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PubMed1996

Study of the amidase signature group.

Computer methods for database search, multiple alignment and cluster analysis indicated significant homology between amino-acid sequences of 21 amidases or amidohydrolases (EC 3.5). All of them were found to be involved in the reduction of organic nitrogen compounds and ammonia production. A conserved motif was found which may be important in amide binding and in catalytic mechanisms. Homology studies between these amidases and some ureases, nitrilases and acyl-transferases or enzymes with unknown functions provided new insight into the evolution of these proteins. Dissemination of these genes seemed to be facilitated by transfer of genetic elements such as transposons and plasmids.

Amidohydrolases↗

PubMed1998

Pharmacological evidence for anandamide amidase in human cardiac and vascular tissues.

The present report demonstrates the presence of antianandamide and anticannabinoid receptor 1 immunopositive material on the saphenous vascular endothelium. The endogenous cannabinoid, anandamide, in a dose-dependent manner stimulated the release of nitric oxide (NO) from saphenous vein, internal thoracic artery and right atrium tissue segments in vitro. This process can be antagonized by the nitric oxide synthase (NOS) inhibitor, N-omega-nitro-L-arginine methyl ester (L-NAME) (10(-4) M; 3.4+/-0.9 nM NO; P<0.01 compared to anandamide alone), as well as by the cannabinoid receptor I antagonist SR 141716A (2.9+/-1.0 nM NO; P<0.01). Furthermore, in the presence of varying concentrations of methylarachidonylfluorophosphonate, an anandamide amidase inhibitor, 10(-8) M anandamide stimulates a higher peak level of NO that remains elevated for a longer period of time (P<0.05) compared to anandamide alone, demonstrating the presence of anandamide amidase in human vascular tissues. Morphine, as anandamide, can stimulate the release of NO from right atria. This process can be inhibited by the opiate receptor antagonist naloxone and the NOS inhibitor L-NAME. As expected SR 141716A (10(-6) M; 26+3.8 NO nM in the presence of 10(-7) M morphine) did not antagonize morphine's ability to release NO. Taken together, the data demonstrate that cannabinoid signalling is involved with the regulation of the microvascular environment.

Amidohydrolases↗

PubMed1998

Substrate-dependent enantioselectivity of a novel hydantoinase from Arthrobacter aurescens DSM 3745: purification and characterization as new member of cyclic amidases.

A hydantoinase from Arthrobacter aurescens DSM 3745 has been purified to homogeneity with a yield of 77% using a three-step purification procedure. The active enzyme is a tetramer consisting of four identical subunits, each with a molecular mass of 49670 Da as determined by mass spectrometry. The N-terminal amino acid sequence of the enzyme indicates sequence identities to cyclic amidases involved in the nucleotide metabolism as the D-hydantoinase from Agrobacterium radiobacter (53%), the D-selective dihydropyrimidinase from Bacillus stearothermophilus (38%), the allantoinase from Rana catesbeiana (26%), as well as to the catalytic subunit of the urease from Helicobacter pylori (50%). However, all studies based on substrate-dependent growth, induction and catalytic behavior documented the novelty of the bacterial hydantoinase and that its physiological role is not related to any of these enzymes or known metabolic pathways. Its substrate specificity differs from hydantoinases listed in Enzyme Nomenclature and is rather more predominant for the cleavage of aryl- than for alkyl-hydantoin derivatives. It is shown that the stereoselectivity of this enzyme depends on the substrate used for bioconversion: although it is strictly L-selective for the cleavage of D,L-5-indolylmethylhydantoin, it appears to be D-selective for the hydrolysis of D,L-methylthioethylhydantoin. Due to these findings we conclude that this novel bacterial hydantoinase should be classified as a new member of the EC-group 3.5.2 of cyclic amidases.

Amidohydrolases↗

PubMed2004

Probing the Ser-Ser-Lys catalytic triad mechanism of peptide amidase: computational studies of the ground state, transition state, and intermediate.

Peptide amidase (Pam), a hydrolytic enzyme that belongs to the amidase signature (AS) family, selectively catalyzes the hydrolysis of the C-terminal amide bond (CO-NH(2)) of peptides. The recent availability of the X-ray structures of Pam, fatty acid amide hydrolase, and malonamidase E2 has led to the proposal of a novel Ser-Ser-Lys catalytic triad mechanism for the amide hydrolysis by the AS enzymes. The molecular dynamics (MD) simulations using the CHARMM force field were performed to explore the catalytic mechanism of Pam. The 1.8 A X-ray crystal structure of Pam in complex with the amide analogue of chymostatin was chosen for the initial coordinates for the MD simulations. The five systems that were investigated are as follows: (i) enzyme.substrate with Lys123-NH(2), (ii) enzyme.substrate with Lys123-NH(3)(+), (iii) enzyme.substrate with Lys123-NH(3)(+) and Ser226-O(-), (iv) enzyme.transition state, and (v) enzyme.tetrahedral intermediate. Our data support the presence of the hydrogen bonding network among the catalytic triad residues, Ser226, Ser202, and Lys123, where Ser226 acts as the nucleophile and Ser202 bridges Ser226 and Lys123. The MD simulation supports the catalytic role of the crystallographic waters, Wat1 and Wat2. In all the systems that have been studied, the backbone amide nitrogens of Asp224 and Thr223 create an oxyanion hole by hydrogen bonding to the terminal amide oxygen of the substrate, and stabilize the oxyanion tetrahedral intermediate. The results from both our computational investigation and previously published experimental pH profile support two mechanisms. In a mechanism that is relevant at lower pH, the Lys123-NH(3)(+)-Ser202 dyad provides structural support to the catalytic residue Ser226, which in turn carries out a nucleophilic attack at the substrate amide carbonyl in concert with Wat1-mediated deprotonation and stabilization of the tetrahedral transition state by the oxyanion hole. In the mechanism operating at higher pH, the Lys123-NH(2)-Ser202 catalytic dyad acts as a general base to assist addition of Ser226 to the substrate amide carbonyl. The results from the MD simulation of the tetrahedral intermediate state show that both Ser202 and Lys123 are possible candidates for protonation of the leaving group, NH(2), to form the acyl-enzyme intermediate.

Amidohydrolases↗

PubMed2004

Mandelamide hydrolase from Pseudomonas putida: characterization of a new member of the amidase signature family.

A recently discovered enzyme in the mandelate pathway of Pseudomonas putida, mandelamide hydrolase (MAH), catalyzes the hydrolysis of mandelamide to mandelic acid and ammonia. Sequence analysis suggests that MAH is a member of the amidase signature family, which is widespread in nature and contains a novel Ser-cis-Ser-Lys catalytic triad. Here we report the expression in Escherichia coli, purification, and characterization of both wild-type and His(6)-tagged MAH. The recombinant enzyme was stable, exhibited a pH optimum of 7.8, and was able to hydrolyze both enantiomers of mandelamide with little enantiospecificity. The His-tagged variant showed no significant change in kinetic constants. Phenylacetamide was found to be the best substrate, with changes in chain length or replacement of the phenyl group producing greatly decreased values of k(cat)/K(m). As with another member of this family, fatty acid amide hydrolase, MAH has the uncommon ability to hydrolyze esters and amides at similar rates. MAH is even more unusual in that it will only hydrolyze esters and amides with little steric bulk. Ethyl and larger esters and N-ethyl and larger amides are not substrates, suggesting that the MAH active site is very sterically hindered. Mutation of each residue in the putative catalytic triad to alanine resulted in total loss of activity for S204A and K100A, while S180A exhibited a 1500-fold decrease in k(cat) and significant increases in K(m) values. Overall, the MAH data are similar to those of fatty acid amide hydrolase and support the suggestion that there are two distinct subgroups within the amidase signature family.

Amidohydrolases↗

PubMed2006

An unusual beta-vinyl effect leading to high efficiency and enantioselectivity of the amidase, nitrile biotransformations for the preparation of enantiopure 3-arylpent-4-enoic acids and amides and their applications in synthesis.

Biotransformations of 3-arylpent-4-enenitriles catalyzed by Rhodococcus erythropolis AJ270, a nitrile hydratase/amidase-containing microbial whole-cell catalyst were studied, and an unusual beta-vinyl effect of the substrate on the biocatalytic efficiency and enantioselectivity of the amidase was observed. While 3-arylpent-4-enenitriles and 3-phenylpentanenitrile were efficiently hydrated by the action of the less R-enantioselective nitrile hydratase, the amidase showed greater activity and higher enantioselectivity against 3-arylpent-4-enoic acid amides than 3-arylpentanoic acid amides. Under very mild conditions, nitrile biotransformations provided an efficient synthesis of highly enantiopure (R)-3-arylpent-4-enoic acids and (S)-3-arylpent-4-enoic acid amides, and their applications were demonstrated by the synthesis of chiral gamma-amino acid, 2-pyrrolidinone, and 2-azepinone derivatives.

Amides↗

PubMed2000

Gene cloning, nucleotide sequencing, and purification and characterization of the D-stereospecific amino-acid amidase from Ochrobactrum anthropi SV3.

The gene encoding the D-stereospecific amino-acid amidase from Ochrobactrum anthropi SV3 was cloned and sequenced. Analysis of 7.3 kb of genomic DNA revealed the presence of six ORFs, one of which (daaA) encodes the D-amino-acid amidase. This enzyme, DaaA, is composed of 363 amino-acid residues (molecular mass 40 082 Da), and the deduced amino-acid sequence exhibits homology to alkaline D-peptidase from Bacillus cereus DF4-B (32% identity), DD-peptidase from Streptomyces R61 (29% identity), and other penicillin-recognizing proteins. The DaaA protein contains the typical SXXK, YXN, and H(K)XG active-site motifs identified in the penicillin-binding proteins and beta-lactamases. The daaA gene modified in the nucleotide sequence upstream from its start codon was overexpressed in Escherichia coli. The activity of the recombinant DaaA enzyme in cell-free extracts of E. coli was 33.6 U. mg-1 with D-phenylalaninamide as substrate, which is about 350-fold higher than in extracts of O. anthropi SV3. This enzyme was purified to electrophoretic homogeneity by ammonium sulfate fractionation and three column chromatography steps. On gel-filtration chromatography, DaaA appeared to be a monomer with a molecular mass of 40 kDa. It had maximal activity at 45 degrees C and pH 9.0, and was completely inactivated in the presence of phenylmethanesulfonyl fluoride or Zn2+. DaaA had hydrolyzing activity toward D-amino-acid amides with aromatic or hydrophobic side chains, but did not act on the substrates for the DD-peptidase and beta-lactamase, despite their sequence similarity to DaaA. The characteristics of the recombinant DaaA are similar to those found for the native enzyme partially purified from O. anthropi SV3.

Amidohydrolases↗

PubMed1998

Anchor structure of staphylococcal surface proteins. II. Cooh-terminal structure of muramidase and amidase-solubilized surface protein.

Surface proteins of the Gram-positive organism Staphylococcus aureus are anchored to the bacterial cell wall by a transpeptidation mechanism during which the polypeptide is cleaved between the threonine (T) and the glycine (G) of the LPXTG motif. The carboxyl of threonine is subsequently amide linked to the amino of the pentaglycyl cross-bridge within the staphylococcal peptidoglycan. Previous work examined the anchor structure of surface proteins solubilized from the peptidoglycan by treatment with lysostaphin or phi11 hydrolase and identified COOH-terminally linked triglycyl or L-Ala-D-iGln-L-Lys(Gly5)-D-Ala and MurNAc-[L-Ala-D-iGln-L-Lys(Gly5)-D-Ala](beta1-4)-GlcNAc, respectively. Here, we report the anchor structure of surface proteins solubilized with N-acetylmuramidase and N-acetylmuramyl-L-alanine amidase. N-Acetylmuramidase-released surface protein was linked to MurNAc-[L-Ala-D-iGln-L-Lys(Gly5)-D-Ala](beta1-4)-GlcNAc, whereas N-acetylmuramyl-L-alanine amidase treatment of the cell wall solubilized surface proteins linked to L-Ala-D-iGln-L-Lys(Gly5)-D-Ala. Most, but not all, anchor structures were cross-linked to other cell wall subunits, in which the D-alanyl at position four was amide linked to the pentaglycyl of a neighboring wall peptide.

Amidohydrolases↗

PubMed1998

Dictyostelium discoideum fatty-acyl amidase II has deacylase activity on Rhizobium nodulation factors.

Dictyostelium discoideum (Amoebidae) secretes cell-lysing enzymes: esterases, amidases, and glycosylases, many of which degrade soil bacteria to provide a source of nutrients. Two of these enzymes, fatty-acyl amidases FAA I and FAA II, act sequentially on the N-linked long chain acyl groups of lipid A, the lipid anchor of Gram-negative bacterial lipopolysaccharide. FAA I selectively hydrolyzes the 3-hydroxymyristoyl group N-linked to the proximal glucosamine residue of de-O-acylated lipid A. Substrate specificity for FAA II is less selective, but does require prior de-N-acylation of the proximal sugar, i.e. bis-N-acylated lipid A is not a substrate. We have synthesized a 14C-labeled substrate analog for FAA II and used this in a novel assay to monitor its purification. Inhibitory studies indicate that FAA II is not a serine protease, but may have a catalytic mechanism similar to metalloprotein de-N-acetylases such as LpxC. Interestingly, rhizobial Nod factor signal oligosaccharides that induce root nodules on leguminous plants have many of the structural requirements for substrate recognition by FAA II. In vitro evidence indicates that Rhizobium fredii Nod factors are selectively de-N-acylated by purified FAA II, suggesting that the enzyme may reduce the N2-fixing efficiency of Rhizobium-legume symbioses. In contrast, N-methylated Nod factors from transgenic R. fredii carrying the rhizobial nodS gene were resistant to FAA II, suggesting a mechanism by which Nod factors may be protected from enzymatic de-N-acylation. Since FAA II and Nod factors are both secreted, and Nod factors that lack the N-acyl group are unable to induce nodules, dictyostelial FAA II may decrease the efficiency of symbiotic nitrogen fixation in the environment by reducing the available biologically active nodule inducer signal.

Amidohydrolases↗

PubMed2000

Steric hindrance regulation of the Pseudomonas aeruginosa amidase operon.

Expression of the amidase operon of Pseudomonas aeruginosa is controlled by AmiC, the ligand sensor and negative regulator, and AmiR the transcription antitermination factor activator. We have titrated out AmiC repression activity in vivo by increased AmiR production in trans and shown AmiC regulation of the antitermination activity of AmiR by a steric hindrance mechanism. In the presence of the co-repressor butyramide we have isolated a stable AmiC.AmiR complex. Addition of the inducing ligand acetamide to the complex trips the molecular switch, causing complex dissociation and release of AmiR. The AmiC.AmiR butyramide complex exhibits acetamide-dependent, sequence-specific RNA binding activity and a K(d) of 1.0 nm has been calculated for the AmiR.RNA interaction. The results show that amidase operon expression is controlled by a novel type of signal transduction system in which activity of a site-specific RNA binding activator is regulated via a sequestration mechanism.

Acetamides↗

PubMed2001

A monoclonal antibody specific for Pseudomonas aeruginosa amidase.

Amidase from Pseudomonas aeruginosa was purified by anionic exchange chromatography and used to immunise female Balb/c mice. Monoclonal antibodies (MAbs) were raised by hybridoma technology using Sp2/0 myeloma cells as fusion partner. A selected IgM subclass MAb was purified from in vitro hybridoma cell line supernatant by a two-step anionic exchange chromatography. The MAb was specific for amidase from P. aeruginosa as determined by Western blotting and recognized the native and denatured forms of the enzyme.

Amidohydrolases↗

PubMed2002

Crystallization and preliminary X-ray crystallographic analysis of malonamidase E2, an amidase signature family member.

Malonamidase E2 from Bradyrhizobium japonicum catalyzes the hydrolysis of malonamate. The enzyme belongs to an amidase signature family which has a highly conserved serine- and glycine-rich sequence over a stretch of approximately 45 amino acids. More than 100 known or predicted members belonging to this family, whose biological functions vary widely, can be identified in sequence databases. Although urgently needed, no three-dimensional structure of any protein of this family is yet available. The crystallization of malonamidase E2 was undertaken as a first step toward the goal of providing information on the canonical structure of the amidase signature family. The enzyme was crystallized using the hanging-drop vapour-diffusion method at 277 K under two different conditions. One crystal form, which is easier to work with than the other form, belongs to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 104.29, b = 95.58, c = 74.90 A. The unit cell is likely to contain two molecules of MAE2, with a crystal volume per protein mass (V(M)) of 2.045 A(3)Da(-1) and solvent content of about 39.9% by volume. A native data set to 1.8 A resolution was obtained from a flash-cooled crystal using synchrotron radiation.

Amidohydrolases↗

PubMed2006

The quaternary structure of the amidase from Geobacillus pallidus RAPc8 is revealed by its crystal packing.

The amidase from Geobacillus pallidus RAPc8, a moderate thermophile, is a member of the nitrilase enzyme superfamily. It converts amides to the corresponding acids and ammonia and has application as an industrial catalyst. RAPc8 amidase has been cloned and functionally expressed in Escherichia coli and has been purified by heat treatment and a number of chromatographic steps. The enzyme was crystallized using the hanging-drop vapour-diffusion method. Crystals produced in the presence of 1.2 M sodium citrate, 400 mM NaCl, 100 mM sodium acetate pH 5.6 were selected for X-ray diffraction studies. A data set having acceptable statistics to 1.96 A resolution was collected under cryoconditions using an in-house X-ray source. The space group was determined to be primitive cubic P4(2)32, with unit-cell parameter a = 130.49 (+/-0.05) A. The structure was solved by molecular replacement using the backbone of the hypothetical protein PH0642 from Pyrococcus horikoshii (PDB code 1j31) with all non-identical side chains substituted with alanine as a probe. There is one subunit per asymmetric unit. The subunits are packed as trimers of dimers with D3 point-group symmetry around the threefold axis in such a way that the dimer interface seen in the homologues is preserved.

Amidohydrolases↗

PubMed1975

Envelope-bound N-acetylmuramyl-L-alanine amidase of Escherichia coli K 12. Purification and properties of the enzyme.

N-Acetylmuramyl-L-alanine amidase activity was detected in Escherichia coli K 12 by usine N-acetylmuramyl-L-alanyl-gamma-D-glutamyl-(L)-meso-[3H]diaminopimelic acid as a radioactive substrate. This activity cleaves the amide bond between the residues of N-acetylmuramyl acid and L-alanine. It was readily obtained in a soluble form either by mechanical disruption of the cells or by spheroplast formation. In the latter case the release of most of the activity into the sucrose medium seems to indicate that it is either periplasmic or associated with the outer membrane of the envelope of E. coli K 12. The enzyme was purified to near homogeneity. A molecular weight of 39 000 was determined by gel filtration and confirmed by polyacrylamide gel electrophoresis. Further characterisation of this N-acetylmuramyl-L-alanine amidase activity was carried out by investigating several of its properties.

Alanine↗

PubMed1994

Cloning of the wide spectrum amidase gene from Brevibacterium sp. R312 by genetic complementation. Overexpression in Brevibacterium sp. and Escherichia coli.

The amiE gene of Brevibacterium sp. R312 encoding wide spectrum amidase was isolated by complementation of a Brevibacterium sp. mutant using a plasmid gene bank of chromosomal DNA. The amiE structural gene and its promoter were localized on a 1.8-kb fragment by subsequent subcloning and complementation studies. Another promoter localized in the pSR 1 fragment of the cloning vector was shown to be able to control amiE gene expression. In Brevibacterium sp., the investigation of amidase activities related to one copy of the gene suggested that the regulation of the amiE gene expression was under negative control. High expression levels have been obtained in Brevibacterium sp. and, after substitution of the amiE promoter by the tac promoter, in Escherichia coli.

Amidohydrolases↗

PubMed1994

Purification and Characterization of an l-Amino Amidase from Mycobacterium neoaurum ATCC 25795.

An l-amino amidase from Mycobacterium neoaurum ATCC 25795 responsible for the enantioselective resolution of dl-alpha-methyl valine amide was purified and characterized. The purification procedure included ammonium sulfate fractionation, gel filtration, and anion-exchange chromatography, which resulted in a homogeneous preparation of the enzyme with a native molecular mass of 136 kDa and a subunit molecular mass of 40 kDa. The purified enzyme displayed the highest activity at 50 degrees C and at pH 8.0 and 9.5. The enzyme was strongly inhibited by the metal-chelating agent 1,10-phenanthroline, the disulfide-reducing agent dithiothreitol, and the cysteine proteinase inhibitor iodoacetamide. The purified amino amidase showed a unique l-enantioselective activity towards a broad range of both alpha-H- and alpha-alkyl-substituted amino acid amides, with the highest activity towards the cyclic amino acid amide dl-proline amide. No activity was measured with dl-mandelic acid amide nor with the dipeptide l-phenylalanine-l-leucine. The highest catalytic efficiency (k(cat)/K(m) ratio) was measured with dl-alpha-allyl alanine amide, dl-alpha-methyl phenylalanine amide, and dl-alpha-methyl leucine amide.

Journal Article↗

PubMed1997

Formation of a Chiral Hydroxamic Acid with an Amidase from Rhodococcus erythropolis MP50 and Subsequent Chemical Lossen Rearrangement to a Chiral Amine.

The amidase from Rhodococcus erythropolis MP50 demonstrated, in the presence of hydroxylamine, acyltransferase activity and catalyzed the formation of hydroxamates from amides and hydroxylamine. The rates of acyltransferase activity of the purified amidase for the substrates acetamide, phenylacetamide, and 2-phenylpropionamide were higher than the corresponding rates for the hydrolysis reactions. With the substrate 2-phenylpropionamide the hydrolysis reaction and the acyltransferase activity were highly enantioselective. The optically active 2-phenylpropionhydroxamate was converted by a chemical Lossen rearrangement in an aqueous medium into the enantiopure S-1-phenylethylamine.

Journal Article↗

PubMed1998

Acyl transfer activity of an amidase from Rhodococcus sp. strain R312: formation of a wide range of hydroxamic acids.

The enantioselective amidase from Rhodococcus sp. strain R312 was produced in Escherichia coli and was purified in one chromatographic step. This enzyme was shown to catalyze the acyl transfer reaction to hydroxylamine from a wide range of amides. The optimum working pH values were 7 with neutral amides and 8 with alpha-aminoamides. The reaction occurred according to a Ping Pong Bi Bi mechanism. The kinetic constants demonstrated that the presence of a hydrophobic moiety in the carbon side chain considerably decreased the Km(amide) values (e.g., Km(amide) = 0.1 mM for butyramide, isobutyramide, valeramide, pivalamide, hexanoamide, and benzamide). Moreover, very high turnover numbers (kcat) were obtained with linear aliphatic amides (e.g., kcat = 333 s-1 with hexanoamide), whereas branched-side-chain-, aromatic cycle- or heterocycle-containing amides were sterically hindered. Carboxylic acids, alpha-amino acids, and methyl esters were not acyl donors or were very bad acyl donors. Only amides and hydroxamic acids, both of which contained amide bonds, were determined to be efficient acyl donors. On the other hand, the highest affinities of the acyl-enzyme complexes for hydroxylamine were obtained with short, polar or unsaturated amides as acyl donors (e.g., KmNH2OH = 20, 25, and 5 mM for acetyl-, alanyl-, and acryloyl-enzyme complexes, respectively). No acyl acceptors except water and hydroxylamine were found. Finally, the purified amidase was shown to be L-enantioselective towards alpha-hydroxy- and alpha-aminoamides.

Acylation↗