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

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, Lphenylalanine, 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.

Edited by Judit Ov adiIn Ruminococcus albus, 4-O-b-D-mannosyl-D-glucose phosphorylase (RaMP1) and b-(1,4)-mannooligosaccharide phosphorylase (RaMP2) belong to two subfamilies of glycoside hydrolase family 130. The two enzymes phosphorolyze b-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-b-D-mannosyl-Dglucose and RaMP2 with/without b-(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.Keywords: 4-O-b-D-mannosyl-D-glucose phosphorylase; glycoside hydrolase family 130; hydrogen bond-network; substrate recognition; X-ray crystallography; b-1,4-mannooligosaccharide phosphorylase Highlights• Reveals the structures of RaMP1 and RaMP2, and their complexes with substrates.• Structural insight into substrate recognition of RaMP1 and RaMP2 is discussed.• RaMP1 is the first GH130 enzyme exhibiting as a homotrimer in its native state.• Three loops are important in substrate recognition at the +1 subsite in RaMP1 and RaMP2.• A hydrogen-bond network in RaMP1 is considered to be important for substrate binding.

Eisenia hydrolysis-enhancing protein (EHEP), which is a novel protein that has been identified in Aplysia kurodai, protects β-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 Å using 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.

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 β-glucosidase (akuBGL) to digest the laminarin in algae to 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 apo and tannic-acid (TNA, a phlorotannin-analog) bound form of EHEP, as well as akuBGL. EHEP consisted of three peritrophin-A domains formed in a triangle 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 the inhibition by binding with phlorotannin to free the akuBGL pocket.