Characterization, Expression Profiling, and Functional Analyses of a 4CL-Like Gene of Populus trichocarpa

Abstract

Adenosine 5 0 -monophosphate (AMP) (adenylate)-forming acetyl-CoA synthetase (ACS) catalyzes the formation of acetyl-coenzyme A (CoA), and the ACS family is closely related to the 4-coumarate CoA ligase (4CL) family. In this study, a 4CL-like gene was cloned from Populus trichocarpa and named Pt4CL-like. Characterization of Pt4CL-like, using bioinformatics, showed that it contained box I and box II domains at the end of the C-terminal sequence, and there is a characteristic sequence of ACS, namely, peroxisome-targeting sequence (PTS). Real-time PCR results showed that the 4CL-like gene was expressed in all tissues tested, and was highly expressed in the stems. A denaturation and renaturation process was conducted, and the recombinant Pt4CL-like protein was purified through HisTrapTM high performance affinity chromatography. It showed Pt4CL-like protein did not catalyze substrates of 4CL, but could significantly catalyzed sodium acetate. These results indicate that Pt4CL-like protein belongs to the ACS family, providing a theoretical basis for further analysis and comparison of the functions of adenylate-forming enzymes and 4CL family.