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crobiol. 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°C, respectively. The K m values were 0.56 and 0.11 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.Among eight stereoisomers of hydroxyprolines, only trans-4-hydroxy-L-proline is abundant in nature as a component of collagens produced by higher organisms (7). It is known that procollagen-proline dioxygenase (prolyl 4-hydroxylase; EC 1.14.11.2) hydroxylates L-proline residues of procollagen posttranslationally to trans-4-hydroxy-L-proline residues during collagen biosynthesis (7,14,21). Procollagen-proline 3-dioxygenase (prolyl 3-hydroxylase; EC 1.14.11.7), which hydroxylates peptidic L-proline residues to trans-3-hydroxy-L-proline, is also involved in collagen biosynthesis (11,17). However, free Lproline is not accepted as a substrate for these prolyl hydroxylases. These enzymes belong to a family of 2-oxoglutaratedependent dioxygenases which requires 2-oxoglutarate and O 2 as cosubstrates and ferrous ion as a cofactor for the reaction.In contrast, hydroxylation of free L-proline to free trans-4-hydroxy-L-proline was reported for microorganisms producing the peptide antibiotic etamycin, which contains 4-hydroxyproline as its component (5, 15). Recently, Lawrence et al. have purified 4-hydroxylase of free L-proline from Streptomyces griseoviridus P8648, which produces etamycin (9). They reported that microbial proline 4-hydroxylase belongs to a family of 2-oxoglutarate-dependent dioxygenases as well as prolyl hydroxylases involved in collagen biosynthesis (9).Independently, we have screened microbial proline 4-hydroxylases from more than 3,000 strains and have found 8 actinomycete strains to produce proline 4-hydroxylase (unpublished results). In this screening, we found a novel activity which hydroxylates free L-proline to cis-3-hydroxy-L-proline in Streptomyces sp. strain TH1, Streptomyces canus AT...

Microbial proline 4-hydroxylases, which hydroxylate freel-proline totrans-4-hydroxy-l-proline, were screened in order to establish an industrial system for biotransformation of l-proline totrans-4-hydroxy-l-proline. Enzyme activities were detected in eight strains, including strains ofDactylosporangium spp. and Amycolatopsis spp. The Dactylosporangium sp. strain RH1 enzyme was partially purified 3,300-fold and was estimated to be a monomer polypeptide with an apparent molecular mass of 31 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Degenerate primers based on the N-terminal amino acid sequence of the 31-kDa polypeptide were synthesized in order to amplify the corresponding 71-bp DNA fragment. A 5.5-kbp DNA fragment was isolated by using the 71-bp fragment labeled with digoxigenin as a probe for a genomic library ofDactylosporangium sp. strain RH1 constructed inEscherichia coli. One of the open reading frames found in the cloned DNA, which encoded a 272-amino-acid polypeptide (molecular mass, 29,715 daltons), was thought to be a proline 4-hydroxylase gene. The gene was expressed in E. coli as a fused protein with the N-terminal 34 amino acids of the β-galactosidase α-fragment. The E. coli recombinant exhibited proline 4-hydroxylase activity that was 13.6-fold higher than the activity in the original strain, Dactylosporangium sp. strain RH1. No homology was detected with other 2-oxoglutarate-dependent dioxygenases when databases were searched; however, the histidine motif conserved in 2-oxoglutarate-dependent dioxygenases was found in the gene.

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During the screening of microbial proline hydroxylases, novel proline 3-hydroxylase activities, which hydroxylate free L-proline to free cis-3-hydroxy-L-proline, were detected in whole cells of Streptomyces sp. strain TH1 and Bacillus sp. strains TH2 and TH3 from 3,000 strains isolated from soil. The reaction product was purified from a reaction mixture of Streptomyces sp. strain TH1, and its chemical structure was identified as cis-3-hydroxy-L-proline by instrumental analyses. Proline 3-hydroxylase activity was also detected in Streptomyces canus ATCC 12647 which produces the 3-hydroxyproline-containing peptide antibiotic telomycin. Bacillus sp. strains TH2 and TH3 were found to accumulate cis-3-hydroxy-L-proline in culture media at 426 and 352 M, respectively. It was suggested that hydroxylation occurred in a highly regio-and stereospecific manner at position 3 of L-proline because no hydroxylation product other than cis-3-hydroxy-L-proline was observed. Proline 3-hydroxylases of these strains were first characterized on crude enzyme preparations. Since 2-oxoglutarate and ferrous ion were required for hydroxylation of L-proline, these 3-hydroxylases were thought to belong to a family of 2-oxoglutarate-related dioxygenases. The reaction was inhibited by Co 2؉ , Zn 2؉ , and Cu 2؉. L-Ascorbic acid accelerated the reaction. The optimum pH and temperature were 7.5 and 35؇C, respectively.