Investigation of the first step of biotin biosynthesis in Bacillus sphaericus. Purification and characterization of the pimeloyl-CoA synthase, and uptake of pimelate.
The pimeloyl-CoA synthase from Bacillus sphaericus has been purified to homogeneity from an overproducing strain of Escherichia coli. The purification yielded milligram quantities of the synthase with a specific activity of 1 unit/mg of protein. Analysis of the products showed that this enzyme catalysed the transformation of pimelate into pimeloyl-CoA with concomitant hydrolysis of ATP to AMP. Using a continuous spectrophotometric assay, we have examined the catalytic properties of the pure enzyme. The pH profile under Vmax. conditions showed a maximum around 8.5. Apparent Km values for pimelate, CoASH, ATP.Mg2- and Mg2+ were respectively 145 microM, 33 microM, 170 microM and 2.3 mM. The enzyme was inhibited by Mg2+ above 10 mM. This acid-CoA ligase exhibited a very sharp substrate specificity, e.g. neither GTP nor pimelate analogues (di- or mono-carboxylic acids) were processed. The bivalent metal ion requirement was also investigated: Mn2+ (73%) and Co2+ (32%) but not Ca2+ could replace Mg2+. The enzyme was inhibited by metal chelators such as 1,10-phenanthroline and EDTA. The synthase was a homodimer with a 28,000-M(r) subunit. N-Terminal sequencing definitely proved that this enzyme was encoded by the bioW gene. A careful study of pimelate uptake by B. sphaericus, E. coli and Pseudomonas dentrificans showed that this metabolite crossed the membrane of these microorganisms by passive diffusion, ruling out the involvement of the bioX gene product as pimelate carrier.
Ploux
O
O
Laboratoire de Chimie Organique Biologique URA CNRS 493, Université Pierre et Marie Curie Tour, Paris, France.
Soularue
P
P
Marquet
A
A
Gloeckler
R
R
Lemoine
Y
Y
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology / Q000378:metabolism*; D001710:Biotin / Q000096:biosynthesis*; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003066:Coenzyme A Ligases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010867:Pimelic Acids / Q000378:metabolism*; D013379:Substrate Specificity
D010867:Pimelic Acids; D001710:Biotin; D003066:Coenzyme A Ligases; C077998:pimeloyl-CoA synthetase