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Updated: Oct 6, 2025

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells
Published on: March 3, 2015
Combined active pocket and hinge region engineering to develop an NADPH-dependent phenylglycine dehydrogenase
Xinjian Yin1, Yujing Zeng2, Jun Chen2
1School of Marine Science, Sun Yat-sen University, Zhuhai 519080, China; Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), Zhuhai 519080, China.
Abstract:
NADPH-dependent amino acid dehydrogenases (AADHs) are favorable enzymes to construct artificial biosynthetic pathways in whole-cell for high-value noncanonical amino acids (NcAAs) production. Glutamate dehydrogenases (GluDHs) represent attractive candidates for the development of novel NADPH-dependent AADHs. Here, we report the development of a novel NADPH-dependent phenylglycine dehydrogenase by combining active pocket engineering and hinge region engineering of a GluDH from Pseudomonas putida (PpGluDH). The active pocket of PpGluDH was firstly tailored to optimize its binding mode with bulky substrate α-oxobenzeneacetic acid (α-OA), and then, the hinge region was further engineered to tune the protein conformational dynamics, which finally resulted in a mutant M3 (T196A/T121I/L123D) with a 103-fold increase of catalytic efficiency (kcat/Km) toward α-OA. The M3 mutant exhibited high catalytic performance in both in vitro biocatalysis preparation and in vivo biosynthesis of l-phenylglycine, indicating its promising practical applications. Our results demonstrated that co-engineering of the active pocket and hinge region is an effective strategy for developing novel NADPH-dependent AADHs from GluDHs for NcAAs production.
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