Rapid and specific identification of nitrile hydratase (NHase)-encoding genes in soil samples by polymerase chain reaction

Precigou, Sylvain; Goulas, Philippe; Duran, Robert

doi:10.1111/j.1574-6968.2001.tb10879.x

Cited by 57 publications

(30 citation statements)

References 25 publications

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“…A total of 73 microbial nitrile hydratase ␣-subunit amino acid sequences and 66 ␤-subunit sequences were aligned. Nitrile hydratases have a highly conserved CXLCSC motif, where "X" corresponds to a T (threonine) residue for cobalt-type and an S (serine) residue for iron-type nitrile hydratases (61). Analysis of the multiple sequence alignment reveals that the metal binding motif was conserved in all ␣-subunit sequences at positions 98 to 103.…”

Section: -H-e-g-r-c-f-v-vmentioning

confidence: 99%

Characterization of a Nitrilase and a Nitrile Hydratase from Pseudomonas sp. Strain UW4 That Converts Indole-3-Acetonitrile to Indole-3-Acetic Acid

Duca

Rose

Glick

2014

Appl Environ Microbiol

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Indole-3-acetic acid (IAA) is a fundamental phytohormone with the ability to control many aspects of plant growth and development. Pseudomonas sp. strain UW4 is a rhizospheric plant growth-promoting bacterium that produces and secretes IAA. While several putative IAA biosynthetic genes have been reported in this bacterium, the pathways leading to the production of IAA in strain UW4 are unclear. Here, the presence of the indole-3-acetamide (IAM) and indole-3-acetaldoxime/indole-3-acetonitrile (IAOx/IAN) pathways of IAA biosynthesis is described, and the specific role of two of the enzymes (nitrilase and nitrile hydratase) that mediate these pathways is assessed. The genes encoding these two enzymes were expressed in Escherichia coli, and the enzymes were isolated and characterized. Substrate-feeding assays indicate that the nitrilase produces both IAM and IAA from the IAN substrate, while the nitrile hydratase only produces IAM. The two nitrile-hydrolyzing enzymes have very different temperature and pH optimums. Nitrilase prefers a temperature of 50°C and a pH of 6, while nitrile hydratase prefers 4°C and a pH of 7.5. Based on multiple sequence alignments and motif analyses, physicochemical properties and enzyme assays, it is concluded that the UW4 nitrilase has an aromatic substrate specificity. The nitrile hydratase is identified as an iron-type metalloenzyme that does not require the help of a P47K activator protein to be active. These data are interpreted in terms of a preliminary model for the biosynthesis of IAA in this bacterium.

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Section: -H-e-g-r-c-f-v-vmentioning

confidence: 99%

Characterization of a Nitrilase and a Nitrile Hydratase from Pseudomonas sp. Strain UW4 That Converts Indole-3-Acetonitrile to Indole-3-Acetic Acid

Duca

Rose

Glick

2014

Appl Environ Microbiol

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“…The fact that 14 of the 16 actinomycetes screened by Precigou et al (69) for NHase genes did not produce expected PCR products, even with Co-type NHase primers, could have been because (i) the primers that were designed did not amplify the presumed NHases present in these bacteria or (ii) NHase genes were not present in these bacteria. Resolution of these questions is difficult, because the authors did not report whether they checked for growth on nitriles.…”

Section: Discussionmentioning

confidence: 91%

“…This finding suggests that these genes, which have adjacent locations in many of the NHaseamidase systems that have been genetically analyzed (42), have undergone coevolution. Comparison of amidase phylogeny with NHase phylogeny revealed similar evolution of these two enzymes (3,15,69). The physical proximity and coevolution of NHase and amidase genes point to the emergence of this gene cluster for a specific metabolic function, namely, the transformation of nitriles, via amides, into acids.…”

Section: Discussionmentioning

confidence: 98%

“…69,2003 NHase AND AMIDASE GENE DIVERSITY IN R. ERYTHROPOLIS 5761 enzymes with unusual characteristics. This strategy has been tested by Precigou et al (69) for rapid and specific identification of NHase-encoding genes in soils.…”

Section: Discussionmentioning

confidence: 99%

“…69,2003 NHase AND AMIDASE GENE DIVERSITY IN R. ERYTHROPOLIS 5755 available in the DDBJ/EMBL/GenBank databases were aligned, but there was a low level of sequence homology between the genes encoding the iron-containing NHase enzymes of rhodococci and the genes encoding the high-and low-molecular-mass cobalt-containing NHase enzymes of R. rhodochrous J-1 (43) and M8 (79). Although we were interested in designing primers that amplified NHase genes from as many rhodococcal strains as possible, we biased the study for se- (62,86), N-774 (33), AJ270 (DDBJ/EMBL/GenBank accession number AJ278349), and ACV2 (DDBJ/ EMBL/GenBank accession number Z48769); the latter strain is a mutant of Brevibacterium sp.…”

Section: Molecular Screeningmentioning

confidence: 99%

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Diversity of Nitrile Hydratase and Amidase Enzyme Genes in Rhodococcus erythropolis Recovered from Geographically Distinct Habitats

Brandão

Clapp

Bull

2003

Appl Environ Microbiol

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A molecular screening approach was developed in order to amplify the genomic region that codes for the ␣-and ␤-subunits of the nitrile hydratase (NHase) enzyme in rhodococci. Specific PCR primers were designed for the NHase genes from a collection of nitrile-degrading actinomycetes, but amplification was successful only with strains identified as Rhodococcus erythropolis. A hydratase PCR product was also obtained from R. erythropolis DSM 43066 T , which did not grow on nitriles. Southern hybridization of other members of the nitrile-degrading bacterial collection resulted in no positive signals other than those for the R. erythropolis strains used as positive controls. PCR-restriction fragment length polymorphism-single-strand conformational polymorphism (PRS) analysis of the hydratases in the R. erythropolis strains revealed unique patterns that mostly correlated with distinct geographical sites of origin. Representative NHases were sequenced, and they exhibited more than 92.4% similarity to previously described NHases. The phylogenetic analysis and deduced amino acid sequences suggested that the novel R. erythropolis enzymes belonged to the iron-type NHase family. Some different residues in the translated sequences were located near the residues involved in the stabilization of the NHase active site, suggesting that the substitutions could be responsible for the different enzyme activities and substrate specificities observed previously in this group of actinomycetes. A similar molecular screening analysis of the amidase gene was performed, and a correlation between the PRS patterns and the geographical origins identical to the correlation found for the NHase gene was obtained, suggesting that there was coevolution of the two enzymes in R. erythropolis. Our findings indicate that the NHase and amidase genes present in geographically distinct R. erythropolis strains are not globally mixed.Nitrile hydratase (NHase) is a soluble bacterial metalloenzyme (41) that catalyzes the hydration of nitrile compounds to the corresponding amides, which may be converted by an amidase to the corresponding acids plus ammonia (40). NHase consists of two subunits (␣ and ␤), each with a molecular mass of approximately 23 kDa; the amino acid sequences of the subunits are not related, and the structural genes are normally adjacent in the same operon, although the ␣-and ␤-coding sequence order is variable (42). NHase is classified into two groups on the basis of the metal ion composing the catalytic center; an Fe-type NHase has a nonheme iron atom (74), and a Co-type NHase has a noncorrinoid cobalt atom (12) at the catalytic center. The Fe-and Co-type NHase enzymes differ in biotransformation activity and substrate specificity, although their amino acid sequences exhibit significant homology.NHase enzymes have considerable practical importance as biocatalysts for the industrial production of acrylamide and nicotinamide (84) and also for environmental bioremediation, where they have been shown to be effective in the removal of nitrile...

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Integron First Gene Cassettes: A Target to Find Adaptive Genes in Metagenomes

Huang¹,

Cagnon²

2011

Handbook of Molecular Microbial Ecology I

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Rapid and specific identification of nitrile hydratase (NHase)-encoding genes in soil samples by polymerase chain reaction

Cited by 57 publications

References 25 publications

Characterization of a Nitrilase and a Nitrile Hydratase from Pseudomonas sp. Strain UW4 That Converts Indole-3-Acetonitrile to Indole-3-Acetic Acid

Characterization of a Nitrilase and a Nitrile Hydratase from Pseudomonas sp. Strain UW4 That Converts Indole-3-Acetonitrile to Indole-3-Acetic Acid

Diversity of Nitrile Hydratase and Amidase Enzyme Genes in Rhodococcus erythropolis Recovered from Geographically Distinct Habitats

Integron First Gene Cassettes: A Target to Find Adaptive Genes in Metagenomes

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