title: Residue Y70 of the Nitrilase Cyanide Dihydratase fromBacillus pumilusIs Critical for Formation and Activity of the Spiral Oligomer.
abstract: Nitrilases pose attractive alternatives to the chemical hydrolysis of nitrile compounds. The activity of bacterial nitrilases towards substrate is intimately tied to the formation of large spiral-shaped oligomers. In the nitrilase CynD (cyanide dihydratase) fromBacillus pumilus, mutations in a predicted oligomeric surface region altered its oligomerization and reduced its activity. One mutant, CynD Y70C, retained uniform oligomer formation however it was inactive, unlike all other inactive mutants throughout that region all of which significantly perturbed oligomer formation. It was hypothesized that Y70 is playing an additional role necessary for CynD activity beyond influencing oligomerization. Here, we performed saturation mutagenesis at residue 70 and demonstrated that only tyrosine or phenylalanine is permissible for CynD activity. Furthermore, we show that other residues at this position are not only inactive, but have altered or disrupted oligomer conformations. These results suggest that Y70's essential role in activity is independent of its role in the formation of the spiral oligomer.
authors: Department of Biology, Texas A&M University, College Station, TX 77843-3258, USA.
authors: Jason M
authors: JM
authors: Park
authors: Department of Biology, Texas A&M University, College Station, TX 77843-3258, USA.
authors: Christian M
authors: CM
authors: Ponder
authors: Structural Biology Research Unit, Institute for Infectious Diseases and Molecular Medicine, University of Cape Town, Cape Town 7925, South Africa.
authors: B Trevor
authors: BT
authors: Sewell
authors: Department of Biology, Texas A&M University, College Station, TX 77843-3258, USA.
authors: Michael J
authors: MJ
authors: Benedik
mesh_terms: D020816:Amino Acid Motifs; D000619:Aminohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001407:Bacillus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009570:Nitriles / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D009570:Nitriles; D006867:Hydrolases; D000619:Aminohydrolases; C083488:cyanide dihydratase; C021867:nitrilase
keywords: Nitrilase; bioremediation; cyanide; cyanide dihydratase; oligomerization surface; quaternary structure