Bacterial β-Glucosidase Reveals the Structural and Functional Basis of Genetic Defects in Human Glucocerebrosidase 2 (GBA2)

Human glucosylcerebrosidase 2 (GBA2) of the CAZy family GH116 is responsible for the breakdown of glycosphingolipids on the cytoplasmic face of the endoplasmic reticulum and Golgi apparatus. Genetic defects in GBA2 result in spastic paraplegia and cerebellar ataxia, while cross-talk between GBA2 and GBA1 glucosylceramidases may affect Gaucher disease. Here, we report the first three-dimensional structure for any GH116 enzyme, Thermoanaerobacterium xylanolyticum TxGH116 β-glucosidase, alone and in complex with diverse ligands. These structures allow identification of the glucoside binding and active site residues, which are shown to be conserved with GBA2. Mutagenic analysis of TxGH116 and structural modeling of GBA2 provide a detailed structural and functional rationale for pathogenic missense mutations of GBA2

Association of Ferredoxin:NADP+ oxidoreductase with the photosynthetic apparatus modulates electron transfer in Chlamydomonas reinhardtii

R.M. acknowledges support from the MEXT (Ministry of Education, Culture, Sports, Science and Technology, 15K21122). T.H. gratefully acknowledges support from the DFG (DIP project cooperation “Nanoengineered optoelectronics with biomaterials and bioinspired assemblies”) and the Volkswagen Foundation (LigH2t). G.K. acknowledges support from CREST, Japan Science and Technology Agency. M.H. acknowledges support from the DFG (Deutsche Forschungsgemeinschaft, HI 739/13-1)

Dynamics and energetics of cyanobacterial photosystem I:ferredoxin complexes in different redox states

Fast turnover of ferredoxin/Fd reduction by photosystem-I/PSI requires that it dissociates rapidly after it has been reduced by PSI:Fd intracomplex electron transfer. The rate constants of Fd dissociation from PSI have been determined by flash-absorption spectroscopy with different combinations of cyanobacterial PSIs and Fds, and different redox states of Fd and of the terminal PSI acceptor (FAFB). Newly obtained values were derived firstly from the fact that the dissociation constant between PSI and redox-inactive gallium-substituted Fd increases upon (FAFB) reduction and secondly from the characterization and elucidation of a kinetic phase following intracomplex Fd reduction to binding of oxidized Fd to PSI, a process which is rate-limited by the foregoing dissociation of reduced Fd from PSI. By reference to the complex with oxidized partners, dissociation rate constants were found to increase moderately with (FAFB) single reduction and by about one order of magnitude after electron transfer from (FAFB)(-) to Fd, therefore favoring turnover of Fd reduction by PSI. With Thermosynechococcus elongatus partners, values of 270, 730 and \textgreater10000 s(-1) were thus determined for (FAFB)Fdoxidized, (FAFB)(-)Fdoxidized and (FAFB)Fdreduced, respectively. Moreover, assuming a conservative upper limit for the association rate constant between reduced Fd and PSI, a significant negative shift of the Fd midpoint potential upon binding to PSI has been calculated (\textless-60 mV for Thermosynechococcus elongatus). From the present state of knowledge, the question is still open whether this redox shift is compatible with a large (\textgreater10) equilibrium constant for intracomplex reduction of Fd from (FAFB)(-)

The ATP-Mediated Regulation of KaiB-KaiC Interaction in the Cyanobacterial Circadian Clock

<div><p>The cyanobacterial circadian clock oscillator is composed of three clock proteins—KaiA, KaiB, and KaiC, and interactions among the three Kai proteins generate clock oscillation <i>in vitro</i>. However, the regulation of these interactions remains to be solved. Here, we demonstrated that ATP regulates formation of the KaiB-KaiC complex. In the absence of ATP, KaiC was monomeric (KaiC^1mer) and formed a complex with KaiB. The addition of ATP plus Mg²⁺ (Mg-ATP), but not that of ATP only, to the KaiB-KaiC^1mer complex induced the hexamerization of KaiC and the concomitant release of KaiB from the KaiB-KaiC^1mer complex, indicating that Mg-ATP and KaiB compete each other for KaiC. In the presence of ATP and Mg²⁺ (Mg-ATP), KaiC became a homohexameric ATPase (KaiC^6mer) with bound Mg-ATP and formed a complex with KaiB, but KaiC hexamerized by unhydrolyzable substrates such as ATP and Mg-ATP analogs, did not. A KaiC N-terminal domain protein, but not its C-terminal one, formed a complex with KaiB, indicating that KaiC associates with KaiB <i>via</i> its N-terminal domain. A mutant KaiC^6mer lacking N-terminal ATPase activity did not form a complex with KaiB whereas a mutant lacking C-terminal ATPase activity did. Thus, the N-terminal domain of KaiC is responsible for formation of the KaiB-KaiC complex, and the hydrolysis of the ATP bound to N-terminal ATPase motifs on KaiC^6mer is required for formation of the KaiB-KaiC^6mer complex. KaiC^6mer that had been hexamerized with ADP plus aluminum fluoride, which are considered to mimic ADP-Pi state, formed a complex with KaiB, suggesting that KaiB is able to associate with KaiC^6mer with bound ADP-Pi. </p> </div

The role of ROC75 as a daytime component of the circadian oscillator in Chlamydomonas reinhardtii.

The circadian clocks in chlorophyte algae have been studied in two model organisms, Chlamydomonas reinhardtii and Ostreococcus tauri. These studies revealed that the chlorophyte clocks include some genes that are homologous to those of the angiosperm circadian clock. However, the genetic network architectures of the chlorophyte clocks are largely unknown, especially in C. reinhardtii. In this study, using C. reinhardtii as a model, we characterized RHYTHM OF CHLOROPLAST (ROC) 75, a clock gene encoding a putative GARP DNA-binding transcription factor similar to the clock proteins LUX ARRHYTHMO (LUX, also called PHYTOCLOCK 1 [PCL1]) and BROTHER OF LUX ARRHYTHMO (BOA, also called NOX) of the angiosperm Arabidopsis thaliana. We observed that ROC75 is a day/subjective day-phase-expressed nuclear-localized protein that associates with some night-phased clock genes and represses their expression. This repression may be essential for the gating of reaccumulation of the other clock-related GARP protein, ROC15, after its light-dependent degradation. The restoration of ROC75 function in an arrhythmic roc75 mutant under constant darkness leads to the resumption of circadian oscillation from the subjective dawn, suggesting that the ROC75 restoration acts as a morning cue for the C. reinhardtii clock. Our study reveals a part of the genetic network of C. reinhardtii clock that could be considerably different from that of A. thaliana

Effects of mutations in the ATPase motifs and phosphorylation sites of KaiC on formation of the KaiB_1-94-KaiC^6mer complex.

<p>A. Native PAGE gels of the reaction products of KaiC^1mer with KaiB_1-94. Reaction mixtures containing 15 μM KaiB_1-94 and 5 μM KaiC^1mer were incubated at 4 °C for the periods indicated. Other conditions were the same as described for Figure 1. B. Native PAGE gels of the reaction products of KaiC^6mer with KaiB_1-94. Reaction mixtures containing 5 μM KaiB_1-94 and 1 μM KaiC^6mer were incubated in the presence of Mg-ATP at 4 °C for the periods indicated. Other conditions were the same as described for Figure 1C. C. A typical 2D SDS-PAGE gel from the native-PAGE gel shown in Figure 4B. The protein bands were excised, and the proteins were extracted from them and subjected to SDS-PAGE. Other conditions were the same as described for Figure 1C. The bands 1 to 3 were the KaiC_CatE2-_/DD^6mer band (control), the upper band of the reaction products of KaiC_CatE2-_/DD^6mer with KaiB_1-94 incubated at 4 °C for 6 h, and the lower band of the reaction products of KaiC_CatE2-_/DD^6mer with KaiB_1-94 incubated similarly. D. Native PAGE gels of the reaction products of KaiC^6mer with KaiB_1-94. Reaction mixtures were incubated at 4 °C for the periods indicated. Other conditions were the same as described for Figure 4B expect that unphosphorylatable mutant KaiCs were used. E. SDS-PAGE gels showing no phosphorylation of KaiC_AA^6mer, KaiC_CatE2-^6mer, and KaiC_K294H^6mer. KaiCs^6mer (0.5 μM) were incubated with Mg-ATP in the presence of 0.5 μM KaiA at 40 °C for the periods indicated and then subjected to SDS-PAGE. p-KaiC, the phosphorylated forms of KaiC; np-KaiC, the unphosphorylated form of KaiC. Other conditions were the same as described for Figure 3. </p

The sequence motifs of KaiC and KaiB-KaiC complex formation.

<p>A. A diagram of the sequence motifs of KaiC. B. Time courses of KaiB-KaiC_DD^6mer complex formation. KaiB_WT (1 μM) and KaiB_1-94 (2 μM) were separately incubated with 1 μM KaiC_DD^6mer in reaction buffer containing Mg-ATP at 4 °C or 40 °C for the various periods indicated, and then aliquots of the reaction mixtures were subjected to native PAGE on 10 % gels. After staining the gels with CBB to visualize proteins, we estimated the amounts of KaiBs-KaiC_DD^6mer complex by densitometry. We used the maximum value obtained at a time of 6 h as the maximum value. KaiBs added and temperature conditions: open circles, KaiB_WT (40 °C); closed circles, KaiB_1-94 (40 °C); open squares, KaiB_1-94 (4 °C). C. A native PAGE gel of the reaction products of KaiC_DD^1mer with KaiB_1-94. Reaction mixtures containing 15 μM KaiB_1-94 and 5 μM KaiC_DD^1mer in reaction buffer were incubated at 4 °C for 6 h and then subjected to native PAGE. Proteins were visualized by CBB staining of the gel. Because KaiB_1-94 has an isoelectric point of 9.7, it moved in the opposite direction and could not detect by native PAGE. D. A native PAGE gel of the reaction products of KaiC_DD^1mer with KaiB_WT. Reaction mixtures containing 7.5 μM KaiB_WT and 5 μM KaiC_DD^1mer in reaction buffer were incubated at 4 °C for 6 h and then subjected to native-PAGE. E. Time course of KaiB_1-94-KaiC_DD^1mer complex formation. Reaction mixtures containing 5 μM KaiB_1-94 and 5 μM KaiC_DD^1mer in reaction buffer were incubated at 4 °C for the periods indicated and then subjected to native PAGE. We used the value obtained at a time of 9 h as the maximum value. A typical experimental data is shown. F. KaiB_1-94 concentration-dependence of the KaiB_1-94-KaiC_DD^1mer complex formation. Reaction mixtures containing 1 μM KaiC_DD^1mer and various amounts (0.5, 0.75, 1.0, 1.25, 1.5, 2.0, 2.5, 3.0, and 4.0 μM) of KaiB_1-94 in reaction buffer were incubated at 4 °C for 6 h and then subjected to native PAGE. We used the value obtained at a KaiB_1-94 concentration of 4 μM as the maximum value. A typical experimental data is shown.</p

Native PAGE gels, gel filtration chromatography elution profiles, and the immunoblots of the KaiB_1-94-KaiC complexes.

<p>A, B. Native PAGE gels. Reaction mixtures containing 15 μM KaiB_1-94 and 5 μM KaiC_N^1mer (A) or KaiC_C/DD^1mer (B) were incubated at 4 °C for the periods indicated (hereafter, unless otherwise stated, 0 h shows data for samples taken at the onset of incubation) and subjected to native PAGE. Other conditions were the same as for Figure 1C legend. C. Gel filtration chromatography elution profiles and an immunoblot. Reaction mixtures containing 7.5 μM KaiB_1-94, 2.5 μM KaiC_N^6mer, and 1 mM ATP plus 5 mM MgCl₂ (Mg-ATP) were incubated at 4 °C for 6 h and then subjected to gel filtration chromatography on a Superdex 200/HR 10/30 column equilibrated with reaction buffer containing 0.5 mM ATP and 5 mM MgCl₂ at 4 °C. We also separately analyzed KaiB_1-94 and KaiC_N^6mer by gel filtration chromatography as controls. The peak fraction samples were subjected to SDS-PAGE, blotted to PVDF membranes, and reacted with an anti-KaiB antiserum. Other conditions were the same as for Figure 1C legend. Black solid line, KaiB_1-94 + KaiC_N^6mer; gray solid line, KaiC_N^6mer; black broken line, KaiB_1-94. </p