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 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.
Williamson
Dael S
DS
Electron Microscope Unit, University of Cape Town, Rondebosch, Cape Town, 7701, South Africa.
Dent
Kyle C
KC
Weber
Brandon W
BW
Varsani
Arvind
A
Frederick
Joni
J
Thuku
Robert N
RN
Cameron
Rory A
RA
van Heerden
Johan H
JH
Cowan
Donald A
DA
Sewell
B Trevor
BT
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
D017931:DNA Primers; D009570:Nitriles; D021122:Protein Subunits; D011994:Recombinant Proteins; D000619:Aminohydrolases; C021867:nitrilase