Carotenoids with cyclic end groups are essential components of the photosynthetic membranes in all plants, algae, and cyanobacteria. These lipid-soluble compounds protect against photooxidation, harvest light for photosynthesis, and dissipate excess light energy absorbed by the antenna pigments. The cyclization of lycopene (v,v-carotene) is a key branch point in the pathway of carotenoid biosynthesis. Two types of cyclic end groups are found in higher plant carotenoids: the p and E rings. Carotenoids with two p rings are ubiquitous, and those with one p and one E ring are common; however, carotenoids with two E rings are rare. We have identified and sequenced cDNAs that encode the enzymes catalyzing the formation of these two rings in Arabidopsis. These p and E cyclases are encoded by related, single-copy genes, and both enzymes use the linear, symmetrical lycopene as a substrate. However, the E cyclase adds only one ring, forming

An Arabidopsis thaliana cDNA encoding the enzyme ␤-carotene hydroxylase was identified by functional complementation in Escherichia coli. The product of this cDNA adds hydroxyl groups to both ␤ rings of the symmetrical ␤-carotene (␤,␤-carotene) to form zeaxanthin (␤,␤-carotene-3,3-diol) and converts the monocyclic ␤-zeacarotene (7,8-dihydro-␤,-carotene) to hydroxy-␤-zeacarotene (7,8-dihydro-␤,-carotene-3-ol). The ⑀ rings of ␦-carotene (⑀,-carotene) and ␣-zeacarotene (7,8-dihydro-⑀,-carotene) are poor substrates for the enzyme. The predicted amino acid sequence of the A. thaliana enzyme resembles the four known bacterial ␤-carotene hydroxylase enzymes (31-37% identity) but is much longer, with an N-terminal extension of more than 130 amino acids. Truncation of the cDNA to produce a polypeptide lacking the first 69 amino acids does not impair enzyme activity in E. coli. Truncation to yield a polypeptide of a length comparable with the bacterial enzymes (lacking 129 N-terminal amino acids) resulted in the accumulation of the monohydroxy intermediate ␤-cryptoxanthin (␤,␤-carotene-3-ol), predominantly, when ␤-carotene was provided as the substrate. It is suggested that amino acid residues 70 -129 of the A. thaliana enzyme may play a role in formation of a functional homodimer.Carotenoids are essential components of the photosynthetic apparatus in plants, algae, and cyanobacteria. These yellow, orange, and red pigments protect against photooxidation, harvest light for photosynthesis, and serve a number of other important functions. Most of the carotenoids important in photosynthetic organisms are xanthophylls or oxygenated carotenoids (1). The dihydroxy carotenoid zeaxanthin (␤,␤-carotene-3,3Ј-diol) 1 is thought to play a central role in the nonradiative dissipation of light energy under conditions of excessive photon capture by the photosynthetic light-harvesting apparatus (2). Zeaxanthin is formed from ␤-carotene (␤,␤-carotene) by hydroxylation at position 3 in both rings of this symmetrical precursor ( Fig. 1). Zeaxanthin, in turn, serves as the substrate for biosynthesis of many other important xanthophylls.Bacterial genes encoding enzymes that convert ␤-carotene to zeaxanthin have been described (3-6), but a gene or cDNA encoding an enzyme with this activity has not yet been identified in any photosynthetic organism. We employed a functional complementation approach to identify a cDNA that encodes ␤-carotene hydroxylase in the higher plant Arabidopsis thaliana. The polypeptide predicted by this cDNA resembles the bacterial enzymes (31-37% amino acid identities), lending strong support to the concept of a common origin for the pathways of carotenoid biosynthesis in photosynthetic and nonphotosynthetic organisms. The A. thaliana enzyme, however, is much longer than the bacterial enzymes and has an N-terminal region that is essential for proper enzyme function and yet has no counterpart in the bacterial enzymes. EXPERIMENTAL PROCEDURESPlasmid Construction-A cDNA encoding an isopentenyl pyrophosphate (IPP) 2 isomeras...

A gene encoding the enzyme lycopene cyclase in the cyanobacterium Synechococcus sp strain PCC7942 was mapped by genetic complementation, cloned, and sequenced. This gene, which we have named crtL, was expressed in strains of Escherichia coli that were genetically engineered to accumulate the carotenoid precursors lycopene, neurosporene, and C-carotene. The crtL gene product converts the acyclic hydrocarbon lycopene into the bicyclic p-carotene, an essentia1 component of the photosynthetic apparatus in oxygen-evolving organisms and a source of vitamin A in human and animal nutrition. The enzyme also converts neurosporene to the monocyclic p-zeacarotene but does not cyclize C-carotene, indicating that desaturation of the 7-8 or 7'-8' carbon-carbon bond is required for cyclization. The bleaching herbicide 2-(4-methylphenoxy)triethylamine hydrochloride (MPTA) effectively inhibits both cyclization reactions. A mutation that confers resistance to MPTA in Synechococcus sp PCC7942 was identified as a point mutation in the promoter region of crtL. The deduced amino acid sequence of lycopene cyclase specifies a polypeptide of 41 1 amino acids with a molecular weight of 46,125 anda p l of 6.0. An amino acid sequence motif indicative of FAD utilization is located at the N terminus of the polypeptide. DNA gel blot hybridization analysis indicated a single copy of crtL in Synechococcus sp PCC7942. Other than the FAD binding motif, the predicted amino acid sequence of the cyanobacterial lycopene cyclase bears little resemblance to the two known lycopene cyclase enzymes from nonphotosynthetic bacteria. Preliminary results from DNA gel blot hybridization experiments suggest that, like two earlier genes in the pathway, the Synechococcus gene encoding lycopene cyclase is homologous to plant and alga1 genes encoding this enzyme.

To allow genome-scale identification of genes that regulate cellular signaling, we cloned >90% of all human full-length protein kinase cDNAs and constructed the corresponding kinase activity-deficient mutants. To establish the utility of this resource, we tested the effect of expression of the kinases on three different cellular signaling models. In all screens, many kinases had a modest but significant effect, apparently due to crosstalk between signaling pathways. However, the strongest effects were found with known regulators and novel components, such as MAP3K10 and DYRK2, which we identified in a mammalian Hedgehog (Hh) signaling screen. DYRK2 directly phosphorylated and induced the proteasome-dependent degradation of the key Hh pathway-regulated transcription factor, GLI2. MAP3K10, in turn, affected GLI2 indirectly by modulating the activity of DYRK2 and the known Hh pathway component, GSK3beta. Our results establish kinome expression screening as a highly effective way to identify physiological signaling pathway components and genes involved in pathological signaling crosstalk.

Carotenoids with cyclic end groups are essential components of the photosynthetic membranes in all plants, algae, and cyanobacteria. These lipid-soluble compounds protect against photooxidation, harvest light for photosynthesis, and dissipate excess light energy absorbed by the antenna pigments. The cyclization of lycopene (psi, psi-carotene) is a key branch point in the pathway of carotenoid biosynthesis. Two types of cyclic end groups are found in higher plant carotenoids: the beta and epsilon rings. Carotenoids with two beta rings are ubiquitous, and those with one beta and one epsilon ring are common; however, carotenoids with two epsilon rings are rare. We have identified and sequenced cDNAs that encode the enzymes catalyzing the formation of these two rings in Arabidopsis. These beta and epsilon cyclases are encoded by related, single-copy genes, and both enzymes use the linear, symmetrical lycopene as a substrate. However, the epsilon cyclase adds only one ring, forming the monocyclic delta-carotene (epsilon, psi-carotene), whereas the beta cyclase introduces a ring at both ends of lycopene to form the bicyclic beta-carotene (beta, beta-carotene). When combined, the beta and epsilon cyclases convert lycopene to alpha-carotene (beta, epsilon-carotene), a carotenoid with one beta and one epsilon ring. The inability of the epsilon cyclase to catalyze the introduction of a second epsilon ring reveals the mechanism by which production and proportions of beta,beta- and beta, epsilon-carotenoids may be controlled and adjusted in plants and algae, while avoiding the formation of the inappropriate epsilon,epsilon-carotenoids.