Oligomeric structure of nitrilases: effect of mutating interfacial residues on activity.
Nitrilases are important industrial enzymes that convert nitriles into their corresponding acids or, occasionally, amides. Atomic resolution structures of four members of the nitrilase superfamily have been determined, but these differ from microbial nitrilases in that they do not form typical large homo-oligomeric complexes. At least two nitrilases, the cyanide dihydratases from Pseudomonas stutzeri AK61 and Bacillus pumilus C1, form unusual spiral structures of 14 and 18 subunits, respectively. Evidence suggests that the formation of the spiral structure is essential for activity. Sequence analysis reveals that the nitrilases differ from the nonspiral-forming homologs by two insertions of between 12 and 14 amino acids and a C-terminal extension of up to 35 amino acids. The insertions are positioned at an intermolecular interface in the spiral and probably contribute to its formation. The other interfaces responsible for the formation and/or stabilization of the spirals can also be identified. Comparative structure modeling enables identification of the residues involved in these interacting surfaces, which are remote from the active site. Mutation of these interacting residues usually leads to loss of activity. The effect of the mutations on activity in most cases can be rationalized in terms of a possible effect on spiral formation.
Sewell
B T
BT
Electron Microscope Unit, IIDMM, University of Cape Town, 7701 Cape Town, South Africa. sewell@uctvms.uct.ac.za
Thuku
R N
RN
Zhang
X
X
Benedik
M J
MJ
D000595:Amino Acid Sequence; D000619:Aminohydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001407:Bacillus / Q000201:enzymology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D044223:Pseudomonas stutzeri / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D000619:Aminohydrolases; C021867:nitrilase