Protein engineering of aldolase LbDERA for enhanced activity toward real substrates with a high-throughput screening method coupled with an aldehyde dehydrogenase.
A new high-throughput method for screening 2-deoxyribose-5-phosphate aldolase variants with a higher activity toward aldol reaction of unnatural aldehydes was established for the first time by coupling with an aldehyde dehydrogenase LeADH. The error-prone PCR and site-directed saturation mutagenesis libraries of aldolase LbDERA were constructed and screened using the high-throughput method. Two improved variants, LbDERAT29Land LbDERAF163Y, were identified and combined, giving a double mutant LbDERAT29L/F163Ywhich showed 7-fold higher activity than the native enzyme. The crystal structure of LbDERAT29L/163Yobtained by X-ray diffraction with 1.77 Å resolution revealed the structural changes responsible for the significant activity improvement.
Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.
Xue-Cheng
XC
Jiao
Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China. Electronic address: panjiang@ecust.edu.cn.
Jiang
J
Pan
Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.
Xu-Dong
XD
Kong
Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China. Electronic address: jianhexu@ecust.edu.cn.
Jian-He
JH
Xu
D000444:Aldehyde Dehydrogenase / Q000138:chemical synthesis* / Q000235:genetics* / Q000648:ultrastructure; D001665:Binding Sites; D015195:Drug Design; D004789:Enzyme Activation; D057166:High-Throughput Screening Assays / Q000379:methods*; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering; D013379:Substrate Specificity
D000444:Aldehyde Dehydrogenase
2-Deoxyribose-5-phosphate aldolase; Aldehyde dehydrogenase; Crystal structure; High-throughput screening; Protein engineering; Statin side chains