Structure of a novel photoreceptor, the BLUF domain of AppA from Rhodobacter sphaeroides.
ABSTRACT The flavin-binding BLUF domain of AppA represents a new class of blue light photoreceptors that are present in a number of bacterial and algal species. The dark state X-ray structure of this domain was determined at 2.3 A resolution. The domain demonstrates a new function for the common ferredoxin-like fold; two long alpha-helices flank the flavin, which is bound with its isoalloxazine ring perpendicular to a five-stranded beta-sheet. The hydrogen bond network and the overall protein topology of the BLUF domain (but not its sequence) bear some resemblance to LOV domains, a subset of PAS domains widely involved in signaling. Nearly all residues conserved in BLUF domains surround the flavin chromophore, many of which are involved in an intricate hydrogen bond network. Photoactivation may induce a rearrangement in this network via reorientation of the Gln63 side chain to form a new hydrogen bond to the flavin O4 position. This shift would also break a hydrogen bond to the Trp104 side chain, which may be critical in induction of global structural change in AppA.
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ABSTRACT: The arabidopsis thaliana HY4 gene encodes CRY1, a 75-kilodalton flavoprotein mediating blue light-dependent regulation of seedling development. CRY1 is demonstrated here to noncovalently bind stoichiometric amounts of flavin adenine dinucleotide (FAD). The redox properties of FAD bound by CRY1 include an unexpected stability of the neutral radical flavosemiquinone (FADH.). The absorption properties of this flavosemiquinone provide a likely explanation for the additional sensitivity exhibited by CRY1-mediated responses in the green region of the visible spectrum. Despite the sequence homology to microbial DNA photolyases, CRY1 was found to have no detectable photolyase activity.Science 09/1995; 269(5226):968-70. · 31.20 Impact Factor
Article: AppA is a blue light photoreceptor that antirepresses photosynthesis gene expression in Rhodobacter sphaeroides.[show abstract] [hide abstract]
ABSTRACT: Photosynthetic bacteria regulate photosystem synthesis in response to alterations in oxygen tension and light intensity. In this study we show that the PpsR repressor from Rhodobacter sphaeroides binds to DNA in a redox-dependent manner through the formation/breakage of an intramolecular disulfide bond. We also demonstrate that PpsR is antagonized by the flavin-containing antirepressor, AppA, that is capable of breaking the disulfide bond in oxidized PpsR as well as forming a stable AppA-PpsR(2) antirepressor-repressor complex. Blue light excitation of AppA induces a photocycle that is characterized by a long-lived red-shifted absorbance of the flavin. Light-excited AppA was found to be incapable of forming the AppA-PpsR(2) antirepressor complex. These results establish AppA as a transcription factor that controls both redox and blue light repression of photosystem gene expression by mediating DNA binding activity of PpsR.Cell 10/2002; 110(5):613-23. · 32.40 Impact Factor
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ABSTRACT: Vanillyl-alcohol oxidase (VAO) is member of a newly recognized flavoprotein family of structurally related oxidoreductases. The enzyme contains a covalently linked FAD cofactor. To study the mechanism of flavinylation we have created a design point mutation (His-61 --> Thr). In the mutant enzyme the covalent His-C8alpha-flavin linkage is not formed, while the enzyme is still able to bind FAD and perform catalysis. The H61T mutant displays a similar affinity for FAD and ADP (K(d) = 1.8 and 2.1 microm, respectively) but does not interact with FMN. H61T is about 10-fold less active with 4-(methoxymethyl)phenol) (k(cat) = 0.24 s(-)(1), K(m) = 40 microm) than the wild-type enzyme. The crystal structures of both the holo and apo form of H61T are highly similar to the structure of wild-type VAO, indicating that binding of FAD to the apoprotein does not require major structural rearrangements. These results show that covalent flavinylation is an autocatalytical process in which His-61 plays a crucial role by activating His-422. Furthermore, our studies clearly demonstrate that in VAO, the FAD binds via a typical lock-and-key approach to a preorganized binding site.Journal of Biological Chemistry 01/2001; 275(49):38654-8. · 4.77 Impact Factor