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.