Abstract
Early in sporulation, the mother cell compartment of Bacillus subtilis transcribes the mother cell metabolic gene (mmg) operon. The gene mmgA was assigned by other workers using sequence homology as an acetyl-CoA acetyltransferase [E.C. 2.3.1.9]. The gene was overexpressed in Escherichia coli, and the protein was purified by Ni2+-affinity chromatography. However, the expected MmgA-catalyzed biosynthesis of acetoacetyl-CoA from acetyl-CoA was undetectable by a standard UV assay, HPLC, and mass spectrometry. These methods indicated a preference for the reverse degradative thiolytic reaction, with a k cat of 80 s−1, and a K m of 70 and 50 μM for CoA and acetoacetyl-CoA, respectively.
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Brock M, Maerker C, Schutz A et al (2002) Oxidation of propionate to pyruvate in Escherichia coli: involvement of methylcitrate dehydratase and aconitase. Eur J Biochem 269:6184–6194
Bryan EM, Beall BW, Moran CP Jr (1996) A σE-dependent operon subject to catabolite repression during sporulation in Bacillus subtilis. J Bacteriol 178:4778–4786
Burns KL, Gelbaum LT, Sullards MC et al (2005) Iso-coenzyme A. J Biol Chem 280:16550–16558
Clinkenbeard KD, Sugiyama T, Moss J et al (1973) Molecular and catalytic properties of cytosolic acetoacetyl coenzyme A thiolase from avian liver. J Biol Chem 248:2275–2284
Doi RH (1989) Sporulation and germination, vol 2. In: Harwood CR (ed) Bacillus. Plenum Press, New York, pp 169–215
Georgiou G, Lin SC, Sharma MM (1992) Surface-active compounds from microorganisms. Bio/Technology 10:60–65
Haapalainen AM, Merilaeinen G, Wierenga RK (2006) The thiolase superfamily: condensing enzymes with diverse reaction specificities. Trends Biochem Sci 31:64–71
Haapalainen AM, Merilaeinen G, Pirila PL et al (2007) Crystallographic and kinetic studies of human mitochondrial acetoacetyl-CoA thiolase: the importance of potassium and chloride ions for its structure and function. Biochemistry 46:4305–4321
Jenkins LS, Nunn WD (1987) Genetic and molecular characterization of the genes involved in short-chain fatty-acid degradation in Escherichia coli—the Ato system. J Bacteriol 169:42–52
Kim SA, Copeland L (1997) Acetyl coenzyme A acetyltransferase of Rhizobium sp. (Cicer) strain CC 1192. Appl Environ Microb 63:3432–3437
Klein W, Weber MHW, Marahiel MA (1999) Cold shock response of Bacillus subtilis: isoleucine-dependent switch in the fatty acid branching pattern for membrane adaptation to low temperatures. J Bacteriol 181:5341–5349
Kobayashi K, Ehrlich SD, Albertini A et al (2003) Essential Bacillus subtilis genes. Proc Natl Acad Sci USA 100:4678–4683
Koburger T, Weibezahn J, Bernhardt J et al (2005) Genome-wide mRNA profiling in glucose starved Bacillus subtilis cells. Mol Genet Gen 274:1–12
Kunst F, Ogasawara N, Moszer I et al (1997) The complete genome sequence of the Gram-positive bacterium Bacillus subtilis. Nature 390:249–256
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2 edn. Cold Spring Harbor Laboratory Press, Plainview, New York
Spratt SK, Black PN, Ragozzino MM et al (1984) Cloning, mapping, and expression of genes involved in the fatty acid-degradative multienzyme complex of Escherichia coli. J Bacteriol 158:535–543
Stein T (2005) Bacillus subtilis antibiotics: structures, syntheses and specific functions. Mol Microbiol 56:845–857
Thompson S, Mayerl F, Peoples OP et al (1989) Mechanistic studies on β-ketoacyl thiolase from Zoogloea ramigera: identification of the active-site nucleophile as Cys89, its mutation to Ser89, and kinetic and thermodynamic characterization of wild-type and mutant enzymes. Biochemistry 28:5735–5742
Tobisch S, Zuhlke D, Bernhardt J et al (1999) Role of CcpA in regulation of the central pathways of carbon catabolism in Bacillus subtilis. J Bacteriol 181:6996–7004
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The authors gratefully acknowledge generous financial support from Research Corporation in the Cottrell College Science Award to JJR (CC6093). The MALDI-TOF-MS instrument was supported by the National Science Foundation under Grant No. 0420292.
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Reddick, J.J., Williams, J.K. The mmgA gene from Bacillus subtilis encodes a degradative acetoacetyl-CoA thiolase. Biotechnol Lett 30, 1045–1050 (2008). https://doi.org/10.1007/s10529-008-9642-4
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DOI: https://doi.org/10.1007/s10529-008-9642-4