An amino acid at position 142 in nitrilase from Rhodococcus rhodochrous ATCC 33278 determines the substrate specificity for aliphatic and aromatic nitriles.
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.
Yeom
Soo-Jin
SJ
Department of Bioscience and Biotechnology, Konkuk University, Seoul, 143-701, Republic of Korea.
Kim
Hye-Jung
HJ
Lee
Jung-Kul
JK
Kim
Dong-Eun
DE
Oh
Deok-Kun
DK
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D000619:Aminohydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001120:Arginine / Q000737:chemistry; D001216:Asparagine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D002384:Catalysis; D018698:Glutamic Acid / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009570:Nitriles / Q000378:metabolism; D012240:Rhodococcus / Q000201:enzymology* / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
D000596:Amino Acids; D009570:Nitriles; D001224:Aspartic Acid; D018698:Glutamic Acid; D001216:Asparagine; D001120:Arginine; D000619:Aminohydrolases; C021867:nitrilase