Purification and cloning of a proline 3-hydroxylase, a novel enzyme which hydroxylates free L-proline to cis-3-hydroxy-L-proline

Proline 3-hydroxylase was purified from Streptomyces sp, strain TH1, and its structural gene was cloned, The purified enzyme hydroxylated free L-proline to cis-3-hydroxy-L-proline and showed properties of a 2-oxoglutarate-dependent dioxygenase (H, Mori, T, Shibasaki, Y, Uosaki, K. Ochiai, and A, Ozaki, Appl, Environ, Microbiol. 62:1903-1907, 1996), The molecular mass of the purified enzyme was 35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, The isoelectric point of the enzyme was 4.3. The optimal pH and temperature were 7.0 and 35 degrees C, respectively, The K-m values were 0.56 and O.11 mM for L-proline and 2-oxoglutarate, respectively, The k(cat) value of hydroxylation was 3.2 s(-1). Determined N-terminal and internal amino acid sequences of the purified protein were not found in the SwissProt protein database, A DNA fragment of 74 bp was amplified by PCR with degenerate primers based on the determined N-terminal amino acid sequence, With this fragment as a template, a digoxigenin-labeled N-terminal probe was synthesized by PCR, A 6.5-kbp chromosome fragment was cloned by colony hybridization with the labeled probe, The determined DNA sequence of the cloned fragment revealed a 870-bp open reading frame (ORF 3), encoding a protein of 290 amino acids with a calculated molecular weight of 33,158, No sequence homolog was found in EMBL, GenBank, and DDBJ databases, ORF 3 was expressed in Escherichia coli DH1, Recombinants showed hydroxylating activity five times higher than that of the original bacterium, Streptomyces sp, strain TH1, It was concluded that the ORF 3 encodes functional proline 3-hydroxylase.