The quaternary structure of the amidase from Geobacillus pallidus RAPc8 is revealed by its crystal packing

Abstract

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 mM NaCl, 100 mM sodium acetate pH 5.6 were selected for X-ray diffraction studies. A data set having acceptable statistics to 1.96 A resolution was collected under cryoconditions using an in-house X-ray source. The space group was determined to be primitive cubic P4(2)32, with unit-cell parameter a = 130.49 (+/-0.05) A. 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 D3 point-group symmetry around the threefold axis in such a way that the dimer interface seen in the homologues is preserved.

Figure 1

SDS–PAGE of amidase purification. Lane M, molecular-weight markers (kDa); lane 1, induced cells; lane 2, clarified cell extract; lane 3, heat-treated supernatant; lane 4, pooled fractions from Phenyl-Sepharose hydrophobic interaction chromatography; lane 5, pooled fractions from Q-Sepharose ion-exchange chromatography; lane 6, pooled fractions from gel-exclusion chromatography.

Figure 2

A crystal of the amidase from G. pallidus RAPc8.

Figure 3

Alignment of the G. pallidus RAPc8 amidase sequence. A, cyanide dihydratase (Pseudomonas stutzeri AK61); B, amidase (G. pallidus RAPc8); C, Pseudomonas aeruginosa amidase model (PDB code 1k17); D, hypothetical protein PH0642 from Pyrococcus horikoshii (PDB code 1j31); E, putative CN hydrolase from yeast (PDB code 1f89); F, Nit domain of the NitFhit fusion protein (Caenorhabditis elegans; PDB code 1ems); G, Agrobacterium sp. strain KNK712 N-carbamoyl-d-amino-acid amidohydrolase (PDB code 1erz). The conserved Glu, Cys, Lys catalytic site is highlighted in pink. The central core of the enzymes is highlighted in yellow; the loop regions are highlighted in brown. The major contact surface leading to dimerization of the enzymes is highlighted in green. The second interacting surface occurring in 1ems and 1erz is highlighted in blue. The sequences of the proteins which have three-dimensional structures are indicated in JOY notation (Mizuguchi et al., 1998 ▶). The amidase has the following sequence identities: 1k17, 80.1%; 1j31, 22.1%; 1f89, 21.4%; 1ems, 16.7%; 1erz, 18.2%.

Figure 4

Stereoviews of the suggested solution hexamer viewed down one of the twofold axes from both directions. The preserved dimer surface is centred in (a). The interacting surfaces depicted in the foreground of (b) are based on the model of the putative CN hydrolase from yeast (PDB code 1f89). 66 C-terminal amino acids for each subunit are not depicted in this model.