Kinetic and Structural Studies of Histidine-tagged Tetrahydrodipicolinate N-succinyltransferase

Abstract

Tetrahydrodipicolinate N-succinyltransferase (THDP succinyltransferase) catalyzes the reaction of tetrahydrodipicolinate (THDP) and succinyl-CoA to form L-2-succinylamino-6-oxopimelate and coenzyme A (CoASH) in the succinylase branch of lysine biosynthesis. Because lysine is an essential amino acid to humans, THDP succinyltransferase is a potential target for designed antibiotics. The DapD gene encodes the enzyme. While DapD from a number of sources has been characterized, no data exists for the enzyme from Serratia marcescens, a pathogen known to frequently cause hospital-acquired infections. In our lab, the crystal structure of THDP in Apo form and in complex with an activator is under investigation. The protein was expressed and purified. Based upon SDS/PAGE, the expressed protein is 95% pure and 3 liters of culture produced 50-80 mg of protein. Initially, the PEG/ION screen was used to find the crystallization condition. Using this screen, several conditions produced crystals. A solution containing ammonium acetate and polyethylene glycol 4000 produced small crystals. Therefore, this crystallization condition is being modified to produce diffraction-quality crystals. *This research is funded by Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103447 and P20GM103640 (OCAST).