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

Microbial nitrilases: versatile, spiral forming, industrial enzymes

The nitrilases are enzymes that convert nitriles to the corresponding acid and ammonia. They are members of a superfamily, which includes amidases and occur in both prokaryotes and eukaryotes. The superfamily is characterized by having a homodimeric building block with a abba–abba sandwich fold and an active site containing four positionally conserved residues: cys, glu, glu and lys. Their high chemical specificity and frequent enantioselectivity makes them attractive biocatalysts for the production of fine chemicals and pharmaceutical intermediates. Nitrilases are also used in the treatment of toxic industrial effluent and cyanide remediation. The superfamily enzymes have been visualized as dimers, tetramers, hexamers, octamers, tetradecamers, octadecamers and variable length helices, but all nitrilase oligomers have the same basic dimer interface. Moreover, in the case of the octamers, tetradecamers, octadecamers and the helices, common principles of subunit association apply. While the range of industrially interesting reactions catalysed by this enzyme class continues to increase, research efforts are still hampered by the lack of a high resolution microbial nitrilase structure which can provide insights into their specificity, enantioselectivity and the mechanism of catalysis. This review provides an overview of the current progress in elucidation of structure and function in this enzyme class and emphasizes insights that may lead to further biotechnological applications

Biochemical and Mutational Studies of the Bacillus cereus CECT 5050T Formamidase Support the Existence of a C-E-E-K Tetrad in Several Members of the Nitrilase Superfamily ▿ †

Formamidases (EC 3.5.1.49) are poorly characterized proteins. In spite of this scarce knowledge, ammonia has been described as playing a central role in the pathogenesis of human pathogens such as Helicobacter pylori, for which formamidase has been shown to participate in the nitrogen metabolic pathway. Sequence analysis has revealed that at least two different groups of formamidases are classified as EC 3.5.1.49: on the one hand, the derivatives of the FmdA-AmdA superfamily, which are the best studied to date, and on the other hand, the derivatives of Helicobacter pylori AmiF. Here we present the cloning, purification, and characterization of a recombinant formamidase from Bacillus cereus CECT 5050T (BceAmiF), the second member of the AmiF subfamily to be characterized, showing new features of the enzyme further supporting its relationship with aliphatic amidases. We also present homology modeling-based mutational studies confirming the importance of the Glu140 and Tyr191 residues in the enzymatic activities of the AmiF family. Moreover, we can conclude that a second glutamate residue is critical in several members of the nitrilase superfamily, meaning that what has consistently been identified as a C-E-K triad is in fact a C-E-E-K tetrad