Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.5.1.4 (deaminase)
 5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Southern hybridization analysis using the genes encoding the alpha- and beta-subunits of nitrile hydratase (NHase) from Rhodococcus sp. N-774 as probe suggested that two R. erythropolis strains, JCM6823 and JCM2892, among 31 strains mainly from Japan Culture of Microorganisms (JCM) have NHase genes. Restriction analysis of DNA fragments showing positive hybridization showed that each fragment carried a nucleotide sequence very similar to that of the NHase genes from Rhodococcus sp. N-774. Nucleotide sequence analysis of the DNA fragment cloned from R. erythropolis JCM6823 showed the presence of the genes encoding the alpha- and beta-subunits of NHase, which show 94.7% and 96.2% identity in amino acid sequence to those of Rhodococcus sp. N-774, respectively, as well as a C-terminal portion of the amidase gene upstream from these genes. Despite the extremely high amino acid sequence similarity in both NHases and amidases from R. erythropolis JCM6823 and Rhodococcus sp. N-774, the NHases and amidases from R. erythropolis strains showed broader substrate specificity when compared to those from Rhodococcus sp. N-774. This suggests that a very limited number of amino acid residues are responsible for the difference in substrate specificity. Although the NHase of Rhodococcus sp. N-774 are constitutively produced, the NHases of both R. erythropolis strains were inducibly produced by addition of epsilon-caprolactam as an inducer.
 ...
PMID:Characterization of nitrile hydratase genes cloned by DNA screening from Rhodococcus erythropolis. 776 17

A new soil isolate, tentatively identified as Rhodococcus equi TG328, was found to be effective in the production of S-(+)-2-phenylpropionic acid from (R,S)-2-phenylpropionitrile. The conversion is catalysed by two enzymes. First, a nitrile hydratase converts the (R,S)-nitrile to (R,S)-2-phenylpropionamide. Second, a stereoselective amidase converts the S-(+)-amide to S-(+)-2-phenylpropionic acid. Conditions for optimal enzyme production and accumulation of S-(+)-2-phenylpropionic acid by resting cells were studied. The reaction of resting cells for 30 h at 10 degrees C with (R,S)-2-phenylpropionitrile resulted in the production of 100 g of S-(+)-2-phenylpropionic acid per litre of reaction mixture. The enantiometric excess of the purified S-(+)-2-phenylpropionic acid was 99.4%. The amount of S-(+)-2-phenylpropionic acid accumulated was enhanced by lower reaction temperatures. In addition, unreacted R-(-)-2-phenylpropionamide with 99.0% enantiometric excess was isolated.
 ...
PMID:Production of S-(+)-2-phenylpropionic acid from (R,S)-2-phenylpropionitrile by the combination of nitrile hydratase and stereoselective amidase in Rhodococcus equi TG328. 776 17

For improvement of the production of nitrile hydratase (NHase) from Rhodococcus sp. N-774 by recombinant DNA techniques, several plasmids, each of which had a deletion of the upstream or downstream region of the genes encoding the alpha and beta subunits of NHase, were constructed. Enzyme assays of recombinant R. rhodochrous and Escherichia coli cells showed that a downstream region of the NHase genes was indispensable for the production of active NHase in both cells, but for the production of the active amidase, no genes other than the amidase structural gene were required. The nucleotide sequence of the downstream region contained a single open reading frame (Orf1188) with 396 amino acids. Orf1188 showed similarity in amino acid sequence to P47K, an open reading frame found downstream of the NHase genes from Pseudomonas chlororaphis B23, and also to the cobW gene product, which may be involved in cobalamin biosynthesis in Pseudomonas denitrificans. Because the distance between the TGA stop codon for the NHase beta-subunit and the ATG codon for Orf1188 is only 98 bp, and because production of both Orf1188 and NHase is dependent on a promoter upstream of the amidase gene, these genes appear to be co-transcribed in a polycistronic manner, forming an operon. By optimization of the culture conditions of R. rhodochrous carrying pKRNH2, which contained the amidase, NHase, and Orf1188 genes, the transformant showed the NHase activity 6-fold higher than that of the original strain, Rhodococcus sp. N-774.
 ...
PMID:Nitrile hydratase gene from Rhodococcus sp. N-774 requirement for its downstream region for efficient expression. 776 11

The bacterial strain Rhodococcus butanica (ATCC 21197), which exhibits nitrilase and nitrile hydratase/amidase activities, catalyses the enantioselective hydrolysis of racemic naproxen nitrile (R/S)-1 to furnish a moderate enantiomeric excess of (S)-naproxen (S)-3. Racemic naproxen amide (R/S)-2 is not a good substrate for this strain. Resting cells of the newly selected bacterial strain Rhodococcus sp. C3II catalyse the enantioselective hydrolyses of racemic naproxen nitrile (R/S)-1 and naproxen amide (R/S)-2 as well, to give (S)-3 in excellent optical (99% e.e.) and good chemical yields in aqueous medium and in the biphasic system of phosphate buffer/hexane.
 ...
PMID:Enzyme-catalysed enantioselective hydrolysis of racemic naproxen nitrile. 785 80

The cloned 9.4-kb insert of plasmid pNHJ20L containing low-molecular-mass nitrile hydratase (L-NHase) gene from Rhodococcus rhodochrous J1 [Kobayashi, M. et al. (1991) Biochim. Biophys. Acta 1129, 23-33] was digested with various restriction enzymes, and the trimmed fragments were inserted into pUC18 or pUC19. A 1.96-kb EcoRI-SphI region located 1.9-kb downstream of the L-NHase gene was found to be essential for the expression of amidase activity in Escherichia coli; the gene arrangement of the amidase and the NHase in R. rhodochrous J1 differed from those in Rhodococcus species including N-774 and Pseudomonas chlororaphis B23. The nucleotide-determined sequence indicated that the amidase consists of 515 amino acids (54626 Da) and the deduced amino acid sequence of the amidase had high similarity to those of amidases from Rhodococcus species including N-774 and P. chlororaphis B23 and to indole-3-acetamide hydrolase from Pseudomonas savastanoi. The amidase gene modified in the nucleotide sequence upstream from its start codon expressed 8% of the total soluble protein in E. coli under the control of lac promoter. The level of amidase activity in cell-free extracts of E. coli was 0.468 unit/mg using benzamide as a substrate. This amidase was purified to homogeneity from extracts of the E. coli transformant with 30.4% overall recovery. The molecular mass of the enzyme estimated by HPLC was about 110 kDa and the enzyme consists of two subunits identical in molecular mass (55 kDa). The enzyme acted upon aliphatic amides such as propionamide and also upon aromatic amides such as benzamide. The apparent Km values for propionamide and benzamide were 0.48 mM and 0.15 mM, respectively. This amidase was highly specific for the S-enantiomer of 2-phenylpropionamide, but could not recognize the configuration of 2-chloropropionamide. It also catalyzed the transfer of an acyl group from an amide to hydroxylamine to produce the corresponding hydroxamate.
 ...
PMID:Amidase coupled with low-molecular-mass nitrile hydratase from Rhodococcus rhodochrous J1. Sequencing and expression of the gene and purification and characterization of the gene product. 791 90

A procedure for the assay of nitrile hydratase and amidase activity by high-performance liquid chromatography is described. The method can be used to assay the intermediate compounds resulting from the hydrolysis of adiponitrile into adipic acid, and to determine the kinetics of the hydrolysis of these compounds using whole cells and enzyme extracts. The precision of the method makes it suitable for the determination of the enzyme parameters: Km and Vm (nitrile hydratase and amidase). Using cyanovaleramide as substrate, Km and Vm were respectively 370 mM and 2060 U/mg for nitrile hydratase and 6.6 mM and 33 U/mg for amidase.
 ...
PMID:Application of high-performance liquid chromatography to the study of the biological transformation of adiponitrile. 795 29

The enantiomerically pure (S)-cyano acids 3 and 4 can be obtained by biotransformation with Brevibacterium sp. R 312 of the corresponding prochiral dinitriles 5 and 6, respectively. The hydrolysis is probably a two step process involving a nitrile hydratase and an amidase. In connection with these investigations a facile method for the synthesis of racemic 4-cyano-3-hydroxybutanoic acid derivatives was developed.
 ...
PMID:Microbial hydrolysis of glutaronitrile derivatives with Brevibacterium sp. R 312. 800 Aug 67

The two restriction enzymes AsnI and DraI were found to produce DNA fragment sizes that could be used for mapping the Rhodococcus sp. R312 (formerly Brevibacterium sp. R312) genome by pulsed-field gel electrophoresis. AsnI produced 24 fragments (4 to 727 kb) and DraI yielded 15 fragments (8.5 to 2400 kb). The fragment lengths in each digest were summed, indicating that the size of the chromosome ranged from 6.31 to 6.56 Mb, with a mean of 6.44 Mb. In addition, the wide-spectrum amidase gene (amiE) and the operon containing the enantiomer-selective amidase gene (amdA) and the nitrile hydratase structural gene (nthA, nthB) were localized on the AsnI and DraI fragments.
 ...
PMID:Sizing of the Rhodococcus sp. R312 genome by pulsed-field gel electrophoresis. Localization of genes involved in nitrile degradation. 854 55

The 4.6-kb region 5'-upstream from the gene encoding a cobalt-containing and amide-induced high molecular mass-nitrile hydratase (H-NHase) from Rhodococcus rhodochrous J1 was found to be required for the expression of the H-NHase gene with a host-vector system in a Rhodococcus strain. Sequence analysis has revealed that there are at least five open reading frames (H-ORF1 approximately 5) in addition to H-NHase alpha- and beta-subunit genes. Deletion of H-ORF1 and H-ORF2 resulted in decrease of NHase activity, suggesting a positive regulatory role of both ORFs in the expression of the H-NHase gene. H-ORF1 showed significant similarity to a regulatory protein, AmiC, which is involved in regulation of amidase expression by binding an inducer amide in Pseudomonas aeruginosa. H-ORF4, which has been found to be uninvolved in regulation of H-NHase expression by enzyme assay for its deletion transformant and Northern blot analysis for R. rhodochrous J1, showed high similarity to transposases from insertion sequences of several bacteria. Determination of H-NHase activity and H-NHase mRNA levels in R. rhodochrous J1 has indicated that the expression of the H-NHase gene is regulated by an amide at the transcriptional level. These findings suggest the participation of H-ORF4 (IS1164) in the organization of the H-NHase gene cluster and the involvement of H-ORF1 in unusual induction mechanism, in which H-NHase is formed by amides (the products in the NHase reaction), but not by nitriles (the substrates).
 ...
PMID:Characterization of the gene cluster of high-molecular-mass nitrile hydratase (H-NHase) induced by its reaction product in Rhodococcus rhodochrous J1. 863 53

The 3.5 kilobases (kb) of the 5'-upstream region from nhlBA encoding a cobalt-containing low molecular mass nitrile hydratase (L-NHase) from Rhodococcus rhodochrous J1 was found to be required for the amide-dependent expression of nhlBA in experiments using a Rhodococcus transformation system. Sequence analysis of the 3.5-kb fragment revealed the presence of two open reading frames (nhlD and nhlC) in this fragment. NhlD has similarity to regulators MerR, CadC, and ArsR. NhlC has similarity to the regulators AmiC, for the expression of an aliphatic amidase from Pseudomonas aeruginosa, and NhhC, for the expression of a high molecular mass nitrile hydratase from R. rhodochrous J1. Assays of NHase activity of transformants carrying nhlD deletion or nhlC deletion mutations suggest a negative regulatory role for nhlD and a positive regulatory role for nhlC in the process of the L-NHase formation. Assays of NHase and amidase activities and Western blot analyses of each Rhodococcus transformant carrying various deletion plasmids, have shown that nhlBA and amdA encoding an amidase, which is located 1.9 kb downstream of nhlBA, were regulated in the same manner. These findings present the genetic evidence for a novel gene cluster controlling the expression of L-NHase, which is induced by the reaction product (amide) in the "practical microorganism" R. rhodochrous J1.
 ...
PMID:A novel gene cluster including the Rhodococcus rhodochrous J1 nhlBA genes encoding a low molecular mass nitrile hydratase (L-NHase) induced by its reaction product. 866 59


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>