E1 enzymes facilitate conjugation of ubiquitin and ubiquitin-like proteins through adenylation, thioester transfer within E1, and thioester transfer from E1 to E2 conjugating proteins. Structures of human heterodimeric Sae1/Sae2-Mg . ATP and Sae1/Sae2-SUMO-1-Mg . ATP complexes were determined at 2.2 and 2.75 Å resolution, respectively. Despite the presence of Mg . ATP, the Sae1/Sae2-SUMO-1-Mg . ATP structure reveals a substrate complex insomuch as the SUMO C-terminus remains unmodified within the adenylation site and 35 Å from the catalytic cysteine, suggesting that additional changes within the adenylation site may be required to facilitate chemistry prior to adenylation and thioester transfer. A mechanism for E2 recruitment to E1 is suggested by biochemical and genetic data, each of which supports a direct role for the E1 Cterminal ubiquitin-like domain for E2 recruitment during conjugation.

SummaryPlant isoprenoids represent a heterogeneous group of compounds which play essential roles not only in growth and development, but also in the interaction of plants with their environment. Higher plants contain two pathways for the biosynthesis of isoprenoids: the mevalonate pathway, located in the cytosol/endoplasmic reticulum, and the recently discovered mevalonate-independent pathway (Rohmer pathway), located in the plastids. In order to evaluate the function of the Rohmer pathway in the regulation of the synthesis of plastidial isoprenoids, we have isolated a tomato cDNA encoding 1-deoxy-D-xylulose 5-phosphate synthase (DXS), the ®rst enzyme of the pathway. We demonstrate in vivo activity and plastid targeting of plant DXS. Expression analysis of the tomato DXS gene indicates developmental and organ-speci®c regulation of mRNA accumulation and a strong correlation with carotenoid synthesis during fruit development. 1-Deoxy-D-xylulose feeding experiments, together with expression analysis of DXS and PSY1 (encoding the fruit-speci®c isoform of phytoene synthase) in wildtype and yellow¯esh mutant fruits, indicate that DXS catalyses the ®rst potentially regulatory step in carotenoid biosynthesis during early fruit ripening. Our results change the current view that PSY1 is the only regulatory enzyme in tomato fruit carotenogenesis, and point towards a coordinated role of both DXS and PSY1 in the control of fruit carotenoid synthesis.

For many years it was accepted that isopentenyl diphosphate, the common precursor of all isoprenoids, was synthesized through the well known acetate͞mevalonate pathway. However, recent studies have shown that some bacteria, including Escherichia coli, use a mevalonate-independent pathway for the synthesis of isopentenyl diphosphate. The occurrence of this alternative pathway has also been reported in green algae and higher plants. The first reaction of this pathway consists of the condensation of (hydroxyethyl)thiamin derived from pyruvate with the C1 aldehyde group of D-glyceraldehyde 3-phosphate to yield D-1-deoxyxylulose 5-phosphate. In E. coli, D-1-deoxyxylulose 5-phosphate is also a precursor for the biosynthesis of thiamin and pyridoxol. Here we report the molecular cloning and characterization of a gene from E. coli, designated dxs, that encodes D-1-deoxyxylulose-5-phosphate synthase. The dxs gene was identified as part of an operon that also contains ispA, the gene that encodes farnesyl-diphosphate synthase. D-1-Deoxyxylulose-5-phosphate synthase belongs to a family of transketolase-like proteins that are highly conserved in evolution.Isoprenoids are ubiquitous compounds found in all living organisms. Some isoprenoids play essential roles in particular cell functions such as sterols, contributing to eukaryotic membrane architecture, acyclic polyprenoids found in the side chain of ubiquinone, plastoquinone, and chlorophylls, sugar carriers for polysaccharide biosynthesis, or carotenoids in photosynthetic organisms. Although the physiological role of other isoprenoids is less evident, like that of the vast array of plant secondary metabolites, some are known to play key roles in the adaptative responses to different environmental challenges. In spite of the remarkable diversity of structure and function, all isoprenoids originate from a single metabolic precursor, isopentenyl diphosphate (1, 2).For many years, it was accepted that isopentenyl diphosphate was synthesized through the well known acetate͞mevalonate pathway. However, recent studies have demonstrated that the mevalonate-dependent pathway does not operate in all living organisms (3, 4). An alternative mevalonate-independent pathway for isopentenyl diphosphate biosynthesis was initially characterized in bacteria (4, 5) and later also in green algae (6) and higher plants (7-11). The first reaction of the novel mevalonateindependent pathway involves the condensation of (hydroxyethyl)thiamin derived from pyruvate with the C1 aldehyde group of D-glyceraldehyde 3-phosphate to yield D-1-deoxyxylulose 5-phosphate (5, 12). In Escherichia coli, D-1-deoxyxylulose (most likely in the form of D-1-deoxyxylulose 5-phosphate) is efficiently incorporated into the prenyl side chain of menaquinone and ubiquinone (12,13). In plants, the incorporation of D-1-deoxyxylulose into isoprenoids has also been reported (11,14). In addition, D-1-deoxyxylulose has also been described as a precursor for the biosynthesis of thiamin and pyridoxol. D-1-Deoxyxylulose is the...

SummaryThe genetic manipulation of both the mevalonic acid (MVA) and methylerythritol-4-phosphate (MEP) pathways, leading to the formation of isopentenyl diphosphate (IPP), has been achieved in tomato using 3-hydroxymethylglutaryl CoA ( hmgr-1 ) and 1-deoxy-Dxylulose-5-phosphate synthase ( dxs ) genes, respectively. Transgenic plants containing an additional hmgr-1 from Arabidopsis thaliana , under the control of the cauliflower mosaic virus (CaMV) 35S constitutive promoter, contained elevated phytosterols (up to 2.4-fold), but IPP-derived isoprenoids in the plastid were unaltered. Transgenic lines containing a bacterial dxs targeted to the plastid with the tomato dxs transit sequence resulted in an increased carotenoid content (1.6-fold), which was inherited in the next generation.Phytoene and β -carotene exhibited the greatest increases (2.4-and 2.2-fold, respectively).Extra-plastidic isoprenoids were unaffected in these lines. These data are discussed with respect to the regulation, compartmentalization and manipulation of isoprenoid biosynthetic pathways and their relevance to plant biotechnology.

Post-translational modification of proteins by small polypeptides, such as ubiquitin, has emerged as a common and important mechanism for regulating protein function. Small ubiquitin-like modifier (SUMO) is a small protein that is structurally related to but functionally different from ubiquitin. We report the identification and functional analysis of AtSUMO1, AtSUMO2, and AtSCE1a as components of the SUMO conjugation (sumoylation) pathway in Arabidopsis. In yeast-two hybrid assays, AtSUMO1/2 interacts specifically with a SUMO-conjugating enzyme but not with a ubiquitin-conjugating enzyme. AtSCE1a, the Arabidopsis SUMO-conjugating enzyme ortholog, conjugates SUMO to RanGAP in vitro. AtSUMO1/2 and AtSCE1a colocalize at the nucleus, and AtSUMO1/2 are conjugated to endogenous SUMO targets in vivo. Analysis of transgenic plants showed that overexpression of AtSUMO1/2 does not have any obvious effect in general plant development, but increased sumoylation levels attenuate abscisic acid (ABA)-mediated growth inhibition and amplify the induction of ABA-and stress-responsive genes such as RD29A . Reduction of AtSCE1a expression levels accentuates ABA-mediated growth inhibition. Our results suggest a role for SUMO in the modulation of the ABA signal transduction pathway.