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 (K_i(slope) 1.3 µM; K_i(intercept) 44 µM). 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.     

N-Carbamyl-L-Amino Acid Amidohydrolase of Pseudomonas sp. Strain NS671: Purification and Some Properties of the Enzyme Expressed in Escherichia coli

An N-carbamyl-L-amino acid amidohydrolase was purified from cells of Escherichia coli in which the gene for N-carbamyl-L-amino acid amidohydrolase of Pseudomonas sp. strain NS671 was expressed. The purified enzyme was homogeneous by the criterion of SDS–polyacrvlamide gel electrophoresis. The results of gel filtration chromatography and SDS–polyacrylamide gel electrophoresis suggested that the enzyme was a dimeric protein with 45-kDa identical subunits. The enzyme required Mn²⁺ ion (above 1 mM) for the activity. The optimal pH and temperature were 7.5 and around 40°C, respectively, with N-carbamyl-L-methionine as the substrate. The enzyme activity was inhibited by ATP and was iost completely with p-chloromercuribenzoate (1 mM). The enzyme was strictly L-specific and showed a broad substrate specificity for N-carbamyl-L-α-amino acids.     

Chaperone-Assisted Overexpression of an Active -Carbamoylase from Agrobacterium tumefaciens AM 10

The N-carbamoyl- -amino acid amidohydrolase ( -carbamoylase) gene (dcb) from Agrobacterium tumefaciens AM 10 was cloned by polymerase chain reaction in plasmid pET28a and was overexpressed in Escherichia coli JM109 (DE3). However, almost 80% of the enzyme remained trapped in inclusion bodies. To facilitate the expression of the properly folded active enzyme, the chaperones GroEL/ES were coexpressed in plasmid pKY206. This resulted in a 43-fold increase in active enzyme production compared to the wild-type strain. The histidyl-tagged -carbamoylase was purified by a single step nickel-affinity chromatography to a specific activity of 9.5 U/mg protein.     

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°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°C for the N-carbamoylase enzyme while the hydantoinase activity was optimal at 50°C.     

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.     

Purification and properties of d-hydantoin hydrolase and N-carbamoyl-d-amino acid amidohydrolase from Flavobacterium sp. AJ11199 and Pasteurella sp. AJ11221

We characterized recombinant d-hydantoin hydrolase (DHHase) and N-carbamoyl-d-amino acid amidohydrolase (DCHase) from Flavobacterium sp. AJ11199 and Pasteurella sp. AJ11221. The DHHases from these two strains showed a wide range of hydrolytic activity for various 5-monosubstituted d-hydantoin compounds, including a very high level activity for d-hydantoin compounds corresponding to d-aromatic amino acids such as d-tryptophan d-phenylalanine and d-tyrosine. The DCHases, in turn, were capable of catalyzing the hydrolysis of various N-carbamoyl-d-amino acids (NCD-A.A.) corresponding to d-aliphatic and d-aromatic amino acids. The combination of these enzymes was found to be applicable for the production of various d-amino acids.     

d-Amino acid production by E. coli co-expressed three genes encoding hydantoin racemase, d-hydantoinase and N-carbamoyl-d-amino acid amidohydrolase

Three genes respectively encoding d-specific hydantoinase (DHHase), N-carbamoyl-d-amino acid amidohydrolase (DCHase) and hydantoin racemase (HRase) were co-expressed in E. coli in a system designed for the efficient enzymatic production of d-amino acids via a combination of hydantoin hydrolysis and hydantoin racemization. With the use of whole cells, the d-forms of eight amino acids – d-phenylalanine, d-tyrosine, d-tryptophan, O-benzyl-d-serine, d-valine, d-norvaline, d-leucine and d-norleucine – were efficiently converted from the corresponding dl-5-monosubtituted hydantoin compounds.     

Encapsulation of an Agrobacterium radiobacter extract containing d-hydantoinase and d-carbamoylase activities into alginate–chitosan polyelectrolyte complexes: Preparation of the biocatalyst

Alginate–chitosan polyelectrolyte complexes (PECs) have been used for the first time as a suitable matrix for coimmobilisation of enzymes to reproduce a multistep enzymatic route for production of d-amino acids. Encapsulation of a crude cell extract from Agrobacterium radiobacter containing d-hydantoinase and d-carbamoylase activities into the PECs with negligible leakage from the formed capsules was accomplished. All results in this study indicate that the preparation of the biocatalyst (preparation method and chitosan characteristics) play a key role in the biocatalyst's properties. The most suitable biocatalysts were prepared using a chitosan with a medium molecular weight (600 kDa) and a degree of deacetylation of 0.9. For all of the preparation conditions under study, an encapsulation yield of around 60% was achieved and the enzymatic activity yields ranged from 30 to 80% for d-hydantoinase activity and from 40 to 128% for d-carbamoylase activity relative to the activities of the soluble extract. All of the biocatalysts were able to hydrolyze l,d-hydroxyphenylhydantoin into p-hydroxyphenylglycine with yields ranging from 30 to 80%.     

Identification of novel mimicry epitopes for cardiac myosin heavy chain-α that induce autoimmune myocarditis in A/J mice

Myocarditis is one cause of sudden cardiac death in young adolescents, and individuals affected with myocarditis can develop dilated cardiomyopathy, a frequent reason for heart transplantation. Exposure to environmental microbes has been suspected in the initiation of heart autoimmunity, but the direct causal link is lacking. We report here identification of novel mimicry epitopes that bear sequences similar to those in cardiac myosin heavy chain (MYHC)-α 334–352. These epitopes represent Bacillus spp., Magnetospirillum gryphiswaldense, Cryptococcus neoformans and Zea mays. The mimicry peptides induced varying degrees of myocarditis in A/J mice reminiscent of the disease induced with MYHC-α 334–352. We demonstrate that the mimics induce cross-reactive T cell responses for MYHC-α 334–352 as verified by MHC class II IA^k/tetramer staining and Th-1 and Th-17 cytokines similar to those of MYHC-α 334–352. The data suggest that exposure to environmental microbes which are otherwise innocuous can predispose to heart autoimmunity by molecular mimicry.