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PubMed1997

Arachidonylethanolamide (anandamide) binds with low affinity to dihydropyridine binding sites in brain membranes.

The purpose of this study was to explore the hypothesis that the dihydropyridine (DHP) binding site of the L-type calcium channel is a high affinity binding site for the cannabimimetic arachidonylethanolamide (AEA). Binding affinities were determined from competition isotherms using the DHP analog [3H]PN-200. AEA competed for [3H]PN-200 binding with a K(I) of 40 +/- 4 microM. Inclusion of phenylmethylsulfonyl fluoride to inhibit an amidohydrolase that converts AEA to arachidonic acid had little effect on the K(I) of AEA (48 +/- 6 microM). Arachidonic acid had a slightly higher K(I) (120 +/- 11 microM) and other N-acylethanolamides examined (linolenylethanolamide, dihomo-gamma-linolenylethanolamide, docosatetraenylethanolamide, and palmitoylethanolamide) had no effect on [3H]PN-200 binding at concentrations as high as 10 microM. Our conclusions are that AEA binds to the DHP binding site with relatively low affinity and its conversion to arachidonic acid is not required for binding.

Amides↗

PubMed2000

Fatty acid amide hydrolase substrate specificity.

Fatty acid amide hydrolase (FAAH), also referred to as oleamide hydrolase and anandamide amidohydrolase, is a serine hydrolase responsible for the degradation of endogenous oleamide and anandamide, fatty acid amides that function as chemical messengers. FAAH hydrolyzes a range of fatty acid amides, and the present study examines the relative rates of hydrolysis of a variety of natural and unnatural fatty acid primary amide substrates using pure recombinant rat FAAH.

Amidohydrolases↗

PubMed1999

Trifluoromethyl ketone inhibitors of fatty acid amide hydrolase: a probe of structural and conformational features contributing to inhibition.

The examination of a series of trifluoromethyl ketone inhibitors of Fatty Acid Amide Hydrolase (FAAH, oleamide hydrolase, anandamide amidohydrolase) is detailed in efforts that define structural and conformational properties that contribute to enzyme inhibition and substrate binding. The results imply an extended bound conformation, highlight a role for the presence, position, and stereochemistry of a delta cis double bond, and suggest little apparent role for C11-C18/C22 of the fatty acid amide substrates.

Amidohydrolases↗

PubMed1998

The 1.8 A crystal structure of the ycaC gene product from Escherichia coli reveals an octameric hydrolase of unknown specificity.

BACKGROUND: The ycaC gene comprises a 621 base pair open reading frame in Escherichia coli. The ycaC gene product (ycaCgp) is uncharacterized and has no assigned function. The closest sequence homologs with an assigned function belong to a family of bacterial hydrolases that catalyze isochorismatase-like reactions, but these have only low sequence similarity to ycaCgp (approximately 20% amino acid identity). The ycaCgp was obtained and identified during crystallization trials of an unrelated E. coli protein with which it co-purified. RESULTS: The 1.8 A crystal structure of ycaCgp reveals an octameric complex comprised of two tetrameric rings. A large three-layer (alphabetaalpha) sandwich domain and a small helical domain form the folded structure of the monomeric unit. Comparisons with sequence and structure databases suggest that ycaCgp belongs to a diverse family of bacterial hydrolases. The most closely related three-dimensional structure is that of the D2 tetrameric N-carbamoylsarcosine amidohydrolase (CSHase) from an Arthrobacter species. A conspicuous cleft between two ycaCgp subunits contains several conserved residues including Cys118, which we propose to be catalytic. In the active site, a nonprolyl cis peptide bond precedes Val114 and coincides with a cis peptide bond in CSHase in a region of dissimilar sequence. The crystal structure reveals a probable error or mutation relative to the reported genomic sequence. CONCLUSIONS: Although the specific function of ycaCgp is not yet known, structural studies solidify the relationship of this protein to other hydrolases and illuminate its active site and key elements of the catalytic mechanism.

Amidohydrolases↗

PubMed2001

Sequence analysis of the aminoacylase-1 family. A new proposed signature for metalloexopeptidases.

The amino acid sequence analysis of the human and porcine aminoacylases-1, the carboxypeptidase S precursor from Saccharomyces cerevisiae, the succinyl-diaminopimelate desuccinylase from Escherichia coli, Haemophilus influenzae and Corynebacterium glutamicum, the acetylornithine deacetylase from Escherichia coli and Dictyostelium discoideum and the carboxypeptidase G(2) precursor from Pseudomonas strain, using the Basic Local Alignment Search Tool (BLAST) and the Position-Specific Iterated BLAST (PSI-BLAST), allowed us to suggest that all these enzymes, which share common functional and biochemical features, belong to the same structural family. The three amino acid blocks which were found to be highly conserved, using the CLUSTAL W program, could be assigned to the catalytic active site, based on the general three-dimensional structure of the carboxypeptidase G(2) from the Pseudomonas strain precursor. Six additional proteins with the same signature have been retrieved after performing two successive PSI-BLAST iterations using the sequence of the conserved motif, namely Lactobacillus delbrueckii aminoacyl-histidine dipeptidase, Streptomyces griseus aminopeptidase, Saccharomyces cerevisiae aminopeptidase Y precursor, two Bacillus stearothermophilus N-carbamyl-L-amino acid amidohydrolases and Pseudomonas sp. hydantoin utilization protein C. The three conserved amino acid motifs corresponded to the following blocks: (i) [S, G, A]-H-x-D-x-V; (ii) G-x-x-D; and (iii) x-E-E. This new sequence signature is clearly different from that commonly reported in the literature for proteins belonging to the ArgE/DapE/CPG2/YscS family.

Amidohydrolases↗

PubMed1999

Equilibrium in the hydrolysis and synthesis of cannabimimetic anandamide demonstrated by a purified enzyme.

Anandamide, an endogenous ligand for cannabinoid receptors, loses its biological activities when it is hydrolyzed to arachidonic acid and ethanolamine by anandamide amidohydrolase. We overexpressed a recombinant rat enzyme with a hexahistidine tag in a baculovirus-insect cell expression system, and purified the enzyme with the aid of a Ni-charged resin to a specific activity as high as 5.7 micromol/min/mg protein. The purified recombinant enzyme catalyzed not only the hydrolysis of anandamide and palmitoylethanolamide, but also their reverse synthetic reactions. In order to attain an equilibrium of the anandamide hydrolysis and its reverse reaction within 10 min, we utilized a large amount of the purified enzyme. The equilibrium constant ([arachidonic acid][ethanolamine])/([anandamide][water]) was calculated as 4x10(-3) (37 degrees C, pH 9.0). These experimental results with a purified enzyme preparation quantitatively confirmed the reversibility of the enzyme reaction previously observed with crude enzyme preparations.

Amidohydrolases↗

PubMed1991

Immobilized enzyme electrode for creatinine determination in serum.

An immobilized enzyme electrode for continuous creatinine determination in blood serum is described. The enzymes creatinine amidohydrolase, creatine amidinohydrolase, and sarcosine oxidase are coimmobilized to the surface of the polypropylene membrane of a Clark-type electrode responsive to oxygen. The immobilized enzymes catalyze the decomposition of creatinine with the consumption of oxygen and thus permit the creatinine measurement. The whole assay takes less than 1 min. Effects of pH and temperature on electrode response are also described. The proposed technique offers a rapid, simple, and inexpensive means to determine creatinine in blood serum within the normal and abnormal ranges. The repeatability of the creatine determination in serum is 2.5% (relative standard deviation), and the detection limit is 3 x 10(-6) mol L-1. The results obtained by this method were compared to those obtained with the Technicon AutoAnalyzer SMAC system based on the Jaffé reaction; the correlation factor between the two methods was found to be r = 0.9997.

Amidohydrolases↗

PubMed2001

Crystal structure and mechanism of catalysis of a pyrazinamidase from Pyrococcus horikoshii.

Bacterial pyrazinamidase (PZAase)/nicotinamidase converts pyrazinamide (PZA) to ammonia and pyrazinoic acid, which is active against Mycobacterium tuberculosis. Loss of PZAase activity is the major mechanism of pyrazinamide-resistance by M. tuberculosis. We have determined the crystal structure of the gene product of Pyrococcus horikoshii 999 (PH999), a PZAase, and its complex with zinc ion by X-ray crystallography. The overall fold of PH999 is similar to that of N-carbamoylsarcosine amidohydrolase (CSHase) of Arthrobacter sp. and YcaC of Escherichia coli, a protein with unknown physiological function. The active site of PH999 was identified by structural features that are also present in the active sites of CSHase and YcaC: a triad (D10, K94, and C133) and a cis-peptide (between V128 and A129). Surprisingly, a metal ion-binding site was revealed in the active site and subsequently confirmed by crystal structure of PH999 in complex with Zn(2+). The roles of the triad, cis-peptide, and metal ion in the catalysis are proposed. Because of extensive homology between PH999 and PZAase of M. tuberculosis (37% sequence identity), the structure of PH999 provides a structural basis for understanding PZA-resistance by M. tuberculosis harboring PZAase mutations.

Amidohydrolases↗

PubMed2004

Manipulation of the active site loops of D-hydantoinase, a (beta/alpha)8-barrel protein, for modulation of the substrate specificity.

We previously proposed that the stereochemistry gate loops (SGLs) constituting the substrate binding pocket of D-hydantoinase, a (beta/alpha)(8)-barrel enzyme, might be major structural determinants of the substrate specificity [Cheon, Y. H., et al. (2002) Biochemistry 41, 9410-9417]. To construct a mutant D-hydantoinase with favorable substrate specificity for the synthesis of commercially important non-natural amino acids, the SGL loops of the enzyme were rationally manipulated on the basis of the structural analysis and sequence alignment of three hydantoinases with distinct substrate specificities. In the SGLs of D-hydantoinase from Bacillus stearothermophilus SD1, mutations of hydrophobic and bulky residues Met 63, Leu 65, Phe 152, and Phe 159, which interact with the exocyclic substituent of the substrate, induced remarkable changes in the substrate specificities. In particular, the substrate specificity of mutant F159A toward aromatic substrate hydroxyphenylhydantoin (HPH) was enhanced by approximately 200-fold compared with that of the wild-type enzyme. Saturation mutagenesis at position 159 revealed that k(cat) for aromatic substrates increased gradually as the size of the amino acid side chain decreased, and this seems to be due to reduced steric hindrance between the bulky exocyclic group of the substrate and the amino acid side chains. When site-directed random mutagenesis of residues 63 and 65 was conducted with the wild type and mutant F159A, the selected enzymes (M63F/L65V and L65F/F159A) exhibited approximately 10-fold higher k(cat) values for HPH than the wild-type counterpart, which is likely to result from reorganization of the active site for efficient turnover. These results indicate that the amino acid residues of SGLs forming the substrate binding pocket are critical for the substrate specificity of D-hydantoinase, and the results also imply that substrate specificities of cyclic amidohydrolase family enzymes can be modulated by rational design of these SGLs.

Amidohydrolases↗

PubMed2005

Purification and characterization of enzymes involved in the degradation of chemotactic N-formyl peptides.

N-Formyl peptides are derived from proteolytic degradation/processing of bacterial and mitochondrial proteins and serve as potent chemoattractants for mammalian phagocytic leukocytes. A response to the chemotactic N-formyl peptides released by commensal bacteria in the gut region could be detrimental, leading to unwanted inflammation. Here, two enzymes that act sequentially to degrade N-formyl peptides were purified from the rat intestinal mucosal layer and biochemically characterized. The first enzyme cleaves chemotactic peptide f-MLF to release N-formylmethionine (f-Met) and dipeptide leucylphenylalanine, with a k(cat) value of 14 s(-)(1), a K(M) value of 0.60 mM, and a k(cat)/K(M) value of 22 500 M(-)(1) s(-)(1). In-gel tryptic digestion followed by mass spectral fingerprinting identified the protein as the alpha-N-acylpeptide hydrolase (or acylamino acid-releasing enzyme, EC 3.4.19.1). The second enzyme hydrolyzes N-formylmethionine into formate and methionine with a k(cat) value of 7.9 s(-)(1), a K(M) value of 3.1 mM, and a k(cat)/K(M) value of 2550 M(-)(1) s(-)(1). This protein was identified as the N-acylase IA (or N(alpha)-acyl-l-amino acid amidohydrolase, EC 3.5.1.14). Together, these two enzymes play a protective role in degrading bacterial and mitochondrial N-formylated peptides.

Amidohydrolases↗

PubMedDate not supplied

Production of D-amino acid using whole cells of recombinant Escherichia coli with separately and coexpressed D-hydantoinase and N-carbamoylase.

We developed a fully enzymatic process employing D-hydantoinase and N-carbamoylase for the production of D-amino acid from 5'-monosubstituted hydantoin. For the comparison of the reaction systems using two sequential enzymes, D-hydantoinase of Bacillus stearothermophilus SD1 and N-carbamoyl-D-amino acid amidohydrolase (N-carbamoylase) of Agrobacterium tumefaciens NRRL B11291 were separately expressed in each host cell and coexpressed in the same host cell. A high level and constitutive expression of both enzymes in Escherichia coli in a soluble form was achieved using a promoter derived from B. stearothermophilus SD1. The expression levels of both enzymes ranged from 17% to 23% of the total soluble protein, depending on the expression system. In the case of employing separately expressed enzymes, the product yield of D-hydroxyphenylglycine from D,L-p-hydroxyphenylhydantoin and productivity were 71% and 2.57 mM/g-cell/h in 15 h, respectively. The accumulation of N-carbamoyl-D-hydroxyphenylglycine was significant over the reaction time. On the other hand, use of coexpressed enzymes resulted in 98% product yield of D-hydroxyphenylglycine in 15 h, minimizing the level of intermediates in the reaction mixture. The productivity of coexpressed whole cell reaction was estimated to be 6.47 mM/g-cell/h in 15 h. The coexpressed system was tested for an elevated concentration of D,L-p-hydroxyphenylhydantoin, and efficient production can be achieved.

Amidohydrolases↗

PubMed2006

Tight binding inhibitors of N-acyl amino sugar and N-acyl amino acid deacetylases.

Very potent inhibitors were synthesized for the enzymatic deacetylation of N-acetyl-d-glucosamine-6-phosphate (NagA) and N-acetyl-d-glutamate (DGD). The methyl phosphonamidate derivative of d-glucosamine-6-phosphate bound to N-acetyl-d-glucosamine-6-phosphate deacetylase with an equilibrium dissociation constant of 34 +/- 5 nM at pH 7.5 and an association rate constant of 6.1 x 103 M-1 s-1. The inhibition constant is 4000-fold lower than the Michaelis constant for the substrate N-acetyl-d-glucosamine-6-phosphate. N-Acetyl-d-glutamate deacetylase was inhibited by the methyl phosphonamidate derivative of d-glutamate with an inhibition constant of 460 +/- 70 pM at pH 7.6. The inhibitor bound to the enzyme 500 000-fold tighter than the Michaelis constant for N-formyl-d-glutamate. These compounds mimic the putative tetrahedral intermediate formed upon nucleophilic attack of an activated water molecule on the amide bond of the target substrate. These inhibitors should prove useful in the elucidation of the enzyme-substrate interactions for enzymes within the amidohydrolase superfamily.

Alcaligenes faecalis↗

PubMed1997

Dihydropyrimidinase deficiency and congenital microvillous atrophy: coincidence or genetic relation?

We describe a boy of consanguineous parents who suffered from intractable diarrhoea due to congenital microvillous atrophy, a recessively inherited autosomal disorder. He developed severe cholestatis starting at 2 weeks of age and leading to liver cirrhosis. His psychomotor development appeared only slightly delayed. At the age of 7 months he died due to septicaemia. In addition to disturbances of electrolyte balance and renal tubular function, which could be attributed to microvillous atrophy, marked elevations of dihydrouracil and dihydrothymine as well as moderately elevated excretion of uracil and thymine in urine were repeatedly demonstrated, suggesting a disorder of pyrimidine degradation. An enzymatic defect of 5,6-dihydropyrimidine amidohydrolase (EC 3.5.2.2, dihydropyrimidinase, DHP) was demonstrated in liver biopsy. As both of these recessive disorders seem to be extremely rare, it remains speculative, whether he suffered from two independently inherited metabolic diseases or whether this represents a hitherto undescribed contiguous gene syndrome.

Amidohydrolases↗

PubMed2003

Enhanced hydantoinase and N-carbamoylase activity on immobilisation of Agrobacterium tumefaciens.

Cell extracts of Agrobacterium tumefaciens, immobilised in calcium alginate beads, had a 7-fold increase in N-carbamoylase (N-carbamylamino acid amidohydrolase E.C. 3.5.1) activity on reaction with N-carbamylglycine. The hydantoinase (dihydropyrimidinase E.C. 3.5.2.2) and N-carbamoylase activities remained stable over 4 weeks storage at 4 degrees C relative to the non-immobilised enzymes, with the hydantoinase activity showing a 5-fold increase in activity relative to the non-immobilised hydantoinase. The pH optima of the immobilised hydantoinase and N-carbamoylase enzymes decreased to pH 7 and pH 8, respectively. The temperature optimum remained at 40 degrees C for the N-carbamoylase enzyme while the hydantoinase activity was optimal at 50 degrees C.

Agrobacterium tumefaciens↗

PubMed2001

Effects of homologues and analogues of palmitoylethanolamide upon the inactivation of the endocannabinoid anandamide.

1. The ability of a series of homologues and analogues of palmitoylethanolamide to inhibit the uptake and fatty acid amidohydrolase (FAAH)-catalysed hydrolysis of [(3)H]-anandamide ([(3)H]-AEA) has been investigated. 2. Palmitoylethanolamide and homologues with chain lengths from 12 - 18 carbon atoms inhibited rat brain [(3)H]-AEA metabolism with pI(50) values of approximately 5. Homologues with chain lengths < or = eight carbon atoms gave < 20% inhibition at 100 microM. 3. R-palmitoyl-(2-methyl)ethanolamide, palmitoylisopropylamide and oleoylethanolamide inhibited [(3)H]-AEA metabolism with pI(50) values of 5.39 (competitive inhibition), 4.89 (mixed type inhibition) and 5.33 (mixed type inhibition), respectively. 4. With the exception of oleoylethanolamide, the compounds did not produce dramatic inhibition of [(3)H]-WIN 55,212-2 binding to human CB(2) receptors expressed on CHO cells. Palmitoylethanolamide, palmitoylisopropylamide and R-palmitoyl-(2-methyl)ethanolamide had modest effects upon [(3)H]-CP 55,940 binding to human CB(1) receptors expressed on CHO cells. 5. Most of the compounds had little effect upon the uptake of [(3)H]-AEA into C6 and/or RBL-2H3 cells. However, palmitoylcyclohexamide (100 microM) and palmitoylisopropylamide (30 and 100 microM) produced more inhibition of [(3)H]-AEA uptake than expected to result from inhibition of [(3)H]-AEA metabolism alone. 6. In intact C6 cells, palmitoylisopropylamide and oleoylethanolamide inhibited formation of [(3)H]-ethanolamine from [(3)H]-AEA to a similar extent as AM404, whereas palmitoylethanolamide, palmitoylcyclohexamide and R-palmitoyl-(2-methyl)ethanolamide were less effective. 7. These data provide useful information upon the ability of palmitoylethanolamide analogues to act as 'entourage' compounds. Palmitoylisopropylamide may prove useful as a template for design of compounds that reduce the cellular accumulation and metabolism of AEA without affecting either CB(1) or CB(2) receptors.

Amides↗

PubMed2004

Characterization of an anandamide degradation system in prostate epithelial PC-3 cells: synthesis of new transporter inhibitors as tools for this study.

The response of anandamide is terminated by a carrier-mediated transport followed by degradation catalyzed by the cloned enzyme fatty acid amidohydrolase (FAAH). In this study, we provide biochemical data showing an anandamide uptake process and the expression of FAAH in human prostate. Anandamide was accumulated in PC-3 cells by a saturable and temperature-dependent process. Kinetic studies of anandamide uptake, determined in the presence of cannabinoid and vanilloid antagonists, revealed apparent parameters of KM=4.7+/-0.2 microm and Vmax=3.3+/-0.3 pmol min-1 (10(6) cells)-1. The accumulation of anandamide was moderately inhibited by previously characterized anandamide transporter inhibitors (AM404, UCM707 and VDM11) but was unaffected by inhibitors of other lipid transport systems (phloretin or verapamil) and moderately affected by the FAAH inhibitor methyl arachidonyl fluorophosphonate. The presence of FAAH in human prostate epithelial PC-3 cells was confirmed by analyzing its expression by Western blot and measuring FAAH activity. To further study the structural requirements of the putative carrier, we synthesized a series of structurally different compounds 1-8 and evaluated their capacity as uptake inhibitors. They showed different inhibitory capacity in PC-3 cells, with (9Z,12Z)-N-(fur-3-ylmethyl)octadeca-9,12-dienamide (4, UCM119) being the most efficacious, with maximal inhibition and IC50 values of 49% and 11.3+/-0.5 microM, respectively. In conclusion, PC-3 cells possess a complete inactivation system for anandamide formed by an uptake process and the enzyme FAAH. These results suggest a possible physiological function of anandamide in the prostate, reinforcing the role of endocannabinoid system as a neuroendocrine modulator. British Journal of Pharmacology (2004) 141, 457-467. doi:10.1038/sj.bjp.0705628

Amidohydrolases↗

PubMed2004

Experimental parkinsonism alters anandamide precursor synthesis, and functional deficits are improved by AM404: a modulator of endocannabinoid function.

Modulation of the endocannabinoid system might be useful in treating Parkinson's disease. Here, we show that systemic administration of N-(4-hydroxyphenyl)-arachidonamide (AM404), a cannabinoid modulator that enhances anandamide (AEA) availability in the biophase, exerts antiparkinsonian effects in 6-hydroxydopamine-lesioned rats. Local injections of AM404 into denervated striata reduced parkinsonian motor asymmetries, these effects being associated with the reduction of D2 dopamine receptor function together with a positive modulation of 5-HT(1B) serotonin receptor function. Stimulation of striatal 5-HT(1B) receptors alone was observed to ameliorate parkinsonian deficits, supporting the fact that AM404 exerts antiparkinsonian effects likely through stimulation of striatal 5-HT(1B) serotonin receptor function. Hence, modulation of cannabinoid function leading to enhancement of AEA in the biophase might be of therapeutic value in the control of symptoms of Parkinson's disease. On the other hand, reduced levels of N-acyl-transferase (AEA precursor synthesizing enzyme), without changes in fatty acid amidohydrolase (AEA degradative enzyme), were detected in denervated striata in comparison with intact striata. This finding reveals the presence of a homeostatic striatal mechanism emerging after dopaminergic denervation likely tending to enhance low dopamine tone.

Acyltransferases↗

PubMed2006

Genetic impairment of frontocortical endocannabinoid degradation and high alcohol preference.

Endocannabinoid signaling has recently been implicated in ethanol-seeking behavior. We analyzed the expression of endocannabinoid-related genes in key brain regions of reward and dependence, and compared them between the alcohol-preferring AA (Alko Alcohol) and nonpreferring ANA (Alko Non-Alcohol) rat lines. A decreased expression of fatty acid amidohydrolase (FAAH), the main endocannabinoid-degrading enzyme, was found in prefrontal cortex (PFC) of AA rats, and was accompanied by decreased enzyme activity in this region. Binding of the endocannabinoid-cannabinoid 1 (CB1) receptor ligand (3)[H]SR141716A, and [35S]GTPgammaS incorporation stimulated by the CB1 agonist WIN 55,212-2 were downregulated in the same area. Together, this suggests an overactive endocannabinoid transmission in the PFC of AA animals, and a compensatory downregulation of CB1 signaling. The functional role of impaired FAAH function for alcohol self-administration was validated in two independent ways. The CB1 antagonist SR141716A potently and dose-dependently suppressed self-administration in AA rats when given systemically, or locally into the PFC, but not in the striatum. Conversely, intra-PFC injections of the competitive FAAH inhibitor URB597 increased ethanol self-administration in nonselected Wistar rats. These results show for the first time that impaired FAAH function may confer a phenotype of high voluntary alcohol intake, and point to a FAAH both as a potential susceptibility factor and a therapeutic target.

Alcohol Drinking↗