The PHOT1 (NPH1) gene from Avena sativa specifies the blue light receptor for phototropism, phototropin, which comprises two FMN-binding LOV domains and a serine͞threonine protein kinase domain. Light exposure is conducive to autophosphorylation of the protein kinase domain. We have reconstituted a recombinant LOV2 domain of A. sativa phototropin with various 13 C͞ 15 N-labeled isotopomers of the cofactor, FMN. The reconstituted protein samples were analyzed by NMR spectroscopy under dark and light conditions. Blue light irradiation is shown to result in the addition of a thiol group (cysteine 450) to the 4a position of the FMN chromophore. The adduct reverts spontaneously in the dark by elimination. The light-driven flavin adduct formation results in conformational modification, which was diagnosed by 1 H and 31 P NMR spectroscopy. This conformational change is proposed to initiate the transmission of the light signal via conformational modulation of the protein kinase domain conducive to autophosphorylation of NPH1. N umerous phenomena in the life cycle of plants, including phototropism, stomatal opening, and circadian periodicity, are modulated by light. Photoreceptors responsive to UV, blue, red, and far red light have been reported. Together, they span the spectral range of about 280-800 nm.Blue light-responsive processes have been known for a long time, but cognate receptors have been characterized only recently. Thus, cryptochromes characterized by sequence similarity to DNA photolyases are now believed to be involved in the synchronization of the circadian clock (1-3). Phototropins are involved in phototropism (4, 5) and have no sequence similarity with the cryptochrome group. FAD and FMN serve as chromophores for cryptochromes and phototropins, respectively (6).Phototropin of Avena sativa is a protein with 923 amino acids specified by the NPH1 gene (ref. 7; for review, see also ref. 8).The protein comprises two FMN-binding LOV domains and a serine͞threonine protein kinase domain.The LOV domains of phototropin are similar to PAS domains involved in light, oxygen, or voltage sensing (4) in a variety of regulatory as well as sensor proteins exhibiting diverse functions (4, 9, 10). Recombinant LOV1 and LOV2 domains of phototropins from different plants have been shown to bind FMN (11). The crystal structure of the LOV2 domain of PHY3 protein of Adiantum capillus-veneris has been published recently (12).In this study, we reconstituted the LOV2 apoprotein from A. sativa with Experimental ProceduresMaterials. Stable isotope-labeled FMN samples were prepared by published procedures (13-15). Recombinant LOV2 Domain of Phototropin.A recombinant fusion protein comprising the calmodulin-binding domain from myosin light chain kinase and the LOV2 domain from NPH1 protein of A. sativa was prepared as described earlier (16,17). The fusion protein is subsequently designated recombinant LOV2 domain.Reconstitution of LOV2 Domain with Isotope-Labeled FMN. Recombinant LOV2 domain (fusion protein) was depleted of FMN and su...

The biosynthesis of one riboflavin molecule requires one molecule of GTP and two molecules of ribulose 5-phosphate as substrates. The imidazole ring of GTP is hydrolytically opened, yielding a 4, 5-diaminopyrimidine which is converted to 5-amino-6-ribitylamino-2, 4(1H,3H)-pyrimidinedione by a sequence of deamination, side chain reduction and dephosphorylation. Condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione with 3, 4-dihydroxy-2-butanone 4-phosphate obtained from ribulose 5-phosphate affords 6,7-dimethyl-8-ribityllumazine. Dismutation of the lumazine derivative yields riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, which is recycled in the biosynthetic pathway. The structure of the biosynthetic enzyme, 6,7-dimethyl-8-ribityllumazine synthase, has been studied in considerable detail.

The blue-light sensitive photoreceptor, phototropin, is a flavoprotein which regulates the phototropism response of higher plants. The photoinduced triplet state and the photoreactivity of the flavin-mononucleotide (FMN) cofactor in two LOV domains of Avena sativa, Adiantum capillus-veneris, and Chlamydomonas reinhardtii phototropin have been studied by time-resolved electron paramagnetic resonance (EPR) and UV-vis spectroscopy at low temperatures (T < or = 80 K). Differences in the electronic structure of the FMN as reflected by altered zero-field splitting parameters of the triplet state could be correlated with changes in the amino acid composition of the binding pocket in wild-type LOV1 and LOV2 as well as in mutant LOV domains. Even at cryogenic temperatures, time-resolved EPR experiments indicate photoreactivity of the wild-type LOV domains, which was further characterized by UV-vis spectroscopy. Wild-type LOV1 and LOV2 were found to form an adduct between the FMN cofactor and the functional cysteine with a yield of 22% and 68%, respectively. The absorption maximum of the low-temperature photoproduct of wild-type LOV2 is red-shifted by about 15 nm as compared with the FMN C(4a)-cysteinyl adduct formed at room temperature. In light of these observations, we discuss a radical-pair reaction mechanism for the primary photoreaction in LOV domains.