期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 149, 期 -, 页码 1-6出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2018.04.009
关键词
Hydroxylation; Fe(II)/alpha-Ketoglutarate-dependent dioxygenase; Sulfoxidation; Enzyme activity
类别
资金
- National Key Research and Development Project of China [2017YFD0400304]
- National Natural Science Foundation of China [31771911]
- Natural Science Foundation of Tianjin [16JCQNJC09200]
- Overseas High-level Talents Program of Tianjin University of Science and Technology
Hydroxy amino acids are produced by Fe(II)/alpha KG-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)/alpha KG-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 alpha KG as cosubstrate, respectively.
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