Identification of Active Site Residues Implies a Two-step Catalytic Mechanism for Acyl-ACP Thioesterase

In plants and bacteria that use a Type II fatty acid synthase (FAS), isozymes of acyl-acyl carrier protein (ACP) thioesterase (TEs) hydrolyze the thioester bond of acyl-ACPs, terminating the process of fatty acid biosynthesis. These TEs are therefore critical in determining the fatty acid profiles produced by these organisms. Past characterizations of a limited number of plant-sourced acyl-ACP TEs have suggested a thiol-based, papain-like catalytic mechanism, involving a triad of Cys, His, and Asn residues. In this study, sequence alignment of 1019 plant and bacterial acyl-ACP TEs revealed that the previously proposed Cys catalytic residue is not universally conserved and therefore may not be a catalytic residue. Systematic mutagenesis of this residue to either Ser or Ala in three plant acyl-ACP TEs, CvFatB1 and CvFatB2 from Cuphea viscosissima and CnFatB2 from Cocos nucifera, resulted in enzymatically active variants, demonstrating that this Cys residue (Cys348 in CvFatB2) is not catalytic. In contrast, the multiple sequence alignment, together with the structure modeling of CvFatB2 suggest that the highly conserved Asp309 and Glu347, in addition to previously proposed Asn311 and His313, may be involved in catalysis. The substantial loss of catalytic competence associated with site-directed mutants at these positions confirmed the involvement of these residues in catalysis. By comparing the structures of acyl-ACP TE and the Pseudomonas 4-hydroxybenzoyl-CoA TE, both of which fold in the same hot-dog tertiary structure and catalyze the hydrolysis reaction of thioester bond, we have proposed a two-step catalytic mechanism for acyl-ACP TE that involves an enzyme-bound anhydride intermediate

Subcellular-level resolution MALDI-MS imaging of maize leaf metabolites by MALDI-linear ion trap-Orbitrap mass spectrometer

A significant limiting factor in achieving high spatial resolution for matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) imaging is the size of the laser spot at the sample surface. Here, we present modifications to the beam-delivery optics of a commercial MALDI-linear ion trap-Orbitrap instrument, incorporating an external Nd:YAG laser, beam-shaping optics, and an aspheric focusing lens, to reduce the minimum laser spot size from ~50 μm for the commercial configuration down to ~9 μm for the modified configuration. This improved system was applied for MALDI-MS imaging of cross sections of juvenile maize leaves at 5-μm spatial resolution using an oversampling method. A variety of different metabolites including amino acids, glycerolipids, and defense-related compounds were imaged at a spatial resolution well below the size of a single cell. Such images provide unprecedented insights into the metabolism associated with the different tissue types of the maize leaf, which is known to asymmetrically distribute the reactions of C4 photosynthesis among the mesophyll and bundle sheath cell types. The metabolite ion images correlate with the optical images that reveal the structures of the different tissues, and previously known and newly revealed asymmetric metabolic features are observed

Articulation of three core metabolic processes in Arabidopsis: Fatty acid biosynthesis, leucine catabolism and starch metabolism

<p>Abstract</p> <p>Background</p> <p>Elucidating metabolic network structures and functions in multicellular organisms is an emerging goal of functional genomics. We describe the co-expression network of three core metabolic processes in the genetic model plant <it>Arabidopsis thaliana</it>: fatty acid biosynthesis, starch metabolism and amino acid (leucine) catabolism.</p> <p>Results</p> <p>These co-expression networks form modules populated by genes coding for enzymes that represent the reactions generally considered to define each pathway. However, the modules also incorporate a wider set of genes that encode transporters, cofactor biosynthetic enzymes, precursor-producing enzymes, and regulatory molecules. We tested experimentally the hypothesis that one of the genes tightly co-expressed with starch metabolism module, a putative kinase AtPERK10, will have a role in this process. Indeed, knockout lines of AtPERK10 have an altered starch accumulation. In addition, the co-expression data define a novel hierarchical transcript-level structure associated with catabolism, in which genes performing smaller, more specific tasks appear to be recruited into higher-order modules with a broader catabolic function.</p> <p>Conclusion</p> <p>Each of these core metabolic pathways is structured as a module of co-expressed transcripts that co-accumulate over a wide range of environmental and genetic perturbations and developmental stages, and represent an expanded set of macromolecules associated with the common task of supporting the functionality of each metabolic pathway. As experimentally demonstrated, co-expression analysis can provide a rich approach towards understanding gene function.</p

Light-Dependent Changes in the Spatial Localization of Metabolites in Solenostemon scutellarioides (Coleus Henna) Visualized by Matrix-Free Atmospheric Pressure Electrospray Laser Desorption Ionization Mass Spectrometry Imaging

The visualization of foliage color in plants provides immediate insight into some of the compounds that exist in the leaf. However, many non-colored compounds are also present; their cellular distributions are not readily identifiable optically. In this study we evaluate the applicability of mass spectrometry imaging (MSI) via electrospray laser desorption ionization (ELDI) to reveal the spatial distribution of metabolites. ELDI-MSI is a matrix free, atmospheric pressure ionization method that utilizes a UV laser coupled with supplemental ionization by electrospray. We specifically applied ELDI-MSI to determine the spatial distribution of metabolites in Coleus Henna half leaves that were grown with half-sections either fully illuminated or shaded. We monitored dynamic changes in the spatial distribution of metabolites in response to the change of illumination every 7 days for a 28 day period. A novel source-sink relationship was observed between the 2 halves of the experimental leaf. Furthermore, Coleus Henna leaves present visually recognizable sectors associated with the differential accumulation of flavonoids. Thus, we correlated the effect of differential illumination and presence or absence of flavonoids with metabolic changes revealed by the accumulation of carbohydrates, amino acids, and organic acids. The results show the potential of ELDI-MSI to provide spatial information for a variety of plant metabolites with little sample preparation

Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity

<p>Abstract</p> <p>Background</p> <p>Acyl-acyl carrier protein thioesterases (acyl-ACP TEs) catalyze the hydrolysis of the thioester bond that links the acyl chain to the sulfhydryl group of the phosphopantetheine prosthetic group of ACP. This reaction terminates acyl chain elongation of fatty acid biosynthesis, and in plant seeds it is the biochemical determinant of the fatty acid compositions of storage lipids.</p> <p>Results</p> <p>To explore acyl-ACP TE diversity and to identify novel acyl ACP-TEs, 31 acyl-ACP TEs from wide-ranging phylogenetic sources were characterized to ascertain their <it>in vivo </it>activities and substrate specificities. These acyl-ACP TEs were chosen by two different approaches: 1) 24 TEs were selected from public databases on the basis of phylogenetic analysis and fatty acid profile knowledge of their source organisms; and 2) seven TEs were molecularly cloned from oil palm (<it>Elaeis guineensis</it>), coconut (<it>Cocos nucifera</it>) and <it>Cuphea viscosissima</it>, organisms that produce medium-chain and short-chain fatty acids in their seeds. The <it>in vivo </it>substrate specificities of the acyl-ACP TEs were determined in <it>E. coli</it>. Based on their specificities, these enzymes were clustered into three classes: 1) Class I acyl-ACP TEs act primarily on 14- and 16-carbon acyl-ACP substrates; 2) Class II acyl-ACP TEs have broad substrate specificities, with major activities toward 8- and 14-carbon acyl-ACP substrates; and 3) Class III acyl-ACP TEs act predominantly on 8-carbon acyl-ACPs. Several novel acyl-ACP TEs act on short-chain and unsaturated acyl-ACP or 3-ketoacyl-ACP substrates, indicating the diversity of enzymatic specificity in this enzyme family.</p> <p>Conclusion</p> <p>These acyl-ACP TEs can potentially be used to diversify the fatty acid biosynthesis pathway to produce novel fatty acids.</p