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.

The First Thermophilic α-Oxoamine Synthase Family Enzyme That Has Activities of 2-Amino-3-ketobutyrate CoA Ligase and 7-Keto-8-aminopelargonic Acid Synthase: Cloning and Overexpression of the Gene from an Extreme Thermophile,Thermus thermophilus, and Characterization of Its Gene Product

“…This is similar to the structural composition previously reported for KAPA synthase from E. coli (41.7 kDa for each subunit), 30) but different from that of B. sphaericus (a monomer of 46 kDa). 12) The optimum temperature of the TTHA1582 protein was about 70 C, as high as the optimum growth temperature (65-72 C) of T. thermophilus HB8, 15) and the enzyme showed high thermostability (> 90%) during incubation at temperatures of up to 70 C. KAPA synthase (EC 2.3.1.47), KBL (EC 2.3.1.29), 5-aminolevulinate synthase (ALAS, EC 2.3.1.37), and serine C-palmitoyltransferase (SPT, EC 2.3.1.50) form theoxoamine synthase family.…”

“…All studies of the biosynthetic pathway of biotin, and related enzymes their regulatory mechanisms, and microbial biotin production have so far been performed exclusively using mesophilic organisms, such as microorganisms, including Bacillus sphaericus, 3,[6][7][8][9][10][11][12] Escherichia coli, 13) B. subtilis, and Saccharomyces cerevisiae, and plants, including Arabidopsis thaliana 14) and Lavandula vera. Moreover, from the standpoint of applied microbiology, there has been no report on the synthesis of biotin by combining enzymes, which have synthetic activities of biotin intermediates even if they are not originally biotin biosynthetic enzymes, from different organisms on a genetic level.…”

7-Keto-8-Aminopelagonic Acid Synthase as a Potential Herbicide Target

“…A number of researchers have widely investigated the biosynthetic pathway of biotin through combined biochemical and genetic studies in the bacteria (Ploux and Marquet, 1992;Alexeev et al, 1998;Huang et al, 1995). Also, there have been numerous studies on the related gene for biotin synthesis in a variety of other microorganisms (Zhang et al, 1994;Fleischmann et al, 1995;Bult et al, 1996).…”

“…For substrate synthesis and enzyme assay in vitro, substrate pimeloyl CoA was synthesized according to the method of Ploux and Marquet (1992). TPTA was purchased from Sigma (USA) and used as a KAPAS-inhibitor.…”

The Identification of Binding Mode for Arabidopsis thaliana 7-Keto-8-aminopelargonic Acid Synthase (AtKAPAS) Inhibitors

“…Pimeloyl CoA was synthesized according to the method described previously. 21 AtKAPAS activity was determined according to the method described previously …”