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Isomer‐specific retinoic acid biosynthesis in HeLa cells expressing recombinant class I aldehyde dehydrogenases
Author(s) -
Bhat Pangala V,
Brodeur Helen,
Chag Samuel,
Parisotto Maxime,
Mader Sylvie
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a997-c
Subject(s) - aldehyde dehydrogenase , retinoic acid , biochemistry , enzyme , in vivo , retinal , chemistry , dehydrogenase , retinoid , hela , recombinant dna , biology , in vitro , stereochemistry , gene , microbiology and biotechnology
Retinal dehydrogenase type 1 (RALDH1) catalyzes the oxidation of all‐trans and 9‐cis retinal to the respective retinoic acids (RAs), whereas another member of the aldehyde dehydrogenase family, the phenobarbital‐induced aldehyde dehydrogenase (PB‐ALDH), is very poorly active. We have previously generated chimeras between these two enzymes that displayed selectivity for retinal isomers in crude bacterial extracts. Here, we have characterized the kinetic properties of the corresponding purified recombinant proteins, and demonstrate that these chimeras catalyze oxidation of retinal isomers with high efficiency and selectivity. To examine whether the selectivity of the recombinant enzymes is retained in vivo, we first assessed whether retinoid‐isomerizing activity is present in cultured eukaryotic cells. Our results demonstrate that the only RA isomers detected in RALDH1‐expressing or non‐expressing cells corresponded to the same steric conformation as the supplied retinoids, indicating a lack of measurable 9‐cis/all‐trans retinoid‐isomerizing activity. Finally, HeLa cells transfected with RALDH1 derivatives that were retinal isomer‐selective in vitro produced only the corresponding isomers of RA in vivo, establishing these enzymes as useful tools to assess the respective roles of the two RA isomers in vivo. (supported by CIHR grant)