoA derivatives (e.g. phenylacetyl-CoA, phenoxyacetyl-CoA) to 6-APA. Other thioesters of phenylacetic acid were also used as substrates. An amino acid sequence similar to that of the active site of thioesterases was found in the isopenicillin-N acyltransferase, suggesting that this site is involved in the transfer of phenylacetyl residues from phenylacetyl thioesters. Purified isopenicillin-N acyltransferase also showed isopenicillin-N amidohydrolase, penicillin transacylase and penicillin amidase activities. The isopenicillin-N amidohydrolase (releasing 6-APA) showed a much lower specific activity than the isopenicillin-N acyltransferase of the same enzyme preparation, suggesting that in the isopenicillin-N acyltransferase reaction the 6-APA is not released and is directly converted into benzylpenicillin. Penicillin transacylase exchanged side chains between two hydrophobic penicillin molecules; or between one penicillin molecule and 6-APA. The penicillin amidase activity is probably the reverse of the biosynthetic acyl-CoA:6-APA acyltransferase. Four P. chrysogenum mutants deficient in acyl-CoA:6-APA acyltransferase lacked the other four related activities. Transformation of these mutants with the penDE gene restored all five enzyme activities.

Acyltransferases↗

PubMed2006

TrzN from Arthrobacter aurescens TC1 Is a zinc amidohydrolase.

TrzN, the broad-specificity triazine hydrolase from Arthrobacter and Nocardioides spp., is reportedly in the amidohydrolase superfamily of metalloenzymes, but previous studies suggested that a metal was not required for activity. To help resolve that conundrum, a double chaperone expression system was used to produce multimilligram quantities of functionally folded, recombinant TrzN. The TrzN obtained from Escherichia coli (trzN) cells cultured with increasing zinc in the growth medium showed corresponding increases in specific activity, and enzyme obtained from cells grown with 500 muM zinc showed maximum activity. Recombinant TrzN contained 1 mole of Zn per mole of TrzN subunit. Maximally active TrzN was not affected by supplementation with most metals nor by EDTA, consistent with previous observations (E. Topp, W. M. Mulbry, H. Zhu, S. M. Nour, and D. Cuppels, Appl. Environ. Microbiol. 66:3134-3141, 2000) which had led to the conclusion that TrzN is not a metalloenzyme. Fully active native TrzN showed a loss of greater than 90% of enzyme activity and bound zinc when treated with the metal chelator 8-hydroxyquinoline-5-sulfonic acid. While exogenously added zinc or cobalt restored activity to metal-depleted TrzN, cobalt supported lower activity than did zinc. Iron, manganese, nickel, and copper did not support TrzN activity. Both Zn- and Co-TrzN showed different relative activities with different s-triazine substrates. Co-TrzN showed a visible absorption spectrum characteristic of other members of the amidohydrolase superfamily replaced with cobalt.

Amidohydrolases↗