Disruption of the precise balance of positive and negative molecular regulators of blood and lymphatic vessels can lead to myriad diseases that affect one in four people worldwide. Although dozens of natural inhibitors of hemangiogenesis have been identified, an endogenous selective inhibitor of lymphatic vessels has not yet been described. We report the existence of a secreted, splice variant of vascular endothelial growth factor receptor-2 (sVegfr-2) that inhibits developmental and reparative lymphangiogenesis by blocking Vegf-c. Tissue-specific loss of sVegfr-2 in mice induced, at birth, spontaneous lymphatic invasion of the normally alymphatic cornea and hyperplasia of skin lymphatics without accompanying changes in blood vasculature. sVegfr-2 inhibited lymphangiogenesis but not hemangiogenesis induced by corneal suture injury or transplantation, enhanced corneal allograft survival, and suppressed lymphangioma cellular proliferation. Naturally occurring sVegfr-2 is a molecular uncoupler of blood and lymphatic vessels whose modulation might have a therapeutic role in lymphatic vascular malformations, transplantation, and potentially in tumor lymphangiogenesis and lymphedema.

Terpenes constitute a distinct class of natural products that attract insects, defend against phytopathogenic microbes and combat human diseases. However, like most natural products, they are usually made by plants and microbes in small amounts and as complex mixtures. Chemical synthesis is often costly and inefficient, and may not yield enantiomerically pure terpenes, whereas large-scale microbial production requires expensive feedstocks. We engineered high-level terpene production in tobacco plants by diverting carbon flow from cytosolic or plastidic isopentenyl diphosphate through overexpression in either compartment of an avian farnesyl diphosphate synthase and an appropriate terpene synthase. Isotopic labeling studies suggest little, if any, metabolite exchange between these two subcellular compartments. The strategy increased synthesis of the sesquiterpenes patchoulol and amorpha-4,11-diene more than 1,000-fold, as well as the monoterpene limonene 10-30 fold, and seems equally suited to generating higher levels of other terpenes for research, industrial production or therapeutic applications.

lllinois 60566 (F.W., J.P., R.C., C.S.)3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMCR) catalyzes the irreversible conversion of 3-hydroxy-3-methylglutaryl coenzyme A to mevalonate and is considered a key regulatory step controlling isoprenoid metabolism in mammals and fungi. The ratelimiting nature of this enzyme for isoprenoid biosynthesis in plants remains controversial. To investigate whether HMCR activity could be limiting in plants, we introduced a constitutively expressing hamster HMCR gene into tobacco (Nicotiana tabacum L.) plants to obtain unregulated HMCR activity. The impact of the resulting enzyme activity on the biosynthesis and accumulation of particular isoprenoids was evaluated. Expression of the hamster HMCR gene led t o a 3-t o 6-fold increase in the total HMCR enzyme activity. Total sterol accumulation was consequently increased 3-to 1 O-fold, whereas end-product sterols such as sitosterol, campesterol, and stigmasterol were increased only 2-fold. l h e leve1 of cycloartenol, a sterol biosynthetic intermediate, was increased more than 100-fold. Although the synthesis of total sterols appears to be limited normally by HMCR activity, these results indicate that the activity of one or more later enzyme(s) i n the pathway must also be involved i n determining the relative accumulation of end-product sterols.The levels of other isoprenoids such as carotenoids, phytol chain of chlorophyll, and sesquiterpene phytoalexins were relatively unaltered in the transgenic plants. It appears from these results that compartmentation, channeling, or other rate-determining enzymes operate to control the accumulation of these other isoprenoid end products.

Terpene synthases are a mechanistically intriguing family of enzymes that catalyze complex, multistep reactions that are capable of generating hundreds of structurally diverse hydrocarbon and oxygenated scaffolds of biological and commercial importance. Interestingly, distantly related terpene synthases from fungi to plants all contain an invariant three-dimensional fold, and molecular comparisons of their active sites indicate that they are enriched with relatively inert amino acid residues that do not react directly with the reaction intermediates. Therefore, catalytic specificity appears to rely on the contour and dynamics of the active site created by the positioning of amino acid backbones and side chains on this catalytic surface and by supporting layers of residues surrounding the synthase active site cavity. Despite the high degree of structural relatedness among terpene synthases, previous studies suggest that no clear relationship between phylogenic organization and catalytic specificities is easily deciphered. We now report on the reciprocal interconversion of catalytic specificities between two distinct yet evolutionarily related terpene synthases based on the systematic identification and mutational replacement of variable residues within and surrounding the active site. Furthermore, we uncover previously undocumented biosynthetic activity during the interconversion, activity that could have been present in a common ancestor of these two highly related synthases. These results provide a simplified means for mapping structural features that are responsible for functional attributes and a strategy for identifying residues that differentiate divergent biosynthetic properties in phylogenetically related terpene synthases.phylogenetic relationships ͉ rational design ͉ sesquiterpene ͉ structure-function T erpenes comprise the most diverse collection of natural products known. Terpene hydrocarbon scaffolds are generated by the action of mono-, sesqui-, and diterpene synthases that catalyze multistep reactions with diphosphorylated substrates of 10 (geranyl diphosphate), 15 [farnesyl diphosphate (FPP)] or 20 (geranylgeranyl diphosphate) carbons (1). The reactions catalyzed by terpene synthases are unparalleled relative to other classes of enzymes because they often consist of a series of stereochemically complex steps. These reactions include ionization of the diphosphate substituent creating an acyclic and reactive carbocation intermediate. In some cases, the isomerization of the all-trans substrate configuration to a cis-trans isomer also occurs before subsequent reactions. Additional steps add increasing complexity and include regio-and stereospecific formation of single or multiple rings, proton eliminations to form double bonds, water quenching of carbocations to create terpene alcohols, and stereospecific hydride, methyl, and methylene migrations. Equally intriguing, the three-dimensional structure of terpene synthases from fungi to plants is highly conserved, including a ''terpene fold'' composed lar...