title: Microbial proline 4-hydroxylase screening and gene cloning.
abstract: Microbial proline 4-hydroxylases, which hydroxylate free L-proline to trans-4-hydroxy-L-proline, were screened in order to establish an industrial system for biotransformation of L-proline to trans-4-hydroxy-L-proline. Enzyme activities were detected in eight strains, including strains of Dactylosporangium 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 of Dactylosporangium sp. strain RH1 constructed in Escherichia 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 beta-galactosidase alpha-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.
authors.affiliation: Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6, Asahimachi, Machida, Tokyo 194-8533, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Shibasaki
authors.forename: H
authors.initials: H
authors.lastname: Mori
authors.forename: S
authors.initials: S
authors.lastname: Chiba
authors.forename: A
authors.initials: A
authors.lastname: Ozaki
mesh_terms: D000192:Actinomycetales / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000378:metabolism; D003001:Cloning, Molecular; D003470:Culture Media; D018942:Macrolides; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011393:Procollagen-Proline Dioxygenase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology; D047630:Depsipeptides
chemical_list: D000900:Anti-Bacterial Agents; D003470:Culture Media; D018942:Macrolides; D011393:Procollagen-Proline Dioxygenase; C004910:etamycin; D047630:Depsipeptides