8HR6

leucine DEHYDROGENASE STRUCTURE IN TERNARY COMPLEX WITH NAD+ from Bacillus thuringiensis

Summary for 8HR6

• Entry DOI: 10.2210/pdb8hr6/pdb
• Descriptor: Leucine dehydrogenase, NICOTINAMIDE-ADENINE-DINUCLEOTIDE, SULFATE ION, ... (4 entities in total)
• Functional Keywords: leucine dehydrogenase structure in ternary complex with nad+ from bacillus thuringiensis, oxidoreductase
• Biological source: Bacillus thuringiensis
• Total number of polymer chains: 2
• Total formula weight: 82115.35

Authors

Li, X.,Song, W. (deposition date: 2022-12-14, release date: 2024-03-20)

Primary citation

Li, X.,Gao, C.,Wei, W.,Song, W.,Meng, W.,Liu, J.,Chen, X.,Gao, C.,Guo, L.,Liu, L.,Wu, J.
A Tri-Enzyme Cascade for Efficient Production of L-2-Aminobutyrate from L-Threonine.
Chembiochem, 24:e202300148-e202300148, 2023

PubMed Abstract: L-2-aminobutyrate (L-ABA) is an important chiral drug intermediate with a key role in modern medicinal chemistry. Here, we describe the development of an efficient method for the asymmetric synthesis of L-ABA in a tri-enzymatic cascade in Escherichia coli BL21 (DE3) using a cost-effective L-Thr. Low activity of leucine dehydrogenase from Bacillus thuringiensis (BtLDH) and unbalanced expression of enzymes in the cascade were major challenges. Mechanism-based protein engineering generated the optimal triple variant BtLDH (A262S/V296C/P150M) with 20.7-fold increased specific activity and 9.6-fold increased k /K compared with the wild type. Optimizing plasmids with different copy numbers regulated enzymatic expression, thereby increasing the activity ratio (0.3 : 1:0.6) of these enzymes in vivo close to the optimal ratio (0.4 : 1 : 1) in vitro. Importing the optimal triple mutant BtLDH into our constructed pathway in vivo and optimization of transformation conditions achieved one-pot conversion of L-Thr to 130.2 g/L L-ABA, with 95 % conversion, 99 % e.e. and 10.9 g L  h productivity (the highest to date) in 12 h on a 500 mL scale. These results describe a potential biosynthesis approach for the industrial production of L-ABA.

PubMed: 36946691
DOI: 10.1002/cbic.202300148

Experimental method

X-RAY DIFFRACTION (3.52 Å)