Redesigning Aldolase Stereoselectivity by Homologous Grafting.
The 2-deoxy-d-ribose-5-phosphate aldolase (DERA) offers access to highly desirable building blocks for organic synthesis by catalyzing a stereoselective C-C bond formation between acetaldehyde and certain electrophilic aldehydes. DERA´s potential is particularly highlighted by the ability to catalyze sequential, highly enantioselective aldol reactions. However, its synthetic use is limited by the absence of an enantiocomplementary enzyme. Here, we introduce the concept of homologous grafting to identify stereoselectivity-determining amino acid positions in DERA. We identified such positions by structural analysis of the homologous aldolases 2-keto-3-deoxy-6-phosphogluconate aldolase (KDPG) and the enantiocomplementary enzyme 2-keto-3-deoxy-6-phosphogalactonate aldolase (KDPGal). Mutation of these positions led to a slightly inversed enantiopreference of both aldolases to the same extent. By transferring these sequence motifs onto DERA we achieved the intended change in enantioselectivity.
Institut für Bioorganische Chemie, Heinrich-Heine-Universität Düsseldorf im Forschungszentrum Jülich, 52426, Jülich, Germany.
Carolin
C
Bisterfeld
Institute of Bio- and Geosciences IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, 52425, Jülich, Germany.
Thomas
T
Classen
Institut für Bioorganische Chemie, Heinrich-Heine-Universität Düsseldorf im Forschungszentrum Jülich, 52426, Jülich, Germany.
Irene
I
Küberl
Institut für Bioorganische Chemie, Heinrich-Heine-Universität Düsseldorf im Forschungszentrum Jülich, 52426, Jülich, Germany.
Birgit
B
Henßen
Institute of Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany.
Alexander
A
Metz
Institute of Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany.
Holger
H
Gohlke
Institut für Bioorganische Chemie, Heinrich-Heine-Universität Düsseldorf im Forschungszentrum Jülich, 52426, Jülich, Germany.|Institute of Bio- and Geosciences IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, 52425, Jülich, Germany.
Jörg
J
Pietruszka
D000446:Aldehyde-Lyases / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D055162:Biocatalysis; D004926:Escherichia coli / Q000201:enzymology; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D010802:Phylogeny; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D011773:Pyruvates / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity
D000596:Amino Acids; D011773:Pyruvates; D000446:Aldehyde-Lyases; D005634:Fructose-Bisphosphate Aldolase; C100131:phospho-2-keto-3-deoxy-gluconate aldolase