Fixation of bioactive compounds to the cuticle of Artemia

Artemia is extensively used in aquaculture to feed early stages of cultured marine species. A problem associated with this practice is that Artemia fails to supply some essential nutrients. As a possible solution, we have devised a procedure to make Artemia a vehicle for exogenous nutrients and other bioactive compounds. It consists of the construction of chimeric proteins composed of a chitin-binding domain, which binds to the cuticle of Artemia, and a carrier domain that conveys a functional property. As confirmatory examples, we describe the successful fixation to Artemia's metanauplii of two hybrid proteins: a β-galactosidase from the thermophilic bacterium Thermotoga maritima and the jellyfish green fluorescent protein (GFP), both linked to the CBM2 chitin-binding domain from the hyperthermophilic archaeon Pyrococcus furiosus. Positive results of experiments carried out ex vivo and in vivo show the validity of this approach. The methodology used could become a general procedure for the attachment of different kinds of bioactive compounds, such as enzymes, hormones, antibiotics, etc., to the cuticle of Artemia as well as other arthropods. Statement of relevance: Our results overcome shortcomings of Artemia as a feedstock.En prensa2,04

The cryo-EM Structure of Thermotoga maritima β-Galactosidase: Quaternary Structure Guides Protein Engineering

10 pags., 5 figs., 1 tab.Lactose intolerance is a common digestive disorder that affects a large proportion of the adult human population. The severity of the symptoms is highly variable, depending on the susceptibility to the sugar and the amount digested. For that reason, enzymes that can be used for the production of lactose-free milk and milk derivatives have acquired singular biotechnological importance. One such case is Thermotoga maritima β-galactosidase (TmLac). Here, we report the cryo-EM structure of TmLac at 2.0 Å resolution. The protein features a newly solved domain at its C-Terminus, characteristic of the genus Thermotoga, which promotes a peculiar octameric arrangement. We have assessed the constraints imposed by the quaternary protein structure on the construction of hybrid versions of this GH2 enzyme. Carbohydrate binding modules (CBM) from the CBM2 and CBM9 families have been added at either the amino or carboxy terminus, and the structural and functional effects of such modifications have been analyzed. The results provide a basis for the rational design of hybrid enzymes that can be efficiently attached to different solid supports.This work was supported by grants from the Spanish Ministry of Economy and Competitiveness BIO2016-76601-C3-3-R (to J.S.A.), BFU2017-87316 (to R.F.L.), and AGL2016-75245-R (to J.P.). We thank the staff of the Synchrotron Radiation Source at Barcelona (Alba, Spain) for providing access and for technical assistance at the BL13-XALOC beamline. We thank the Midlands Regional Cryo-EM Facility at LIBSCB, major funder MRC, and C. Savva for assistance during data acquisition. We thank the EM unit from CNIO and the cryo-EM unit from the CNB−CIB (CSIC) for support using the EM facilities

Structural basis of the inhibition of GH1 β-glucosidases by multivalent pyrrolidine iminosugars

10 pags., 6 figs., 1 tab.The synthesis of multivalent pyrrolidine iminosugars via CuAAC click reaction between different pyrrolidine-azide derivatives and tri- or hexavalent alkynyl scaffolds is reported. The new multimeric compounds, together with the monomeric reference, were evaluated as inhibitors against two homologous GH1 β-glucosidases (BglA and BglB from Paenibacillus polymyxa). The multivalent inhibitors containing an aromatic moiety in the linker between the pyrrolidine and the scaffold inhibited the octameric BglA (µM range) but did not show affinity against the monomeric BglB, despite the similarity between the active site of both enzymes. A modest multivalent effect (rp/n = 12) was detected for the hexavalent inhibitor 12. Structural analysis of the complexes between the monomeric and the trimeric iminosugar inhibitors (4 and 10) and BglA showed the insertion of the inhibitors at the active site of BglA, confirming a competitive mode of inhibition as indicated by enzyme kinetics. Additionally, structural comparison of the BglA/4 complex with the reported BglB/2F-glucose complex illustrates the key determinants responsible for the inhibitory effect and explains the reasons of the inhibition of BglA and the no inhibition of BglB. Potential inhibition of other β-glucosidases with therapeutic relevance is discussed under the light of these observations.This work was supported by the Spanish Ministry of Economyand Competitiveness (CTQ2016-77270-R, BIO2016-76601-C3-3-R,AGL2016-75245-R), the Junta de Andalucía (FQM-345), MIUR-Italy(“Progetto Dipartimenti di Eccellenza 2018-2022” allocated toDepartment of Chemistry “Ugo Schiff”) and Ente Cassa di Risparmio di Firenze (grant n°. 2016/0845). M. Martínez-Bailén acknowledges theSpanish government for a FPU fellowship. We thank CITIUS-Universityof Seville (MS and NMR facilities) and the Synchrotron RadiationSource at Alba (Barcelona, Spain) for assistance at BL13-XALOCbeamlin