Kubota Takaaki, Izumi Yoshikazu
Department of Biotechnology, Tottori University, Tottori, Japan.
Biosci Biotechnol Biochem. 2012;76(4):685-90. doi: 10.1271/bbb.110807. Epub 2012 Apr 7.
By detailed BLAST searches of the genome database of various thermophiles, five ORFs with similarity to the bioF gene, which encodes 7-keto-8-aminopelargonic acid synthase (BioF) involved in biotin biosynthesis, of Escherichia coli were found: AqbioF, CltbioF, GkbioF, SytbioF, and TsebioF, from Aquifex aeolicus VF5, Clostridium thermocellum ATCC27405, Geobacillus kaustophilus JCM12893, Symbiobacterium thermophilum IAM14863, and Thermosynechococcus elongatus BP-1 respectively. The five purified recombinant bioF gene products, which were overexpressed in E. coli, had the enzyme activity of BioF. The optimum temperature range and thermostability of five BioFs, AqBioF, CltBioF, GkBioF, SytBioF, and TseBioF, were higher than those of E. coli BioF. In particular, AqBioF was found to show the highest thermostability of the α-oxoamine synthase family enzymes reported to date. Substrate specificity experiments revealed that SytBioF was also able to catalyze the reaction of 2-amino-3-ketobutyrate CoA ligase, a member of the α-oxoamine synthase family, and that it used acetyl-CoA and glycine as substrates, like the TTHA1582 protein of Thermus thermophilus. The other purified BioFs, AqBioF and GkBioF, did not show any activity with acyl-CoAs and amino acids other than pimeloyl-CoA and L-alanine as substrates.
通过对各种嗜热菌基因组数据库进行详细的BLAST搜索，发现了5个与大肠杆菌中编码参与生物素生物合成的7-酮-8-氨基壬酸合酶（BioF）的bioF基因具有相似性的开放阅读框（ORF）：分别来自嗜泉栖热袍菌VF5的AqbioF、嗜热栖热放线菌ATCC27405的CltbioF、嗜碱嗜热栖热放线菌JCM12893的GkbioF、嗜热栖热放线菌IAM14863的SytbioF以及嗜热栖热放线菌BP-1的TsebioF。在大肠杆菌中过表达的这5种纯化的重组bioF基因产物具有BioF的酶活性。5种BioF（AqBioF、CltBioF、GkBioF、SytBioF和TseBioF）的最适温度范围和热稳定性均高于大肠杆菌BioF。特别是，发现AqBioF具有迄今为止报道的α-氧代胺合酶家族酶中最高的热稳定性。底物特异性实验表明，SytBioF也能够催化α-氧代胺合酶家族成员2-氨基-3-酮丁酸辅酶A连接酶的反应，并且它像嗜热栖热放线菌的TTHA1582蛋白一样，以乙酰辅酶A和甘氨酸为底物。其他纯化的BioF（AqBioF和GkBioF）除了以庚二酰辅酶A和L-丙氨酸为底物外，对其他酰基辅酶A和氨基酸没有任何活性。
