Structure and evolution of Alanine/Serine Decarboxylases and the engineering of theanine production

Abstract

Ethylamine (EA), the precursor of theanine biosynthesis, is synthesized from alanine decarboxylation by Alanine Decarboxylase (AlaDC) in tea plants. AlaDC evolves from Serine Decarboxylase (SerDC) through neofunctionalization and has lower catalytic activity. However, lacking structure information hinders the understanding of the evolution of substrate specificity and catalytic activity. In this study, we solved the X-ray crystal structures of AlaDC from Camellia sinensis (CsAlaDC) and SerDC from Arabidopsis thaliana (AtSerDC). Tyr 341 of AtSerDC or the corresponding Tyr 336 of CsAlaDC is essential for their enzymatic activity. Tyr 111 of AtSerDC and the corresponding Phe 106 of CsAlaDC determine their substrate specificity. Both CsAlaDC and AtSerDC have a distinctive zinc finger that contributes to the activity and has not been identified in any other Group II PLP-dependent amino acid decarboxylases. Based on the structure comparison and evolution analysis AlaDC or SerDC led us to perform mutation screen of CsAlaDC. The results indicated that the mutation of L110F or P114A in the CsAlaDC dimerization interface significantly improved the catalytic activity by 110% and 59%, respectively. Combining double mutant CsAlaDC L110F/P114A with theanine synthetase increased 672% of theanine production in an in vitro system. This study provides the structural basis for the substrate selectivity and catalytic activity of CsAlaDC and AtSerDC and sheds light on a new direction for the efficient industrial synthesis of theanine.