A novel l-leucine 5-hydroxylase from Nostoc piscinale unravels unexpected sulfoxidation activity toward l-methionine

Publication date: September 2018 Source:Protein Expression and Purification, Volume 149 Author(s): Dengyue Sun, Dengke Gao, Panpan Xu, Qianqian Guo, Zhangliang Zhu, Xiaotao Cheng, Song Bai, Hui-Min Qin, Fuping Lu Hydroxy amino acids are produced by Fe(II)/αKG-dependent dioxygenases and used widely as medicinal intermediates for chemical synthesis. A novel l-leucine 5-hydroxylase gene from Nostoc piscinale (NpLDO) was cloned into pET28a (+), pColdI and pQE-80 L plasmids. Using a two-step purification process (Ni-affinity chromatography and gel filtration), highly purified recombinant NpLDO was obtained. Recombinant NpLDO displayed unexpectedly high sulfoxidation activity toward l-methionine. The reaction products were analyzed by high-performance liquid chromatography. Sequence alignment analysis implied that residues of His150, His236 and Asp152 constitute the catalytic triad of NpLDO, which is completely conserved in the Fe(II)/αKG-dependent dioxygenase superfamily. Biochemical data showed that NpLDO catalyzed regio- and stereoselective hydroxylation of l-leucine and sulfoxidation of l-methionine with Fe(II) and l-ascorbic acid as cofactor, and αKG as cosubstrate, respectively.
Source: Protein Expression and Purification - Category: Biochemistry Source Type: research