Cloning, overexpression, purification, and characterization of S-adenosylhomocysteine hydrolase from Corynebacterium efficiens YS-314

Abstract

The gene encoding S-adenosylhomocysteine hydrolase activity (SAHase: EC 3.3.1.1) from Corynebacterium efficiens (YS-314) was cloned and expressed as a fusion protein in Escherichia coli Rosetta (DE3). The analyzed nucleotide sequence of the cloned gene proved to be identical to those reported on the NCBI database. The recombinant enzyme is a tetramer, showing a molecular weight of approximately 210 kDa, as estimated by gel filtration. The KM values of the enzyme for S-adenosylhomocysteine (SAH), adenosine (Ado), and homocysteine (Hcy), were determined to be 1.4, 10, and 45 μM. The overexpression of the recombinant enzyme produced a high level of protein (>40 mg of protein per gram of wet cells) and revealed certain thermostability when characterized at temperatures above 40°C. It also showed a high capacity for the synthesis of SAH, thermal stability, and high kinetic similarity to human SAHase, indicating a high biotechnological and pharmacological potential. © 2008 American Chemical Society and American Institute of Chemical Engineers.