Molecular analysis of the nitrile catabolism operon of the thermophile Bacillus pallidus RAPc8.
The gene cluster containing the nitrile hydratase (NHase) and amidase genes of a moderate thermophile, B. pallidus RAPc8 has been cloned and sequenced. The (5.9 kb) section of cloned DNA contained eight complete open reading frames, encoding (in order), amidase (belonging to the nitrilase related aliphatic amidase family), nitrile hydratase beta and alpha subunits (of the cobalt containing class), a 122-amino acid accessory protein, designated P14K, a homologue of the 2Fe-2S class of ferredoxins and three putative proteins with distinct homology to the cobalt uptake proteins cbiM, cbiN and cbiQ of the S. typhimurium LT2 cobalamin biosynthesis pathway. The amidase and nitrile hydratase genes were subcloned and inducibly expressed in Escherichia coli, to levels of approximately 37 U/mg and 49 U/mg, respectively, without the co-expression of additional flanking genes. However, co-expression of P14K with the NHase structural genes significantly enhanced the specific activity of the recombinant NHase. This is the first description of an accessory protein involved in thermostable NHase expression. Modelling of the P14K protein structure has suggested that this protein functions as a subunit-specific chaperone, aiding in the folding of the NHase alpha subunit prior to alpha-beta subunit association and the formation of alpha(2)beta(2) NHase holoenzyme.
Cameron
Rory A
RA
Advanced Research Centre for Applied Microbiology, Department of Biotechnology, University of the Western Cape, Bellville 7535, Cape Town, South Africa.
Sayed
Muhammed
M
Cowan
Don A
DA
D000581:Amidohydrolases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001407:Bacillus / Q000145:classification / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D003035:Cobalt / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D006836:Hydro-Lyases / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics; D009570:Nitriles / Q000378:metabolism*; D009876:Operon / Q000235:genetics*; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid
D009570:Nitriles; D003035:Cobalt; D000581:Amidohydrolases; D006836:Hydro-Lyases; C048831:nitrile hydratase