Investigation of the first step of biotin biosynthesis in Bacillus sphaericus. Purification and characterization of the pimeloyl-CoA synthase, and uptake of pimelate

Abstract

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.

Cloning, sequencing, and characterization of the Bacillus subtilis biotin biosynthetic operon

“…The early steps of the pathway, those involved in the synthesis of pimeloyl-CoA, are less well understood (27,48). B. sphaericus contains an enzyme, pimeloyl-CoA synthetase (bioW), that converts pimelic acid to pimeloyl-CoA (17,43). E. coli lacks this enzyme and cannot use pimelic acid as an intermediate in biotin synthesis (17,27,48).…”

Purification, characterization, DNA sequence and cloning of a pimeloyl-CoA synthetase from Pseudomonas mendocina 35

“…The average V max for the enzyme was 25 µmol\min per mg of protein, which indicated that the enzyme lost some activity on storage or thawing compared with the freshly prepared enzyme (Table 2). In comparison with the biotin-pathwayspecific enzyme from Bacillus sphaericus (1.0 µmol\min per mg of protein) [4], the Ps. mendocina enzyme is between one and two orders of magnitude more active (77.0 µmol\min per mg of protein for the freshly prepared enzyme).…”

“…It is synthesized either directly from pimelic acid and CoA by pimeloyl-CoA synthetase, or by a modified fatty acid-synthesis pathway [1,2]. The pimeloyl-CoA synthetase from Bacillus megaterium has been partially purified and characterized [3], and that from Bacillus sphaericus has been expressed in Escherichia coli and purified to homogeneity [4]. This enzyme is highly specific for pimelic acid and is the product of the bioW gene, suggesting that it has a unique role in biotin biosynthesis in this bacterium.…”

Genetic Analysis of an IncompletebioOperon in a Biotin Auxotrophic Strain ofBacillus subtilisNatto OK2

“…E. coli contains two genes; bioC, which is located in the bio operon, and bioH, which is not linked to the other bio genes, but the roles of these two genes have yet to be identified. On the other hand, B. subtilis contains the bioW gene encoding pimeloyl-CoA synthetase, which is also found in Bacillus sphaericus, 5) and the bioI gene, which shows no homology to either bioC or bioH but is able to complement in either bioC or bioH of E. coli mutants. 3) B. subtilis natto is a commercially important microorganism used in the fermentation of soybeans to make ''natto'', a popular food in Japan.…”