Abstract

Adenosine 5′-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.

Highlights

  • The genome of Populus trichocarpa contains a large number of genes encoding carboxylic acid-activating enzymes, including long-chain fatty acyl-coenzyme A (CoA) synthetases, 4-coumarate: CoA ligases (4CL), and 4CL-like proteins of unknown biochemical function

  • These results show that 4CL-like genes may belong to the acetyl-CoA synthetase (ACS) family [16]

  • Full-length cDNA of Pt4CL-like was isolated from P. trichocarpa, referring to Pt4CL-like (XP_002307770.2)

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Summary

Introduction

The genome of Populus trichocarpa contains a large number of genes encoding carboxylic acid-activating enzymes, including long-chain fatty acyl-coenzyme A (CoA) synthetases, 4-coumarate: CoA ligases (4CL), and 4CL-like proteins of unknown biochemical function. With further research and complete sequencing of the Arabidopsis genome, researchers have gradually uncovered eight genes in Arabidopsis that are highly similar to the 4CL conserved domain containing box I and box II, which can be classified as 4CL-like genes [12,13,14] These genes have no corresponding 4CL substrates (β-coumaric acid, caffeic acid, ferulic acid, 5-hydroxyferulic acid, and erucic acid), and have no direct relationship with the synthesis of lignin or flavonoid compounds in terms of expression [14]. The At1g20510 protein exhibits cyclopentane-1-octanoic acid (OPC-8) CoA ligase activity, which is an essential step in jasmonate biosynthesis [15] These results show that 4CL-like genes may belong to the acetyl-CoA synthetase (ACS) family [16]. 200 mM NaCl, 200 μM abscisic acid, 2 mM H2 O2 , 4 ◦ C cold stress, or 10% PEG6000

Materials
RNA Isolation and cDNA Synthesis
Vector Construction and Heterologous Expression
Denaturation and Renaturation of Recombinant Protein
Purification of the Recombinant Pt4CL-Like and Western Blotting
Enzymatic Activity of Pt4CL-Like
Transcription of Pt4CL-Like in Various Tissues
Expression of Pt4CL-Like in Response to Abiotic Stress Treatments
Molecular Cloning and Sequence Analyses of Pt4CL-Like
Phylogenetic
Construction
M urea used to characteristics manipulate theof precipitate
3: Inclusion dissolved onceofinthe
Expression
Discussion
Full Text
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