title: Development of a multi-enzymatic cascade reaction for the synthesis of trans-3-hydroxy-L-proline from L-arginine.
abstract: Naturally occurring L-hydroxyproline in its four regio- and stereoisomeric forms has been explored as a possible precursor for pharmaceutical agents, yet the selective synthesis of trans-3-hydroxy-L-proline has not been achieved. Our aim was to develop a novel biocatalytic asymmetric method for the synthesis of trans-3-hydroxy-L-proline. So far, we focused on the rhizobial arginine catabolic pathway: arginase and ornithine cyclodeaminase are involved in L-arginine degradation to L-proline via L-ornithine. We hypothesized that trans-3-hydroxy-L-proline should be synthesized if arginase and ornithine cyclodeaminase act on (2S,3S)-3-hydroxyarginine and (2S,3S)-3-hydroxyornithine, respectively. To test this hypothesis, we cloned the genes of L-arginine 3-hydroxylase, arginase, and ornithine cyclodeaminase and overexpressed them in Escherichia coli, with subsequent enzyme purification. After characterization and optimization of each enzyme, a three-step procedure involving L-arginine 3-hydroxylase, arginase, and ornithine cyclodeaminase (in this order) was performed using L-arginine as a starting substrate. At the second step of the procedure, putative hydroxyornithine was formed quantitatively by arginase from (2S,3S)-3-hydroxyarginine. Nuclear magnetic resonance and chiral high-performance liquid chromatography analyses revealed that the absolute configuration of this compound was (2S,3S)-3-hydroxyornithine. In the last step of the procedure, trans-3-hydroxy-L-proline was synthesized selectively by ornithine cyclodeaminase from (2S,3S)-3-hydroxyornithine. Thus, we successfully developed a novel synthetic route, comprised of three reactions, to convert L-arginine to trans-3-hydroxy-L-proline. The excellent selectivity makes this procedure simpler and more efficient than conventional chemical synthesis.
authors.affiliation: Research Institute for Science and Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo, 169-8555, Japan. ryo_h@aoni.waseda.jp.
authors.forename: Ryotaro
authors.initials: R
authors.lastname: Hara
authors.affiliation: Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo, 169-8555, Japan.
authors.forename: Saki
authors.initials: S
authors.lastname: Kitatsuji
authors.affiliation: Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo, 169-8555, Japan.
authors.forename: Kai
authors.initials: K
authors.lastname: Yamagata
authors.affiliation: Research Institute for Science and Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo, 169-8555, Japan.|Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo, 169-8555, Japan.
authors.forename: Kuniki
authors.initials: K
authors.lastname: Kino
mesh_terms: D000642:Ammonia-Lyases / Q000235:genetics / Q000378:metabolism; D001119:Arginase / Q000235:genetics / Q000378:metabolism; D001120:Arginine / Q000378:metabolism*; D001711:Biotransformation; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006909:Hydroxyproline / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; C026215:3-hydroxyproline; D001120:Arginine; D001119:Arginase; D000642:Ammonia-Lyases; C021914:ornithine cyclodeaminase; D006909:Hydroxyproline
keywords: (2S,3S)-3-Hydroxyornithine; Arginase; Arginine hydroxylase; Mesorhizobium loti; Ornithine cyclodeaminase; trans-3-Hydroxy-L-proline