The signature amidase from Sulfolobus solfataricus belongs to the CX3C subgroup of enzymes cleaving both amides and nitriles. Ser195 and Cys145 are predicted to be the active site nucleophiles (original) (raw)
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The catalytic mechanism of amidase also involves nitrile hydrolysis
FEBS Letters, 1998
The amidase from Rhodococcus rhodochrous J1, which hydrolyzes an amide to an acid and ammonium, was surprisingly found to catalyze the hydrolytic cleavage of the C-N triple bond in a nitrile to form an acid and ammonium stoichiometrically. The amidase exhibited a K m of 3.26 mM for benzonitrile in contrast to that of 0.15 mM for benzamide as the original substrate, but the V mx for benzonitrile was about 1/6000 of that for benzamide. A mutant amidase containing alanine instead of Ser IWS , which is essential for amidase catalytic activity, showed no nitrilase activity, demonstrating that this residue plays a crucial role in the hydrolysis of nitriles as well as amides.
FEBS Journal, 2007
Nitrilases are useful industrial enzymes that convert nitriles to the corresponding carboxylic acids and ammonia. They belong to a superfamily [1] that includes amidases, acyl transferases and N-carbamoyld-amino acid amidohydrolases, and they occur in both prokaryotes and eukaryotes. Their applications include the manufacture of nicotinic acid, ibuprofen and acrylic acid and the detoxification of cyanide waste [2,3]. Although nitrilases hydrolyse a variety of nitriles, their natural substrates are, in general, not known. Environmental sampling and sequence analysis has substantially increased our knowledge of the distribution and specificity of these enzymes [4,5], but detailed structural information on nitrilases, which would enable a correlation between sequence and specificity, is not yet available. Members of this superfamily have a characteristic abba-fold, a conserved Glu, Lys, Cys catalytic triad and divergent N-and C-termini. The atomic structures of five homologous enzymes in the superfamily are known, namely the Nit domain of NitFhit fusion protein (1ems) [6], the N-carbamoyl-d-amino acid amidohydrolase (1erz, 1uf5) [7,8], the putative CN hydrolase from yeast (1f89) [9], the hypothetical protein PH0642 from Pyrococcus horikoshii (1j31) [10] and the amidase from Geobacillus pallidus RAPc8 [11]. All the structures are distant homologues having slightly > 20% identity. All have a twofold symmetry that conserves interactions between two helices at the subunit interface known as the A surface [12] (see supplementary Fig. S3). This leads to an extended abba-abba-fold. Although these nitrilase homologues exist as dimers or tetramers, microbial nitrilases occur as higher homo-oligomers [3]. Only in the case of the cyanide dihydratases [13,14] is there any information about the quaternary structure of the microbial nitrilases. The Pseudomonas stutzeri enzyme is an unusual, 14-subunit, self-terminating, homo-oligomeric spiral, whereas that from Bacillus pumilus shows reversible, pH-dependent, switching between an 18-subunit, self-terminating, spiral form and a variable-length, regular helix. Docking a homology model into the 3D reconstruction of the negative stain envelope of the cyanide dihydratase of
Nitrilase Catalyzes Amide Hydrolysis as Well as Nitrile Hydrolysis
Biochemical and Biophysical Research Communications, 1998
While amides were reported to be completely inert as substrates for all nitrilases reported to date, the nitrilase from Rhodococcus rhodochrous J1, which catalyzes the hydrolytic cleavage of the C-N triple bond in nitrile to form acid and ammonium, was surprisingly found to catalyze hydrolysis of amide to acid and ammonium stoichiometrically. This nitrilase exhibited a K m of 2.94 mM for benzamide, similar to that for benzonitrile as the original substrate (2.10 mM), but the V max for benzamide was six orders of magnitude lower than that for benzonitrile. Benzamide inhibited the nitrilase reaction in a reversible, apparently competitive manner. A mutant nitrilase containing alanine or serine instead of Cys165, which is essential for nitrilase catalytic activity, showed no amidase activity. This observation demonstrated that Cys165 plays a crucial role in the hydrolysis of amides as well as nitriles. Together with some reports that certain nitrilases were previously noted to produce low amounts of amide as a by-product from nitrile, the above unexpected findings suggested the existence of a common tetrahedral intermediate in the nitrilase reaction involving nitrile or amide as a substrate.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2010
In this study, we have structurally characterized the amidase of a nitrile-degrading bacterium, Rhodococcus sp. N-771 (RhAmidase). RhAmidase belongs to amidase signature (AS) family, a group of amidase families, and is responsible for the degradation of amides produced from nitriles by nitrile hydratase. Recombinant RhAmidase exists as a dimer of about 107 kDa. RhAmidase can hydrolyze acetamide, propionamide, acrylamide and benzamide with kcat/Km values of 1.14 ± 0.23 mM − 1 s − 1 , 4.54 ± 0.09 mM − 1 s − 1 , 0.087 ± 0.02 mM − 1 s − 1 and 153.5 ± 7.1 mM − 1 s − 1 , respectively. The crystal structures of RhAmidase and its inactive mutant complex with benzamide (S195A/benzamide) were determined at resolutions of 2.17 Å and 2.32 Å, respectively. RhAmidase has three domains: an N-terminal α-helical domain, a small domain and a large domain. The N-terminal α-helical domain is not found in other AS family enzymes. This domain is involved in the formation of the dimer structure and, together with the small domain, forms a narrow substratebinding tunnel. The large domain showed high structural similarities to those of other AS family enzymes. The Ser-cis Ser-Lys catalytic triad is located in the large domain. But the substrate-binding pocket of RhAmidase is relatively narrow, due to the presence of the helix α13 in the small domain. The hydrophobic residues from the small domain are involved in recognizing the substrate. The small domain likely participates in substrate recognition and is related to the difference of substrate specificities among the AS family amidases.
Applied Microbiology and Biotechnology, 2010
The nitrile hydratase (NHase, EC 4.2.1.84) genes (α and β subunit) and the corresponding activator gene from Rhodococcus equi TG328-2 were cloned and sequenced. This Fe-type NHase consists of 209 amino acids (α subunit, M r 23 kDa) and 218 amino acids (β subunit, M r 24 kDa) and the NHase activator of 413 amino acids (M r 46 kDa). Various combinations of promoter, NHase and activator genes were constructed to produce active NHase enzyme recombinantly in E. coli. The maximum enzyme activity (844 U/mg crude cell extract towards methacrylonitrile) was achieved when the NHase activator gene was separately co-expressed with the NHase subunit genes in E. coli BL21 (DE3). The overproduced enzyme was purified with 61% yield after French press, His-tag affinity chromatography, ultrafiltration and lyophilization and showed typical Fe-type NHase characteristics: besides aromatic and heterocyclic nitriles, aliphatic ones were hydrated preferentially. The purified enzyme had a specific activity of 6,290 U/mg towards methacrylonitrile. Enantioselectivity was observed for aromatic compounds only with E values ranging 5-17. The enzyme displayed a broad pH optimum from 6 to 8.5, was most active at 30°C and showed the highest stability at 4°C in thermal inactivation studies between 4°C and 50°C.
An Alternative Mechanism for Amidase Signature Enzymes
Journal of Molecular Biology, 2002
The peptide amidase from Stenotrophomonas maltophilia catalyses predominantly the hydrolysis of the C-terminal amide bond in peptide amides. Peptide bonds or amide functions in amino acid side-chains are not hydrolysed. This specificity makes peptide amidase (Pam) interesting for different biotechnological applications. Pam belongs to the amidase signature (AS) family. It is the first protein within this family whose tertiary structure has been solved. The structure of the native Pam has been determined with a resolution of 1.4A and in complex with the competitive inhibitor chymostatin at a resolution of 1.8A. Chymostatin, which forms acyl adducts with many serine proteases, binds non-covalently to this enzyme.Pam folds as a very compact single-domain protein. The AS sequence represents a core domain that is covered by alpha-helices. This AS domain contains the catalytic residues. It is topologically homologous to the phosphoinositol phosphatase domain. The structural data do not support the recently proposed Ser-Lys catalytic dyad mechanism for AS enzymes. Our results are in agreement with the role of Ser226 as the primary nucleophile but differ concerning the roles of Ser202 and Lys123: Ser202, with direct contact both to the substrate molecule and to Ser226, presumably serves as an acid/bases catalyst. Lys123, with direct contact to Ser202 but no contact to Ser226 or the substrate molecule, most likely acts as an acid catalyst.
The gene for an enantioselective amidase was cloned from Rhodococcus erythropolis MP50, which utilizes various aromatic nitriles via a nitrile hydratase/amidase system as nitrogen sources. The gene encoded a protein of 525 amino acids which corresponded to a protein with a molecular mass of 55.5 kDa. The deduced complete amino acid sequence showed homology to other enantioselective amidases from different bacterial genera. The nucleotide sequence approximately 2.5 kb upstream and downstream of the amidase gene was determined, but no indications for a structural coupling of the amidase gene with the genes for a nitrile hydratase were found. The amidase gene was carried by an approximately 40-kb circular plasmid in R. erythropolis MP50. The amidase was heterologously expressed in Escherichia coli and shown to hydrolyze 2-phenylpropionamide, ␣-chlorophenylacetamide, and ␣-methoxyphenylacetamide with high enantioselectivity; mandeloamide and 2-methyl-3-phenylpropionamide were also converted, but only with reduced enantioselectivity. The recombinant E. coli strain which synthesized the amidase gene was shown to grow with organic amides as nitrogen sources. A comparison of the amidase activities observed with whole cells or cell extracts of the recombinant E. coli strain suggested that the transport of the amides into the cells becomes the rate-limiting step for amide hydrolysis in recombinant E. coli strains.