Structural and biochemical characterization of a nitrilase from the thermophilic bacterium, Geobacillus pallidus RAPc8
Geobacillus pallidus RAPc8 (NRRL: B-59396) is a moderately thermophilic gram-positive bacterium, originally isolated from Australian lake sediment. The G. pallidus RAPc8 gene encoding an inducible nitrilase was located and cloned using degenerate primers coding for well-conserved nitrilase sequences, coupled with inverse PCR. The nitrilase open reading frame was cloned into an expression plasmid and the expressed recombinant enzyme purified and characterized. The protein had a monomer molecular weight of 35,790 Da, and the purified functional enzyme had an apparent molecular weight of ~600 kDa by size exclusion chromatography. Similar to several plant nitrilases and some bacterial nitrilases, the recombinant G. pallidus RAPc8 enzyme produced both acid and amide products from nitrile substrates. The ratios of acid to amide produced from the substrates we tested are significantly different to those reported for other enzymes, and this has implications for our understanding of the mechanism of the nitrilases which may assist with rational design of these enzymes. Electron microscopy and image classification showed complexes having crescent-like, “c-shaped”, circular and “figure-8” shapes. Protein models suggested that the various complexes were composed of 6, 8, 10 and 20 subunits, respectively.
KeywordsNitrilase Geobacillus pallidus Tetrahedral intermediate
We would like to thank Roger Hunter for stimulating discussions concerning the chemistry of the tetrahedral intermediate. DW was supported by a NRF and DAAD bursary. KD was supported by a Harry Crossley fellowship. Much of the work was funded by the Carnegie Corporation of New York through the Joint UCT/UWC Masters programme in structural biology. DAC also thanks the South African National Research Foundation (Institutional Research Development Program) for financial support. AV was supported by the Carnegie Corporation of New York.
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