title: Characterization of novel 2-oxoglutarate dependent dioxygenases converting L-proline to cis-4-hydroxy-l-proline.
abstract: Hydroxyprolines are valuable chiral building blocks for organic synthesis of pharmaceuticals. Several microorganisms producing L-proline trans-4- and cis-3-hydroxylase were discovered and these enzymes were applied to the industrial production of trans-4- and cis-3-hydroxy-L-proline, respectively. Meanwhile, other hydroxyproline isomers, cis-4- and trans-3-hydroxy-L-proline, were not easily available because the corresponding hydroxylase have not been discovered. Herein we report novel L-proline cis-4-hydroxylases converting free L-proline to cis-4-hydroxy-L-proline. Two genes encoding uncharacterized proteins from Mesorhizobium loti and Sinorhizobium meliloti were cloned and overexpressed in Escherichia coli, respectively. The functions of purified proteins were investigated in detail, and consequently we detected L-proline cis-4-hydroxylase activity in both proteins. Likewise L-proline trans-4-, cis-3-hydroxylase and prolyl hydroxylase, these enzymes belonged to a 2-oxoglutarate dependent dioxygenase family and required a non-heme ferrous ion. Although their reaction mechanisms were similar to other hydroxylases, the amino acid sequence homology was not observed (less than 40%).
authors.affiliation: Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University, Ohkubo 3-4-1, Shinjuku-ku, Tokyo 169-8555, Japan.
authors.forename: Ryotaro
authors.initials: R
authors.lastname: Hara
authors.forename: Kuniki
authors.initials: K
authors.lastname: Kino
mesh_terms: D020561:Alphaproteobacteria / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D006909:Hydroxyproline / Q000096:biosynthesis*; D007655:Ketoglutarate Dehydrogenase Complex / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011392:Proline / Q000378:metabolism*; D016962:Sinorhizobium meliloti / Q000201:enzymology / Q000235:genetics; D013696:Temperature
chemical_list: D011392:Proline; D007655:Ketoglutarate Dehydrogenase Complex; D006909:Hydroxyproline