Abstract
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.
Highlights
Protein engineering offers the opportunity to alter protein properties such as stability, substrate specificity, and conversion rates, respectively
We identified amino acid positions responsible for enantioselectivity and activity by comparing homologous aldolases, the enantiocomplementary pyruvate-dependent 2-keto3-deoxy-6-phosphogluconate aldolase (KDPG) and 2-keto-3-deoxy-6-phosphogalactonate aldolase (KDPGal)
We set out to learn from the homologous aldolases keto-3-deoxy-6-phosphogluconate aldolase (KDPG) and KDPGal from E. coli how these enzymes accomplish enantioselectivity and use this knowledge to invert the enantioselectivity of DERA
Summary
Protein engineering offers the opportunity to alter protein properties such as stability, substrate specificity, and conversion rates, respectively. Addressing stereoselectivity still remains challenging for such endeavors, and this holds especially true in cases with no enantiocomplementary enzyme being available. In a summary of successful changes of enzyme enantioselectivity, Faber, Kazlauskas and coworkers described that these were achieved in most cases by altering the substrate binding mode [1]. Even a single amino acid exchange can be sufficient to invert stereoselectivity as demonstrated, e.g., for thiamine diphosphatedependent enzymes [2, 3]. According to a study of Kazlauskas et al, amino acids close to the active site are often the more effective points of attack for changing enantioselectivity [4]. A fruitful approach to identify these stereoselectivity-determining positions is the analysis of enzyme families to expose conserved amino acids as summarized by Pleiss [5]. PLOS ONE | DOI:10.1371/journal.pone.0156525 June 21, 2016
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
