Structural and biochemical characterization of a nitrilase from the thermophilic bacterium, Geobacillus pallidus RAPc8

Dael S. Williamson, Kyle C. Dent, Brandon W. Weber, Arvind Varsani, Joni Frederick, Robert N. Thuku, Rory A. Cameron, Johan H. Van Heerden, Donald A. Cowan, B. Trevor Sewell

Research output: Contribution to journalArticle

18 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)143-153
Number of pages11
JournalApplied Microbiology and Biotechnology
Volume88
Issue number1
DOIs
StatePublished - Sep 1 2010
Externally publishedYes

Keywords

  • Geobacillus pallidus
  • Nitrilase
  • Tetrahedral intermediate

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology

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    Williamson, D. S., Dent, K. C., Weber, B. W., Varsani, A., Frederick, J., Thuku, R. N., Cameron, R. A., Van Heerden, J. H., Cowan, D. A., & Sewell, B. T. (2010). Structural and biochemical characterization of a nitrilase from the thermophilic bacterium, Geobacillus pallidus RAPc8. Applied Microbiology and Biotechnology, 88(1), 143-153. https://doi.org/10.1007/s00253-010-2734-9