title: Imidase catalyzing desymmetric imide hydrolysis forming optically active 3-substituted glutaric acid monoamides for the synthesis of gamma-aminobutyric acid (GABA) analogs.
abstract: The recent use of optically active 3-substituted gamma-aminobutyric acid (GABA) analogs in human therapeutics has identified a need for an efficient, stereoselective method of their synthesis. Here, bacterial strains were screened for enzymes capable of stereospecific hydrolysis of 3-substituted glutarimides to generate (R)-3-substituted glutaric acid monoamides. The bacteria Alcaligenes faecalis NBRC13111 and Burkholderia phytofirmans DSM17436 were discovered to hydrolyze 3-(4-chlorophenyl) glutarimide (CGI) to (R)-3-(4-chlorophenyl) glutaric acid monoamide (CGM) with 98.1% enantiomeric excess (e.e.) and 97.5% e.e., respectively. B. phytofirmans DSM17436 could also hydrolyze 3-isobutyl glutarimide (IBI) to produce (R)-3-isobutyl glutaric acid monoamide (IBM) with 94.9% e.e. BpIH, an imidase, was purified from B. phytofirmans DSM17436 and found to generate (R)-CGM from CGI with specific activity of 0.95 U/mg. The amino acid sequence of BpIH had a 75% sequence identity to that of allantoinase from A. faecalis NBRC13111 (AfIH). The purified recombinant BpIH and AfIH catalyzed (R)-selective hydrolysis of CGI and IBI. In addition, a preliminary investigation of the enzymatic properties of BpIH and AfIH revealed that both enzymes were stable in the range of pH 6-10, with an optimal pH of 9.0, stable at temperatures below 40 °C, and were not metalloproteins. These results indicate that the use of this class of hydrolase to generate optically active 3-substituted glutaric acid monoamide could simplify the production of specific chiral GABA analogs for drug therapeutics.
authors.affiliation: Biotechnology Development Laboratories, Kaneka Corporation, 1-8 Miyamae, Takasago, Hyogo, 676-8688, Japan.
authors.forename: Masutoshi
authors.initials: M
authors.lastname: Nojiri
authors.affiliation: Industrial Microbiology, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto, 606-8502, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Hibi
authors.affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto, 606-8502, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Shizawa
authors.affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto, 606-8502, Japan.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Horinouchi
authors.affiliation: Biotechnology Development Laboratories, Kaneka Corporation, 1-8 Miyamae, Takasago, Hyogo, 676-8688, Japan.
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Yasohara
authors.affiliation: Industrial Microbiology, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto, 606-8502, Japan.
authors.forename: Satomi
authors.initials: S
authors.lastname: Takahashi
authors.affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto, 606-8502, Japan. ogawa@kais.kyoto-u.ac.jp.
authors.forename: Jun
authors.initials: J
authors.lastname: Ogawa
mesh_terms: D042481:Alcaligenes faecalis / Q000201:enzymology*; D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D042521:Burkholderiaceae / Q000201:enzymology*; D004795:Enzyme Stability; D005977:Glutarates / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007094:Imides / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
chemical_list: D005977:Glutarates; D007094:Imides; D011994:Recombinant Proteins; D005680:gamma-Aminobutyric Acid; D000581:Amidohydrolases; C020047:dihydropyrimidinase; C035736:glutaric acid
keywords: 3-substituted glutarimide; Bacterial screening; Desymmetrization; GABA synthesis; Imidase; Stereoselective hydrolysis