Cloning, Sequencing, Heterologous Expression, Purification, and Characterization of Adenosylcobalamin-dependentd-Ornithine Aminomutase from Clostridium sticklandii
作者:Hao-Ping Chen、Shih-Hsiung Wu、Ya-Lin Lin、Chih-Ming Chen、San-San Tsay
DOI:10.1074/jbc.m108365200
日期:2001.11
D-Ornithine aminomutase from Clostridium sticklandii catalyzes the reversible rearrangement of d-ornithine to (2R,4S)-2,4-diaminopentanoic acid. The two genes encoding d-ornithine aminomutase have been cloned, sequenced, and expressed in Escherichia coli. The oraS gene, which encodes a protein of 121 amino acid residues with M(r) 12,800, is situated upstream of the oraE gene, which encodes a protein
来自粘梭菌的D-鸟氨酸氨基变位酶催化D-鸟氨酸向(2R,4S)-2,4-二氨基戊酸的可逆重排。编码d-鸟氨酸氨基变位酶的两个基因已在大肠杆菌中克隆,测序和表达。oraS基因(编码具有121个氨基酸残基的蛋白具有M(r)12,800)位于oraE基因的上游,其编码具有753个氨基酸残基的蛋白具有M(r)82,900。全酶似乎包含α(2)β(2)-异四聚体。OraS与Swiss-Prot数据库中的其他蛋白质没有明显的同源性。OraE推导的氨基酸序列包括保守的碱基间隔/组氨酸上的钴胺素结合基序DXHXXG。OraE在大肠杆菌中表达为包涵体。在OraE上进行重折叠实验表明,OraS和OraE之间的相互作用以及磷酸吡ido醛或腺苷钴胺素的结合在重折叠过程中起着重要作用。d-鸟氨酸,5'-脱氧腺苷钴胺素(AdoCbl)和吡ido醛5'-磷酸酯(PLP)的K(m)值分别为44.5 +/- 2.8、0.43