Kai Yang, Yueshan Huang, Charles Amanze, Liyi Yao, Richmond Anaman, Bin Huang, Weimin Zeng
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引用次数: 0
Abstract
The access to the enantiopure noncanonical amino acid l-phosphinothricin (l-PPT) by applying biocatalysts is highly appealing in organic chemistry. In this study, a NADH-dependent glutamate dehydrogenase from Lachnospiraceae bacterium (LbGluDH) was chosen for the asymmetric synthesis of l-PPT. Three flexible loops undergoing big conformational shifts during the catalysis were identified and rationally engineered following the initial mutagenesis. The enzyme's specific activity toward the key precursor of l-PPT, 2-oxo-4-[(hydroxy) (methyl) phosphinyl] butyric acid (PPO), was improved from negligible to 9 U/mg, and the Km value was reduced to 17 mM. The computational analysis showed that the modified loops broadened the enzyme's narrow tunnels, allowing the substrate to access the binding pocket and get closer to the crucial residue D165, thereby enhancing the catalytic process. Utilizing the variant as the catalyst, the preparation of l-PPT achieved a 100% conversion rate within 60 min, coupled with a stereoselectivity exceeding 99.9%, demonstrating its practical capacity for industrial application. Similar enhancement in catalytic activity was obtained applying the same strategy to a typical NADH-dependent GluDH from Pyrobaculum islandicum (PisGluDH), indicating the effectiveness of our strategy for the protein engineering of GluDHs targeted to the biosynthesis of unnatural compounds.
期刊介绍:
The Journal of Agricultural and Food Chemistry publishes high-quality, cutting edge original research representing complete studies and research advances dealing with the chemistry and biochemistry of agriculture and food. The Journal also encourages papers with chemistry and/or biochemistry as a major component combined with biological/sensory/nutritional/toxicological evaluation related to agriculture and/or food.