Development of an Engineered Sugar Aminotransferase with Simultaneously Improved Stability and Non-Natural Substrate Activity to Synthesize the Glucosidase Inhibitor Valienamine

• Published in: Engineering (Beijing, China) Vol. 42; no. 11; pp. 185 - 195
• Main Authors: Wang, Runxi, Qiao, Lu, Liu, Mufei, Ran, Yanpeng, Wang, Jun, Yan, Wupeng, Feng, Yan, Cui, Li
• Format: Journal Article
• Language: English
• Published: Elsevier Ltd 01.11.2024; State Key Laboratory of Microbial Metabolism & Joint International Research Laboratory of Metabolic and Developmental Sciences,School of Life Science and Biotechnology,Shanghai Jiao Tong University,Shanghai 200240,China
• Subjects: Artificial reaction; Combinatorial active-site saturation test; Iterative saturation mutagenesis; Stability-activity trade-off; Sugar aminotransferase; Valienamine; Artificial reaction; Stability-activity trade-off; Valienamine; Sugar aminotransferase; Combinatorial active-site saturation test; Iterative saturation mutagenesis
• DOI: 10.1016/j.eng.2024.04.026
• ISSN: 2095-8099

[Display omitted] Sugar aminotransferases (SATs) catalyze the installation of chiral amines onto specific keto sugars, producing bioactive amino sugars. Their activity has been utilized in artificial reactions, such as using the SAT WecE to transform valienone into the valuable α-glucosidase inhibitor valienamine. However, the low thermostability and limited activity on non-natural substrates have hindered their applications. Simultaneously improving stability and enzyme activity is particularly challenging owing to the acknowledged inherent trade-off between stability and activity. A customized combinatorial active-site saturation test–iterative saturation mutagenesis (CAST-ISM) strategy was used to simultaneously enhance the stability and activity of WecE toward valienone. Fourteen hotspots related to improving the stability–\activity trade-off were identified based on evolutionary conservation and the average mutation folding energy assessment of 57 residues in the active site of WecE. Positive mutagenesis and combinatorial mutations of these specific residues were accomplished via site-directed saturation mutagenesis (SSM) and iterative evolution cycles. Compared with those of the wild-type (WT) WecE, the quadruple mutant M4 (Y321F/K209F/V318R/F319V) displayed a 641.49-fold increase in half-life (t1/2) at 40 °C and a 31.37-fold increase in activity toward the non-natural substrate valienone. The triple mutant M3 (Y321F/K209F/V318R) demonstrated an 83.04-fold increase in (t1/2) at 40 °C and a 37.77-fold increase in activity toward valienone. The underlying mechanism was dependent on the strengthened interface interactions and shortened transamination reaction catalytic distance, compared with those of the WT, which improved the stability and activity of the obtained mutants. Thus, we accomplished a general target-oriented strategy for obtaining stable and highly active SATs for artificial amino-sugar biosynthesis applications.