Structural basis of EHEP-mediated offense against phlorotannin-induced defense from brown algae to protect akuBGL activity.

The defensive-offensive associations between algae and herbivores determine marine ecology. Brown algae utilize phlorotannin as their chemical defense against the predator Aplysia kurodai, which uses ß-glucosidase (akuBGL) to digest the laminarin in algae into glucose. Moreover, A. kurodai employs Eisenia hydrolysis-enhancing protein (EHEP) as an offense to protect akuBGL activity from phlorotannin inhibition by precipitating phlorotannin. To underpin the molecular mechanism of this digestive-defensive-offensive system, we determined the structures of the apo and tannic acid (TNA, a phlorotannin analog) bound forms of EHEP, as well as the apo akuBGL. EHEP consisted of three peritrophin-A domains arranged in a triangular shape and bound TNA in the center without significant conformational changes. Structural comparison between EHEP and EHEP-TNA led us to find that EHEP can be resolubilized from phlorotannin precipitation at an alkaline pH, which reflects a requirement in the digestive tract. akuBGL contained two GH1 domains, only one of which conserved the active site. Combining docking analysis, we propose the mechanisms by which phlorotannin inhibits akuBGL by occupying the substrate-binding pocket, and EHEP protects akuBGL against this inhibition by binding with phlorotannin to free the akuBGL pocket.

Potential Correlation between Changes in Serum FGF21 Levels and Lenvatinib-Induced Appetite Loss in Patients with Unresectable Hepatocellular Carcinoma.

Lenvatinib, used for unresectable hepatocellular carcinoma (HCC), causes appetite loss, but the underlying mechanisms, clinical impact, and predictive factors have been unclear. The endocrine factor FGF21 modulates appetite and is involved in cachexia. We evaluated the association between FGF21 level changes during lenvatinib treatment for unresectable HCC and appetite loss. Sixty-three eligible unresectable HCC patients who started lenvatinib treatment between 2018 and 2021 were included. We analyzed FGF21 levels at baseline; 1, 2, and 4 weeks after lenvatinib initiation, and before the onset of appetite loss. Grade ≥ 2 lenvatinib-induced appetite loss led to liver functional reserve deterioration at disease progression and a poor prognosis. Baseline characteristics and serum FGF21 levels were similar between patients with and without appetite loss. However, the serum FGF21 change rate increased significantly at 4 weeks post-lenvatinib initiation in patients with grade ≥ 2 appetite loss, as compared to those without appetite loss. Similar significant increases in the serum FGF21 level change rate were observed prior to grade ≥ 2 appetite loss onset. This suggests that changes in FGF21 levels can be used to predict patients with a greater risk of marked appetite loss and provides insights into the mechanisms underlying lenvatinib-induced appetite loss in patients with HCC.

Crystallographic analysis of Eisenia hydrolysis-enhancing protein using a long wavelength for native-SAD phasing.

Eisenia hydrolysis-enhancing protein (EHEP), which is a novel protein that has been identified in Aplysia kurodai, protects ß-glucosidases from phlorotannin inhibition to facilitate the production of glucose from the laminarin abundant in brown algae. Hence, EHEP has attracted attention for its potential applications in producing biofuel from brown algae. In this study, EHEP was purified from the natural digestive fluid of A. kurodai and was crystallized using the sitting-drop vapor-diffusion method. Native and SAD (single-wavelength anomalous diffraction) data sets were successfully collected at resolutions of 1.20 and 2.48 Šusing wavelengths of 1.0 and 2.1 Å, respectively, from crystals obtained in initial screening. The crystals belonged to space group P212121 and contained one EHEP molecule in the asymmetric unit. All 20 S-atom sites in EHEP were located and the phases were determined by the SAD method using the S atoms in the natural protein as anomalous scatterers (native-SAD). After phase improvement, interpretable electron densities were obtained and 58% of the model was automatically built.

Structural insights into the difference in substrate recognition of two mannoside phosphorylases from two GH130 subfamilies.

In Ruminococcus albus, 4-O-ß-D-mannosyl-D-glucose phosphorylase (RaMP1) and ß-(1,4)-mannooligosaccharide phosphorylase (RaMP2) belong to two subfamilies of glycoside hydrolase family 130. The two enzymes phosphorolyze ß-mannosidic linkages at the nonreducing ends of their substrates, and have substantially diverse substrate specificity. The differences in their mechanism of substrate binding have not yet been fully clarified. In the present study, we report the crystal structures of RaMP1 with/without 4-O-ß-D-mannosyl-d-glucose and RaMP2 with/without ß-(1→4)-mannobiose. The structures of the two enzymes differ at the +1 subsite of the substrate-binding pocket. Three loops are proposed to determine the different substrate specificities. One of these loops is contributed from the adjacent molecule of the oligomer structure. In RaMP1, His245 of loop 3 forms a hydrogen-bond network with the substrate through a water molecule, and is indispensible for substrate binding.

The c-di-GMP recognition mechanism of the PilZ domain of bacterial cellulose synthase subunit A.

In some Proteobacteria and Firmicutes such as Pseudomonas aeruginosa, Vibrio cholerae, Xanthomonas campestris, and Clostridium difficile, cyclic dimeric guanosine monophosphate (c-di-GMP) is known to regulate cellular processes, including motility, biofilm formation, and virulence, as a second messenger. Cellulose production in Acetobacter xylinum, a model organism of cellulose biosynthesis, also depends on by cellular c-di-GMP level. In cellulose-synthesizing bacteria, a terminal complex localized in the cell membrane synthesizes cellulose and regulates the production of cellulose sensed by c-di-GMP. Although previous studies indicated that the PilZ domain conserved in cellulose synthase subunit A (CeSA) was part of a receptor for c-di-GMP, the recognition mechanism by PilZ domain of CeSA remains unclear. In the present study, we studied the interaction between c-di-GMP and the PilZ domain of CeSA from a structural viewpoint. First, we solved the crystal structure of the PilZ domain of CeSA from A. xylinum (AxCeSA-PilZ) at 2.1Å resolution. Then, comparison of the sequence and structure of AxCeSA-PilZ to those of known structures of PilZ, such as VCA0042, PP4397, and PA4608, indicated the involvement of Lys573 and Arg643 of AxCeSA-PilZ in the recognition of c-di-GMP besides the RxxxR motif. Finally, the binding characteristics of c-di-GMP to AxCeSA-PilZ and mutants were determined with isothermal titration calorimetry, indicating that the residues corresponding to Lys573 and Arg643 in AxCeSA-PilZ generally contribute to the binding of c-di-GMP to PilZ.

Eukaryotic-type aromatic amino acid decarboxylase from the root colonizer Pseudomonas putida is highly specific for 3,4-dihydroxyphenyl-L-alanine, an allelochemical in the rhizosphere.

Aromatic amino acid decarboxylases (AADCs) are found in various organisms and play distinct physiological roles. AADCs from higher eukaryotes have been well studied because they are involved in the synthesis of biologically important molecules such as neurotransmitters and alkaloids. In contrast, bacterial AADCs have received less attention because of their simplicity in physiology and in target substrate (tyrosine). In the present study, we found that Pseudomonas putida KT2440 possesses an AADC homologue (PP_2552) that is more closely related to eukaryotic enzymes than to bacterial enzymes, and determined the genetic and enzymic characteristics of the homologue. The purified enzyme converted 3,4-dihydroxyphenyl-l-alanine (DOPA) to dopamine with K(m) and k(cat) values of 0.092 mM and 1.8 s(-1), respectively. The enzyme was essentially inactive towards other aromatic amino acids such as 5-hydroxy-l-tryptophan, l-phenylalanine, l-tryptophan and l-tyrosine. The observed strict substrate specificity is distinct from that of any AADC characterized so far. The proposed name of this enzyme is DOPA decarboxylase (DDC). Expression of the gene was induced by DOPA, as revealed by quantitative RT-PCR analysis. DDC is encoded in a cluster together with a LysR-type transcriptional regulator and a major facilitator superfamily transporter. This genetic organization is conserved among all sequenced P. putida strains that inhabit the rhizosphere environment, where DOPA acts as a strong allelochemical. These findings suggest the possible involvement of this enzyme in detoxification of the allelochemical in the rhizosphere, and the potential occurrence of a horizontal gene transfer event between the pseudomonad and its host organism.