Structural studies of acetyl esterase and malt from Escherichia coli

Abstract

Scope and Method of Study: An acetyl esterase, also known as Aes from Escherichia coli, belongs to the hormone sensitive lipase family and down regulates MalT which is the transcriptional activator of the maltose regulon. Moreover, a recent study suggests an interaction between Aes and alpha-galactosidase which is also involved in carbohydrate metabolism. Since Aes interacts with several important proteins, it plays critical roles in carbohydrate metabolism in E. coli. Therefore, the purpose of this study was to determine crystal structures of Aes, DT1, DT1-DT2, and MalT in order to understand the remarkable maltose system in E. coli. To achieve this, cloning, over-expression, purification, and crystallization for each gene were carried out. Moreover, structural studies of Aes were performed.

Findings and Conclusions: The E. coli aes, DT1, DT1-DT2, and malT genes have been cloned with an N-terminal histidine tag and over-expressed. Aes, DT1, and MalT have been successfully purified and a crystal structure of Aes has been determined in this study. X-ray crystallography revealed that Aes contained an alpha/beta hydrolase fold, the central beta-strands being surrounded by alpha-helices. Moreover, the catalytic triad of Aes consisted of Ser-165, Asp-262, and His-292, which was stabilized by hydrogen bonds and was hidden in a shallow cleft. Since crystal screening showed some promising results for DT1 and MalT, further optimization in crystallization of these proteins will lead to crystal structure determination of them. Moreover, purification trials of DT1, DT1-DT2, and MalT should be carried out so as to find better conditions for crystal production.