The molecular enzymatics research team of Shanghai Jiaotong University developed an unnatural substrate binding adaptation strategy to achieve efficient evolution of aminotransferases

Recently, the internationally renowned journal ACS Catalysis published the research results of Cui Li, an associate researcher of Professor Feng Yan's team of Shanghai Jiaotong University, "Molecular Evolution of an Aminotransferase Based on Substrate–Enzyme Binding Energy Analysis for Efficient Valienamine Synthesis"
。 In this study, a semi-rational evolutionary method based on "enzyme-substrate binding energy analysis" was proposed, which effectively adapted unnatural small molecule substrates in the large binding pocket of transaminases, and effectively improved the ability
of enzymes to catalyze the synthesis of Jinggangmecenamine, an intermediate of diabetes drugs.
Associate Professor Cui Li of the School of Life Science and Technology was the corresponding author, and Professor Feng Yan and Associate Professor Shao Wenguang participated in the research
.

Synthetic biology provides a transformative model
for improving drug innovation and superior yield through engineered system design and reconstructing the required synthetic systems in biological systems with standardized and modular elements.
The research team successfully established the biosynthesis pathway
of Jinggangenamine by using the glycosidase_{inhibitor-C7N}aminocyclic alcohol compound Jinggangenenamine as the model molecule and the Jinggangmycin biosynthesis intermediates as the design node, coupled with heterologous highly stereoselective aminotransferase.
However, artificially designed pathway heterologous enzymes have low catalytic activity for unnatural substrates and poor pathway adaptability is still a problem
that needs to be broken through for the efficient operation of synthetic biology design pathways.

In this study, based on the realization of heterologous aminotransferase-catalyzed jingomenone synthesis of Jinggangenamine, the structural characteristics of the binding pocket of the candidate aminotransferase substrate and its binding differences with natural substrates and design substrates were systematically analyzed, and unfavorable binding conformations
leading to low catalytic activity of enzymes to unnatural substrates were found 。 Through virtual mutation binding screening and evolutionary conservation analysis of active center amino acids, nine potential amino acid sites affecting the formation of adverse conformations were identified, and the combined active site saturation test/iterative saturation mutation (CAST/ISM) was carried out.
The binding conformation of the obtained excellent mutant to Jinggangenone is more conducive to the occurrence of transamination reaction, and the synthesis ability of Jinggangenamine is increased by 35 times
.

The research team of molecular enzymology and synthetic biology has long been committed to the design and construction of artificial pathways in drug synthetic biology and the reprogramming of microbial synthetic pathways
.
With the funding of the Key Special Project of Synthetic Biology of the Ministry of Science and Technology and the National Natural Science Foundation of China, the design and transformation
of the biosynthetic pathway of Jinggang enzyme enamine, ginsenoside Rh2 and serinol have been completed 。 The "unnatural substrate binding adaptation" strategy developed in this study provides an evolutionary scheme for the interaction between the adaptation enzyme and the substrate, optimizing the binding conformation of the "small substrate" in the "large pocket", and improving the catalytic ability of the candidate enzyme to the unnatural substrate.
This paper further deepens the exploration of efficient adaptation of synthetic biology design pathways, improves the ability to control the catalytic activity of functional enzymes by protein engineering technology, and provides a solution
for applying the concept of synthetic biology to construct synthetic pathways of high-value compounds based on unnatural reactions.

Link to the paper: https://pubs.
acs.
org/doi/10.
1021/acscatal.
2c03784