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Phytochromes regulate central responses of plants and microorganisms such as shade avoidance and photosystem synthesis. Canonical phytochromes comprise a photosensory module of three domains. The C-terminal phytochrome-specific (PHY) domain interacts via a tongue element with the bilin chromophore in the central GAF (cGMP phosphodiesterase/adenylate cyclase/FhlA) domain. The bilin isomerizes upon illumination with red light, transforming the receptor from the Pr state to the Pfr state. The "knotless" phytochrome All2699 from the cyanobacterium Nostoc sp. PCC7120 comprises three GAF domains as a sensory module and a histidine kinase as an effector. GAF1 and GAF3 both bind a bilin, and GAF2 contains a tongue-like element. We studied the response of All2699, GAF1-GAF2, and GAF1 to red light by Fourier transform infrared difference spectroscopy, including a 13C-labeled protein moiety for assignment. In GAF1-GAF2, a refolding of the tongue from β-sheet to α-helix and an upshift of the ring D carbonyl stretch from 1700 to 1712 cm-1 were observed. Therefore, GAF1-GAF2 is regarded as the smallest model system available to study the tongue response and interaction with the chromophore. Replacement of an arginine in the tongue with proline (R387P) did not affect the unfolding of the β-sheet to Pfr but strongly impaired α-helix formation. In contrast, the Y55H mutation close to bilin ring D did not interfere with conversion to Pfr. Strikingly, the presence of GAF3 in the full-length All2699 diminished the response of the tongue and generated the signal pattern found for GAF1 alone. These results point to a regulatory or integrative role of GAF3 in All2699 that is absent in canonical phytochromes.

The hotdog-fold superfamily is a functionally diverse family of evolutionarily related enzymes, which share a common α + β-fold. The founding member of this superfamily is the Escherichia coli β-hydroxydecanoyl-holo acyl carrier protein (ACP) dehydrase/isomerase.a5 Smith and co-workers coined the name “hotdog-fold” to represent the observed tertiary structure consisting of a 5-turn α-helix cradled by a curved, 7-stranded antiparallel β-sheet. The functional unit was reported as a dimer, with the subunit interface joining the two sheets into a continuous 14-stranded β-sheet. The two active sites are located at opposite ends of the interfaced sheets. Soon after the report of the dehydratase/isomerase, the hotdog-fold was observed in the 4-hydroxybenzoyl-coenzyme A thioesterase from Pseudomonas sp. strain CBS3 (Ps 4-HB-CoA TE)1 and shortly thereafter in the 4-HB-CoA thioesterase from Arthrobacter special strain AU (Ar 4-HB-CoA TE)3 (Figure ​(Figure1A1A and B). Both thioesterases catalyze the final step of the 4-chlorobenzoate-degradation pathway.6,7 Surprisingly, the two thioesterases share little sequence identity (

The mammalian brown fat inducible thioesterase variant 2 (BFIT2), also known as ACOT11, is a multi-modular protein containing two consecutive hotdog-fold domains and a C-terminal steroidogenic acute regulatory protein related lipid transfer (START) domain (StarD14). In this study, we demonstrate that the N-terminal region of human BFIT2 (hBFIT2) constitutes a mitochondrial location signal sequence, which undergoes mitochondria-dependent posttranslational cleavage. The mature hBFIT2 is shown to be located in the mitochondrial matrix whereas the paralog “cytoplasmic acetyl-CoA hydrolase” (CACH, also known as ACOT12) was found in the cytoplam. In-vitro activity analysis of full-length hBFIT2 isolated from stably transfected HEK293 cells demonstrates selective thioesterase activity directed towards long chain fatty acyl-CoA thioesters, thus distinguishing BFIT2 catalytic function from that of CACH. The results from a protein-lipid overlay test indicate that the hBFIT2 StarD14 domain binds phosphatidylinositol 4-phosphate.

Thioesters play a central role in the cells where they participate in metabolism, membrane synthesis, signal transduction, and gene regulation. Thioesters are converted to the thiol and carboxylic acid components by thioesterase-catalyzed hydrolysis. Here we examine the biochemical and biological function of the hot dog fold thioesterase YciA (EcYciA) from Escherichia coli and its close sequence homologue HI0827 from Haemophilus influenzae (HiYciA). The quaternary structure of HiYciA was determined, using equilibrium sedimentation techniques, to be a homohexamer. Mass spectral and (31)P NMR analysis of purified HiYciA revealed a bound CoA ligand. Kinetic analyses showed that CoA is a strong feedback inhibitor. YciA thioesterase activity toward acyl-CoA substrates was determined using steady-state kinetic methods. The k cat and k cat/ K m values obtained reveal a striking combination of high catalytic efficiency and low substrate specificity. The substrate activity of propionyl-s- N-acetylcysteine was found to be negligible and that of n-butyryl-pantetheinephosphate low, and therefore, it is evident YciA does not target acylated ACPs or other acylated proteins as substrates. The results from bioinformatic analysis of the biological distribution and genome contexts of yciAs are reported. We conclude that YciA is responsible for the efficient, "seemingly" indiscriminant, CoA-regulated hydrolysis of cellular acyl-CoA thioesters in a wide range of bacteria and hypothesize that this activity may support membrane biogenesis.

Herein, we report on an in vitro kinetic activity analysis that demonstrates that the protein known as the Akt C-terminal modulator protein is a broad-range, high-activity acyl-CoA thioesterase. In vitro tests of possible activity regulation by product inhibition or by Akt1 binding gave negative results. Truncation mutants confined the thioesterase activity to the C-terminal domain, consistent with our threading model. The N-terminal domain of unknown fold and function was found to contribute to solubility.

PecE and PecF jointly catalyze the covalent attachment of phycocyanobilin to Cys-alpha84 of PecA and its concomitant isomerization to phycoviolobilin. (a) An Eschertchia coli supernatant expressing pecF has a residual activity of 6%; compared to the holoenzyme, this activity is lost upon purification. (b) Functional domains of both subunits from the cyanobacterium Mastigocladus laminosus were evaluated by mutageneses and chemical modification of amino acids. When in PecE the two motifs Y29YAAWWL and D263DLL were deleted, the holoenzyme lost its activity; it is also inactivated upon deletion of a central part (R111 to A122). The three conserved cysteines C48, C91, and C161 have only minor effects on catalysis. When in PecF the 20 C-terminal and 56 N-terminal amino acids were truncated, the lyase-isomerase activity in combination with PecE decreased to 12% and 15%, respectively, compared to the native enzyme. The catalytic efficiency (k(cat)/K(m)) decreased 16-fold when the unique four histidine residues in PecF beginning at H53 were deleted. H121 and C122 of PecF are essential for the enzyme activity; they are part of a unique stretch extending from A104 to N125 which is absent in the beta-subunit of related but nonisomerizing lyases. A single histidine and a single tryptophan are required for activity in both PecE and PecF, as judged from diethyl pyrocarbonate and N-bromosuccinimide modification and statistical analyses. Inactivation of PecE and PecF is also possible by arginine-specific reagents, while modifications of lysine, glutamate, and aspartate retained activity. (c) PecE and PecF, as well as most of the mutants, bind PCB covalently in substoichiometric amounts, as assayed by Zn(2+)-induced fluorescence on denaturing gels.

Photochromic biliproteins can be switched by light between two states, initiated by Z/E photoisomerization of the linear tetrapyrrole chromophore. The cyanobacterium Anabaena sp. PCC 7120 contains three genes coding for such biliproteins, two coding for phytochromes (aphA/B) and one for the alpha subunit of phycoerythrocyanin (pecA). (a) aphA was overexpressed in Escherichia coli with N-terminal His and S tags, and the protein was reconstituted by an optimized protocol with phycocyanobilin (PCB), to yield the photochromic chromoprotein, PCB-AphA, carrying the PCB chromophore. (b) AphA chromophorylation is autocatalytic such as in other phytochromes. (c) AphA chromophorylation is also possible by chromophore transfer from the PCB-carrying biliprotein, phycocyanin (CPC). The autocatalytic transfer is very slow, and it is enhanced more than 100-fold by catalysis of PCB:CpcA lyase and alpha-CPC as donor. (d) Through deletion mutations of aphA, a short sequence IQPHGV [amino acids (aa) 26-31] was found essential for the lyase activity of AphA, indicating an interaction of the N terminus with the chromophore-binding domain around cysteine 259. (e) A motif of at least 23 aa, starting with this sequence and located approximately 250 aa N terminal of the chromophore-binding cysteine, is proposed to relate to the lyase function in plant and most prokaryotic phytochromes. (f) Long-range interactions in AphA are further supported by blue-shifted absorptions (