title: The 8-amino-7-oxopelargonate synthase from Bacillus sphaericus. Purification and preliminary characterization of the cloned enzyme overproduced in Escherichia coli.
abstract: The 8-amino-7-oxopelargonate synthase [6-carboxyhexanoyl-CoA:L-alanine carboxyhexanoyltransferase (decarboxylating); EC 2.3.1.47] from Bacillus sphaericus involved in biotin biosynthesis was purified from an Escherichia coli overproducing strain. The purification afforded an electrophoretically homogeneous enzyme with a specific activity of 0.67 unit/mg. The purified enzyme is a monomer of 41 kDa. N-Terminal sequencing of the first 14 amino acid residues showed complete agreement with the predicted sequence from the bioF gene. The pure enzyme showed the characteristic absorption band (425 nm) of pyridoxal 5'-phosphate-dependent enzymes. Furthermore, the holoenzyme was resolved during an affinity step yielding the inactive apoenzyme, which recovered activity and the 425 nm-absorption band on dialysis against pyridoxal 5'-phosphate. Km values for L-alanine and pimeloyl-CoA were respectively 3 mM and 1 microM.
authors.affiliation: Laboratoire de Chimie Organique Biologique U.R.A. C.N.R.S. 493, Université Pierre et Marie Curie, France.
authors.forename: O
authors.initials: O
authors.lastname: Ploux
authors.forename: A
authors.initials: A
authors.lastname: Marquet
mesh_terms: D000217:Acyltransferases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D000596:Amino Acids; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D000217:Acyltransferases; C021929:8-amino-7-oxononanoate synthase