In silico analysis of class I adenylate-forming enzymes reveals family and group-specific conservations.

Luciferases, aryl- and fatty-acyl CoA synthetases, and non-ribosomal peptide synthetase proteins belong to the class I adenylate-forming enzyme superfamily. The reaction catalyzed by the adenylate-forming enzymes is categorized by a two-step process of adenylation and thioesterification. Although all of these proteins perform a similar two-step process, each family may perform the process to yield completely different results. For example, luciferase proteins perform adenylation and oxidation to produce the green fluorescent light found in fireflies, while fatty-acyl CoA synthetases perform adenylation and thioesterification with coenzyme A to assist in metabolic processes involving fatty acids. This study aligned a total of 374 sequences belonging to the adenylate-forming superfamily. Analysis of the sequences revealed five fully conserved residues throughout all sequences, as well as 78 more residues conserved in at least 60% of sequences aligned. Conserved positions are involved in magnesium and AMP binding and maintaining enzyme structure. Also, ten conserved sequence motifs that included most of the conserved residues were identified. A phylogenetic tree was used to assign sequences into nine different groups. Finally, group entropy analysis identified novel conservations unique to each enzyme group. Common group-specific positions identified in multiple groups include positions critical to coordinating AMP and the CoA-bound product, a position that governs active site shape, and positions that help to maintain enzyme structure through hydrogen bonds and hydrophobic interactions. These positions could serve as excellent targets for future research.

Dynamic Expression Profile, Regulatory Mechanism and Correlation with Egg-laying Performance of ACSF Gene Family in Chicken (Gallus gallus).

Acyl-CoA synthetases (ACSs) are responsible for acyl-CoA synthesis from nonpolar hydrophilic fatty acids and play a vital role in many metabolic processes. As a category of ACS isozymes, members of ACS family (ACSF1-3) participate in lipid metabolism; however, their expression patterns, regulatory mechanisms and effects on egg-laying performance in chicken are poorly understood. Our in vivo and in vitro studies showed that ACSF1-3 genes were extensively expressed, and their expression levels changed dynamically in the liver among different development stages. Moreover, ACSF1 expression was upregulated and ACSF2 expression was downregulated by estrogen, but ACSF3 showed no response to estrogen treatment. The regulatory effect of estrogen on ACSF1 expression was mediated via ERα. The ACSF2 was highly expressed in the liver in peak-laying hens compared with pre-laying and late-laying hens, and also highly expressed in the liver continued egg-laying hens compared with inactive egg-laying hens. It is suggested that hepatic ACSF2 expression level might relate to egg-laying performance in chicken. In conclusion, the expression of ACSF1 was upregulated by estrogen via ERα, and the expression of ACSF2 was downregulated by estrogen and might be related to egg-laying performance in chicken.

Sunflower (Helianthus annuus) long-chain acyl-coenzyme A synthetases expressed at high levels in developing seeds.

Long chain fatty acid synthetases (LACSs) activate the fatty acid chains produced by plastidial de novo biosynthesis to generate acyl-CoA derivatives, important intermediates in lipid metabolism. Oilseeds, like sunflower, accumulate high levels of triacylglycerols (TAGs) in their seeds to nourish the embryo during germination. This requires that sunflower seed endosperm supports very active glycerolipid synthesis during development. Sunflower seed plastids produce large amounts of fatty acids, which must be activated through the action of LACSs, in order to be incorporated into TAGs. We cloned two different LACS genes from developing sunflower endosperm, HaLACS1 and HaLACS2, which displayed sequence homology with Arabidopsis LACS9 and LACS8 genes, respectively. These genes were expressed at high levels in developing seeds and exhibited distinct subcellular distributions. We generated constructs in which these proteins were fused to green fluorescent protein and performed transient expression experiments in tobacco cells. The HaLACS1 protein associated with the external envelope of tobacco chloroplasts, whereas HaLACS2 was strongly bound to the endoplasmic reticulum. Finally, both proteins were overexpressed in Escherichia coli and recovered as active enzymes in the bacterial membranes. Both enzymes displayed similar substrate specificities, with a very high preference for oleic acid and weaker activity toward stearic acid. On the basis of our findings, we discuss the role of these enzymes in sunflower oil synthesis.

The VLCFA elongase gene family in Arabidopsis thaliana: phylogenetic analysis, 3D modelling and expression profiling.

As precursors of wax compounds, very long chain fatty acids participate in the limitation of non-stomatal water loss and the prevention of pathogen attacks. They also serve as energy storage in seeds and as membrane building blocks. Their biosynthesis is catalyzed by the acyl-CoA elongase, a membrane-bound enzymatic complex containing four distinct enzymes (KCS, KCR, HCD and ECR). Twenty-one 3-ketoacyl-CoA synthase (KCS) genes have been identified in Arabidopsis thaliana genome. In this paper we present an overview of the acyl-CoA elongase genes in Arabidopsis focusing on the entire KCS family. We show that the KCS family is made up of 8 distinct subclasses, according to their phylogeny, duplication history, genomic organization, protein topology and 3D modelling. The analysis of the subcellular localization in tobacco cells of the different subunits of the acyl-CoA elongase shows that all these proteins are localized in the endoplasmic reticulum demonstrating that VLCFA production occurs in this compartment. The expression patterns in Arabidopsis of the acyl-CoA elongase genes suggest several levels of regulations at the tissular or organ level but also under stress conditions suggesting a complex organization of this multigenic family.

Alternative promotion of the mouse acyl-CoA synthetase 6 (mAcsl6) gene mediates the expression of multiple transcripts with 5'-end heterogeneity: genetic organization of mAcsl6 variants.

We report four variants and alternative promoter usage for the mouse acyl-CoA synthetase 6 (mAcsl6) gene. The variants, which were organized into 26 exons and 25 introns spanning 55 kb of DNA on mouse chromosome 11, were classified according to their 5'-UTRs and alternative splicing of exon 13. Alignment of the nucleotide sequences showed that the mAcsl6 variant 1 (mAcsl6_v1) and mAcsl6_v2 used a different promoter and had different splicing patterns than mAcsl6_v3 and mAcsl6_v4. The results of the promoter analysis suggest that the mAcsl6 promoter 1 (mAcsl6_pr1) region has a negative regulatory function. To verify this result, we constructed id vector constructs that contained the promoter regions mAcsl6_pr1 and 2, and the chimeric transcript. Although the mAcsl6_pr1 region was deleted, the mAcsl6_v1 and 2 transcripts were detected consistently.

Characterization of the monovalent and divalent cation requirements for the xenobiotic carboxylic acid: CoA ligases of bovine liver mitochondria.

The XL-I, XL-II and XL-III forms of xenobiotic/medium-chain fatty acid: CoA ligase were found to be inactive toward benzoate in the absence of either monovalent or divalent cations. The absolute requirement for monovalent cation was satisfied by either K+, Rb+, or NH4+. Na+ only supported a very low rate. Varying the nature of the anion had only a minor effect. For XL-I and XI-II, the optimum concentration of K+ was 50 mM; higher (physiologic) concentrations led to a decrease in activity. K+ did not inhibit XL-III. The absolute requirement for divalent cation was satisfied by Mg2+ or Mn2+, or to a lesser extent by Co2+ or Fe2+. For the XL-I and XL-II, excess uncomplexed Mg2+ or Mn2+ decreased the rate; the optimum concentration of Mn2+ was approximately the same as the concentration of ATP in the assay, and the optimum concentration of Mg2+ was approximately double the concentration of ATP in the assay. This is consistent with the concept that the divalent cation is required to complex with ATP and with the known stability constants for the ATP complexes of these two divalent cations. XL-III was not inhibited by uncomplexed divalent cations. Uncomplexed ATP was a moderate inhibitor of XL-I and XL-II, and a weak inhibitor of XL-III. The data indicate that in vivo benzoate conjugation is K+ and Mg2+ dependent, and that the cation effects are complex and differ for XL-I and XL-II as compared with XL-III.