Abstract
The acetyl-CoA synthetase (ACS) family is a subfamily of adenylate-forming enzymes, which has a close evolutionary relationship with the 4-coumarate:CoA ligase (4CL) family. In this study, two ACS genes were cloned from Populus trichocarpa and were named PtrACS1 and PtrACS2. Bioinformatics characterization of PtrACS1 and PtrACS2 showed that they contained the key ACS residues and a putative peroxisome targeting sequence 1 (PTS1) at the end of the C-terminal sequence. Real-time PCR results showed that PtrACS1 and PtrACS2 were expressed in the phloem, xylem, leaves, and roots of one-year-old P. trichocarpa, but were expressed primarily in the leaves. The ACS enzyme activity was higher in leaves than other tissues in P. trichocarpa. Two overexpressed recombinant proteins showed no catalytic activity toward the substrates of 4CL, but did have notable catalytic activity toward sodium acetate and substrates of ACS. The relative activities of PtrACS1 and PtrACS2 were 194.16 ± 11.23 and 422.25 ± 21.69 μM min−1 mg−1, respectively. The Km and Vmax of PtrACS1 were 0.25 mM and 698.85 μM min−1 mg−1, while those for PtrACS2 were 0.72 mM and 245.96 μM min−1 mg−1, respectively. Our results revealed that both proteins belong to the ACS family, and provide a theoretical foundation for the identification and functional analysis of members of the adenylate-forming enzyme superfamily.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-2532-7) contains supplementary material, which is available to authorized users.
Highlights
The adenylate-forming enzyme superfamily is characterized by the presence of a highly conserved putative AMP-binding domain (PROSITE PS00455) and a shared ATP-dependent, two-step reaction mechanism as follows (Schneider et al 2005): Acid + ATP → Acyl-AMP + PPi (REACTION 1) and Acyl-AMP + CoA → acylCoA + AMP (REACTION 2), which contributes to the biosynthesis or degradation of diverse compounds such as fatty acids, amino acids, and a variety of secondary metabolites
Molecular cloning and characterization of PtrACS1 and PtrACS2 Based on the genomic sequence of the poplar genome, we cloned and characterized two complete cDNA sequences from P. trichocarpa, Ptr4CL6 and Ptr4CL8
The results clearly showed that PtrACS1 and PtrACS2 had high homology (Fig. 4, Additional file 1: Table S1), and had the closest evolutionary relationships with other acetyl-CoA synthetase (ACS) sequences (AAB92552 Arabidopsis thaliana ACS, NP_198504 Arabidopsis thaliana ACS, AED94121 Arabidopsis thaliana ACS, EOY03030 Theobro macacao ACS isoform 1, XP_007032105 Theobro macacao ACS isoform 2, and XP_007032106 Theobro macacao ACS isoform 3)
Summary
The adenylate-forming enzyme superfamily is characterized by the presence of a highly conserved putative AMP-binding domain (PROSITE PS00455) and a shared ATP-dependent, two-step reaction mechanism as follows (Schneider et al 2005): Acid + ATP → Acyl-AMP + PPi (REACTION 1) and Acyl-AMP + CoA → acylCoA + AMP (REACTION 2), which contributes to the biosynthesis or degradation of diverse compounds such as fatty acids, amino acids, and a variety of secondary metabolites. In the Arabidopsis and Populus model plants, a number of genes encoding adenylateforming enzymes are annotated as being closely related to 4CL despite having unknown specific biochemical functions Most of these 4CL-like enzymes contain peroxisome targeting sequence 1 (PTS1) sequences in the C-terminal region, and are predicted to be targeted to the peroxisome (Schneider et al 2005; Koo et al 2006). Since the Populus trichocarpa genome sequence was published, more 4CL-like genes have been found and identified, but most do not have the enzymatic activity of 4CL (Zhang et al 2015) Which family these 4CL-like proteins belong to and what reactions they catalyze should be explored
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