High Acetyltransferase Activity Research Articles

Phylogenomics and functional analysis of Glycerol-3-Phosphate Acyltransferase (GPAT) Genes: A critical role in lipid biosynthesis.

The tung tree (Vernicia fordii Hemsl.), an economically important woody plant, is widely planted for the production of high-quality tung oil. Glycerol-3-phosphate acyltransferases (GPATs), the rate-limiting enzymes in triacylglycerol synthesis, play an important role in seed oil biosynthesis. In this study, we performed a genome-wide analysis of VfGPATs. A total of 9 VfGPATs were identified from the whole tung genome, and phylogenetic analysis divided the VfGPATs into three major clades: clade II (VfGPAT9), clade III (VfATS1) and clade IV (VfGPAT1 ~ 8). Subcellular localization analysis revealed that five VfGPATs (1, 5, 6, 8, and 9) are localized in the endoplasmic reticulum, and four VfGPATs (3-1, 3-2, 3-3, and ATS1) are localized in the chloroplast. Overexpression of VfGPATs in Arabidopsis thaliana revealed that the oil content in VfGPAT8- and VfGPAT9-transgenic plants were significantly increased by 26.60 and 55.94% compared to the wild-type. Transient expression of VfGPAT8 + VfFADX and VfGPAT9 + VfFADX could promote the synthesis of α-eleostearic acid and enhance the accumulation of lipid droplets in tobacco (Nicotiana benthamiana) leaves. We further tested the enzymatic activities of VfGPAT8 and VfGPAT9 with the yeast double mutant strain ZAFU1. The results showed that VfGPAT8 complemented the phosphatidate biosynthetic defect in the double mutant, while VfGPAT9 could not, suggesting that VfGPAT8 has a high acetyltransferase activity. However, altering serine (S) residue at position 113 of VfGPAT9 to threonine (T) could restore its enzymatic activity. This study provided important insights into the evolutionary history of VfGPATs and will promote the genetic improvement of tung trees and related species.

Assembly and Full Functionality of Recombinantly Expressed Dihydrolipoyl Acetyltransferase Component of the Human Pyruvate Dehydrogenase Complex

The dihydrolipoyl acetyltransferase (E2) component of mammalian pyruvate dehydrogenase complex (PDC) consists of 60 COOH-terminal domains as an inner assemblage and sequentially via linker regions an exterior pyruvate dehydrogenase (E1) binding domain and two lipoyl domains. Mature human E2, expressed in a protease-deficient Escherichia coli strain at 27 degrees , was prepared in a highly purified form. Purified E2 had a high acetyltransferase activity, was well lipoylated based on its acetylation, and bound a large complement of bovine E1. Electron micrographs demonstrated that the inner core was assembled in the expected pentagonal dodecahedron shape with E1 binding around the inner core periphery. With saturating E1 and excess dihydrolipoyl dehydrogenase (E3) but no E3-binding protein (E3BP), the recombinant E2 supported the overall PDC reaction at 4% of the rate of bovine E2.E3BP subcomplex. The lipoates of assembled human E2 or its free bilipoyl domain region were reduced by E3 at rates proportional to the lipoyl domain concentration, but those of the E2.E3BP were rapidly used in a concentration-independent manner consistent with bound E3 rapidly using a set of lipoyl domains localized nearby. Given this restriction and the need for E3BP for high PDC activity, directed channeling of reducing equivalents to bound E3 must be very efficient in the complex. The recombinant E2 oligomer increased E1 kinase activity by up to 4-fold and, in a Ca2+-dependent process, increased phospho-E1 phosphatase activity more than 15-fold. Thus the E2 assemblage fully provides the molecular intervention whereby a single E2-bound kinase or phosphatase molecule rapidly phosphorylate or dephosphorylate, respectively, many E2-bound E1. Thus, we prepared properly assembled, fully functional human E2 that mediated enhanced regulatory enzyme activities but, lacking E3BP, supported low PDC activity.

Hamster Monomorphic Arylamine N-Acetyltransferase: Expression in Escherichia coli and Purification

N-Acetylation is a major pathway in the metabolism of hydrazine and arylamine drugs, and has been associated with carcinogen bioactivation. Monomorphic hamster liver N-acetyltransferase (NAT1) cDNA was cloned from hamster liver cells by reverse transcriptase-coupled polymerase chain reaction. The determined nucleotide sequence was identical to that reported for NAT1. The NAT1 coding region was subcloned into the pG1 yeast expression vector, but cell extracts provided only transient acetyltransferase activity. In addition, cDNA was subcloned into the expression vectors pFLAG-ATS and pFLAG-MAC, The latter vectors encoded a tac promoter and appended a low-molecular weight (1 kDa) hydrophilic FLAG; marker peptide to the amino terminus of NAT1. Unexpectedly, periplasmic export of FLAG ATS-NAT1 by the ompA signal peptide of pFLAG-ATS proved to be detrimental to enzyme activity. High acetyltransferase activity, however, was obtained when the fusion protein was expressed in the cytosol. Enzyme purified to homogeneity by immunoaffinity chromatography exhibited substrate specificity comparable to that of the hamster-derived protein.

Histones and histone acetylation during the embryonic development of Drosophila hydei

Histones and histone acetylation have been investigated during three stages of Drosophila hydei embryogenesis — early gastrula, late gastrula and organogenesis. No essential changes in the electrophoretic pattern of the histones have been revealed during the stages examined. However, we established an enhanced level of [ 14C]acetate incorporation at the time of extensive gene activation during gastrulation as well as some quantitative differences in the pattern of acetylation during gastrula and organogenesis. We consider most of them to be related to chromatin assembly during the stage of gastrulation and suggest that the correlation between histone acetylation and gene activity during Drosophila embryogenesis concerns histone H3 acetylation. The involvement of both acetylation and deacetylation in the steady-state acetylation level has been examined as well. We have found that the higher acetyltransferase activity is responsible for the enhanced level of acetate incorporation during gastrulation.