Crystal structure and enzyme mechanism of esterase Est1 from an archaeon, Sulfolobus shibatae

Institute for Protein Research, Osaka University* Graduate School of Engineering, Tohoku University, Japan**
â—‹Masami Kusunoki* Hideaki Unno* Yasuhiro Oshima** Tokuzo Nishino** Toru Nakayama**

Carboxylesterases (esterases) catalyze the hydrolysis and ester transfer reaction of carboxyl esters, playing important roles in the metabolism, decomposition, and detoxification intra-cellular and extra-cellular processes. Most of the esterases belong to a large group of the proteins, a/b hydrolase super-family. Esterases are industrially important enzymes utilized in various fields. The thermal stability and optimal pH of these enzymes are important properties for such applications. Especially, esterases with their maximal activities at acidic pH are not reported yet and its availability is strongly requested. We started the biochemical and crystallographic study of esterase Est1 from Sulfolobus shibatae, a member of the hormone-sensitive lipase family of the a/b hydrolase super-family, in order to clarify the enzyme mechanisms of Est1 and to gain a structural basis for designing the mutant enzyme with the activity at acidic pH.
The recombinant enzyme of Est1 was overexpressed in Escherichia coli cells and was crystallized by the hanging drop vapor diffusion method. The native crystal belongs to the orthorombic space group space I222 with cell dimensions a=58.40 b=71.94, and c=137.33 Å. The X-ray diffraction data to 1.5 Å resolution was collected at the beamline 44XU, SPring-8. The crystal structure was determined by the molecular replacement method with the CCP4 program Molrep and was refined with the program Refmac-5 with Rwork and Rfree values of 0.157 and 0.180, respectively.
The mutant enzymes His274Ala, His274Glu, and His274Asp were also prepared and their crystal structures were determined to gain insight into enzyme mechanisms. We are now examining these structures and will report the detail.