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An amino acid at position 142 in nitrilase from Rhodococcus rhodochrous ATCC 33278 determines the substrate specificity for aliphatic and aromatic nitriles.

ABSTRACT: Nitrilase from Rhodococcus rhodochrous ATCC 33278 hydrolyses both aliphatic and aromatic nitriles. Replacing Tyr-142 in the wild-type enzyme with the aromatic amino acid phenylalanine did not alter specificity for either substrate. However, the mutants containing non-polar aliphatic amino acids (alanine, valine and leucine) at position 142 were specific only for aromatic substrates such as benzonitrile, m-tolunitrile and 2-cyanopyridine, and not for aliphatic substrates. These results suggest that the hydrolysis of substrates probably involves the conjugated pi-electron system of the aromatic ring of substrate or Tyr-142 as an electron acceptor. Moreover, the mutants containing charged amino acids such as aspartate, glutamate, arginine and asparagine at position 142 displayed no activity towards any nitrile, possibly owing to the disruption of hydrophobic interactions with substrates. Thus aromaticity of substrate or amino acid at position 142 in R. rhodochrous nitrilase is required for enzyme activity.

SUBMITTER: Yeom SJ

PROVIDER: S-EPMC2570083 | biostudies-literature | 2008 Nov

REPOSITORIES: biostudies-literature

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An amino acid at position 142 in nitrilase from Rhodococcus rhodochrous ATCC 33278 determines the substrate specificity for aliphatic and aromatic nitriles.

Yeom Soo-Jin SJ Kim Hye-Jung HJ Lee Jung-Kul JK Kim Dong-Eun DE Oh Deok-Kun DK

The Biochemical journal 20081101 3

Nitrilase from Rhodococcus rhodochrous ATCC 33278 hydrolyses both aliphatic and aromatic nitriles. Replacing Tyr-142 in the wild-type enzyme with the aromatic amino acid phenylalanine did not alter specificity for either substrate. However, the mutants containing non-polar aliphatic amino acids (alanine, valine and leucine) at position 142 were specific only for aromatic substrates such as benzonitrile, m-tolunitrile and 2-cyanopyridine, and not for aliphatic substrates. These results suggest th ...[more]

PMID: 18412544

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Project description:BACKGROUND:Nitrilases are nitrile-converting enzymes commonly found within the plant kingdom that play diverse roles in nitrile detoxification, nitrogen recycling, and phytohormone biosynthesis. Although nitrilases are present in all higher plants, little is known about their function in trees. Upon herbivory, poplars produce considerable amounts of toxic nitriles such as benzyl cyanide, 2-methylbutyronitrile, and 3-methylbutyronitrile. In addition, as byproduct of the ethylene biosynthetic pathway upregulated in many plant species after herbivory, toxic ?-cyanoalanine may accumulate in damaged poplar leaves. In this work, we studied the nitrilase gene family in Populus trichocarpa and investigated the potential role of the nitrilase PtNIT1 in the catabolism of herbivore-induced nitriles. RESULTS:A BLAST analysis revealed three putative nitrilase genes (PtNIT1, PtNIT2, PtNIT3) in the genome of P. trichocarpa. While PtNIT1 was expressed in poplar leaves and showed increased transcript accumulation after leaf herbivory, PtNIT2 and PtNIT3 appeared not to be expressed in undamaged or herbivore-damaged leaves. Recombinant PtNIT1 produced in Escherichia coli accepted biogenic nitriles such as ?-cyanoalanine, benzyl cyanide, and indole-3-acetonitrile as substrates in vitro and converted them into the corresponding acids. In addition to this nitrilase activity, PtNIT1 showed nitrile hydratase activity towards ?-cyanoalanine, resulting in the formation of the amino acid asparagine. The kinetic parameters of PtNIT1 suggest that the enzyme utilizes ?-cyanoalanine and benzyl cyanide as substrates in vivo. Indeed, ?-cyanoalanine and benzyl cyanide were found to accumulate in herbivore-damaged poplar leaves. The upregulation of ethylene biosynthesis genes after leaf herbivory indicates that herbivore-induced ?-cyanoalanine accumulation is likely caused by ethylene formation. CONCLUSIONS:Our data suggest a role for PtNIT1 in the catabolism of herbivore-induced ?-cyanoalanine and benzyl cyanide in poplar leaves.

Dataset Information

An amino acid at position 142 in nitrilase from Rhodococcus rhodochrous ATCC 33278 determines the substrate specificity for aliphatic and aromatic nitriles.

Publications

An amino acid at position 142 in nitrilase from Rhodococcus rhodochrous ATCC 33278 determines the substrate specificity for aliphatic and aromatic nitriles.

Similar Datasets

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