Development of a multi-enzymatic cascade reaction for the synthesis of trans-3-hydroxy-L-proline from L-arginine.

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.