Structure of an aliphatic amidase from Geobacillus pallidus RAPc8

Kimani, S W; Agarkar, V B; Cowan, D A; Sayed, M F R; Sewell, B T

Structure of an aliphatic amidase from Geobacillus pallidus RAPc8

Journal Article

2007

Journal Title

Acta Crystallographica. Section D, Biological Crystallography

Link to Journal

http://journals.iucr.org/d/index.html

Publisher

International Union of Crystallography

Publisher

University of Cape Town

Abstract

The amidase from Geobacillus pallidus RAPc8, a moderate thermophile, is a member of the nitrilase superfamily and catalyzes the conversion of amides to the corresponding carboxylic acids and ammonia. It shows both amide-hydrolysis and acyl-transfer activities and also exhibits stereoselectivity for some enantiomeric substrates, thus making it a potentially important industrial catalyst. The crystal structure of G. pallidus RAPc8 amidase at a resolution of 1.9 Å was solved by molecular replacement from a crystal belonging to the primitive cubic space group P4232. G. pallidus RAPc8 amidase is homohexameric in solution and its monomers have the typical nitrilase-superfamily [alpha]-[beta]-[beta]-[alpha] fold. Association in the hexamer preserves the eight-layered [alpha]-[beta]-[beta]-[alpha]:[alpha]-[beta]-[beta]-[alpha] structure across an interface which is conserved in the known members of the superfamily. The extended carboxy-terminal tail contributes to this conserved interface by interlocking the monomers. Analysis of the small active site of the G. pallidus RAPc8 amidase suggests that access of a water molecule to the catalytic triad (Cys, Glu, Lys) side chains would be impeded by the formation of the acyl intermediate. It is proposed that another active-site residue, Glu142, the position of which is conserved in the homologues, acts as a general base to catalyse the hydrolysis of this intermediate. The small size of the substrate-binding pocket also explains the specificity of this enzyme for short aliphatic amides and its asymmetry explains its enantioselectivity.