title: Structural and biochemical characterization of a nitrilase from the thermophilic bacterium, Geobacillus pallidus RAPc8.
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 approximately 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.
authors.affiliation: Electron Microscope Unit, University of Cape Town, Rondebosch, Cape Town, 7701, South Africa.
authors.forename: Dael S
authors.initials: DS
authors.lastname: Williamson
authors.forename: Kyle C
authors.initials: KC
authors.lastname: Dent
authors.forename: Brandon W
authors.initials: BW
authors.lastname: Weber
authors.forename: Arvind
authors.initials: A
authors.lastname: Varsani
authors.forename: Joni
authors.initials: J
authors.lastname: Frederick
authors.forename: Robert N
authors.initials: RN
authors.lastname: Thuku
authors.forename: Rory A
authors.initials: RA
authors.lastname: Cameron
authors.forename: Johan H
authors.initials: JH
authors.lastname: van Heerden
authors.forename: Donald A
authors.initials: DA
authors.lastname: Cowan
authors.forename: B Trevor
authors.initials: BT
authors.lastname: Sewell
mesh_terms: D000595:Amino Acid Sequence; D000619:Aminohydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D016000:Cluster Analysis; D017931:DNA Primers / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D005822:Genetic Vectors; D056506:Geobacillus / Q000201:enzymology*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009570:Nitriles / Q000378:metabolism*; D010802:Phylogeny; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D055503:Protein Multimerization; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017385:Sequence Homology
chemical_list: D017931:DNA Primers; D009570:Nitriles; D021122:Protein Subunits; D011994:Recombinant Proteins; D000619:Aminohydrolases; C021867:nitrilase