Open AccessThe quaternary structure of the amidase from Geobacillus pallidus RAPc8 is revealed by its crystal packing
Author(s) -
Agarkar Vinod B.,
Kimani Serah W.,
Cowan Donald A.,
Sayed Muhammed F.R.,
Sewell B. Trevor
Publication year - 2006
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309106043855
Subject(s) - molecular replacement , dimer , geobacillus stearothermophilus , crystallography , thermophile , amidase , chemistry , multiple isomorphous replacement , crystal structure , stereochemistry , enzyme , peptide sequence , organic chemistry , biochemistry , gene
The amidase from Geobacillus pallidus RAPc8, a moderate thermophile, is a member of the nitrilase enzyme superfamily. It converts amides to the corresponding acids and ammonia and has application as an industrial catalyst. RAPc8 amidase has been cloned and functionally expressed in Escherichia coli and has been purified by heat treatment and a number of chromatographic steps. The enzyme was crystallized using the hanging‐drop vapour‐diffusion method. Crystals produced in the presence of 1.2 M sodium citrate, 400 m M NaCl, 100 m M sodium acetate pH 5.6 were selected for X‐ray diffraction studies. A data set having acceptable statistics to 1.96 Å resolution was collected under cryoconditions using an in‐house X‐ray source. The space group was determined to be primitive cubic P 4 2 32, with unit‐cell parameter a = 130.49 (±0.05) Å. The structure was solved by molecular replacement using the backbone of the hypothetical protein PH0642 from Pyrococcus horikoshii (PDB code 1j31 ) with all non‐identical side chains substituted with alanine as a probe. There is one subunit per asymmetric unit. The subunits are packed as trimers of dimers with D 3 point‐group symmetry around the threefold axis in such a way that the dimer interface seen in the homologues is preserved.