The crystal structure of Canavalia brasiliensis lectin suggests a correlation between its quaternary conformation and its distinct biological properties from Concanavalin A

AbstractCanavalia brasiliensis lectin was isolated from the seeds of a Brazilian autochthonous Leguminosae plant. Despite extensive amino acid sequence similarity with Concanavalin A, C. brasiliensis lectin exerts in vitro and in vivo cellular effects that are markedly different from those displayed by Concanavalin A. We have solved the crystal structure of the C. brasiliensis lectin at 3.0 Å resolution. The three-dimensional structure of the lectin monomer can be superimposed onto that of Concanavalin A with a root-mean-square deviation for all Cα atoms of 0.65 Å. However, this parameter is 0.84 and 1.62 Å when the C. brasiliensis lectin dimer and tetramer, respectively, are compared with the same structures of Concanavalin A. We suggest that these differences in quaternary structure may account for the different biological properties of these two highly related Leguminosae lectins.© 1997 Federation of European Biochemical Societies

Structure and evolutionary trace-assisted screening of a residue swapping the substrate ambiguity and chiral specificity in an esterase

11 pags., 6figs., 3 pags.Our understanding of enzymes with high substrate ambiguity remains limited because their large active sites allow substrate docking freedom to an extent that seems incompatible with stereospecificity. One possibility is that some of these enzymes evolved a set of evolutionarily fitted sequence positions that stringently allow switching substrate ambiguity and chiral specificity. To explore this hypothesis, we targeted for mutation a serine ester hydrolase (EH) that exhibits an impressive 71-substrate repertoire but is not stereospecific (e.e. 50%). We used structural actions and the computational evolutionary trace method to explore specificity-swapping sequence positions and hypothesized that position I244 was critical. Driven by evolutionary action analysis, this position was substituted to leucine, which together with isoleucine appears to be the amino acid most commonly present in the closest homologous sequences (max. identity, ca. 67.1%), and to phenylalanine, which appears in distant homologues. While the I244L mutation did not have any functional consequences, the I244F mutation allowed the esterase to maintain a remarkable 53-substrate range while gaining stereospecificity properties (e.e. 99.99%). These data support the possibility that some enzymes evolve sequence positions that control the substrate scope and stereospecificity. Such residues, which can be evolutionarily screened, may serve as starting points for further designing substrate-ambiguous, yet chiral-specific, enzymes that are greatly appreciated in biotechnology and synthetic chemistry.MF acknowledges the grant ‘INMARE’ from the EuropeanUnion’s Horizon 2020 (grant agreement no. 634486), the grantsPCIN-2017-078 (within the Marine Biotechnology ERA-NET) and BIO2017-85522-R from the Ministerio de Economía, Industria y Competitividad, Agencia Estatal de Investigación (AEI), Fondo Eur-opeo de Desarrollo Regional (FEDER) and the European Union (EU),and the grant 2020AEP061 from the Agencia Estatal CSIC. J.S-A.acknowledges grant PID2019-105838RB-C33 from the Ministeriode Ciencia e Innovación, Agencia Estatal de Investigación (AEI),Fondo Europeo de Desarrollo Regional (FEDER) and the EuropeanUnion (EU). P.N.G. acknowledges the support of the Era-Net IB Pro-ject MetaCat funded through UK Biotechnology and BiologicalSciences Research Council (BBSRC), grant No. BB/M029085/1, andthe Centre for Environmental Biotechnology Project, co-fundedby European Regional Development Fund (ERDF) via the WelshGovernment (WEFO); R.B. acknowledges the Supercomputing Wales project, co-funded by ERDF via WEFO. OL and PK were sup-ported by the National Institutes of Health (NIH) grants 5R01AG061105, 5R01GM066099, and 5R01GM079656. C. Coscolínthanks the Ministerio de Economía y Competitividad and FEDER fora PhD fellowship (Grant BES-2015-073829). Staff of the Synchrotron Radiation Source at Alba (Barcelona, Spain) for assistance at the BL13-XALOC beamlin

Synthesis and chromatographic-separation of the stereoisomers of furnidipine

The four stereoisomers of methyl tetrahydrofuran-2-ylmethyl 2,6-dimethyl-4-(o-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxilate (furnidipine), have been synthesized and separated by chiral chromatography using D-phenylglycine as chiral stationary phase. Enantiomeric purity of stereoisomers is determined by HPLC-CSP technique and configurations deduced via X-ray crystallography

Structural Basis of the Inhibition of GH1 β-glucosidases by Multivalent Pyrrolidine Iminosugars

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.Ministerio de Economía y Competitividad CTQ2016-77270-R, BIO2016-76601-C3-3-R, AGL2016-75245-RJunta de Andalucía FQM-345Fondazione CR Firenze 2016/084

Enzymatic Synthesis of Phloretin alpha-Glucosides Using a Sucrose Phosphorylase Mutant and its Effect on Solubility, Antioxidant Properties and Skin Absorption

Glycosylation of polyphenols may increase their aqueous solubility, stability, bioavailability and pharmacological activity. Herein, we used a mutant of sucrose phosphorylase from Thermoanaerobacterium thermosaccharolyticum engineered to accept large polyphenols (variant TtSPP_R134A) to produce phloretin glucosides. The reaction was performed using 10% (v/v) acetone as cosolvent. The selective formation of a monoglucoside or a diglucoside (53% and 73% maximum conversion percentage, respectively) can be kinetically controlled. MS and 2D-NMR determined that the monoglucoside was phloretin 4¿-O-¿-D-glucopyranoside and the diglucoside phloretin-4¿-O-[¿-D-glucopyranosyl-(1¿3)-O-¿-D-glucopyranoside], a novel compound. The molecular features that determine the specificity of this enzyme for 4¿-OH phenolic group were analysed by induced-fit docking analysis of each putative derivative, using the crystal structure of TtSPP and changing the mutated residue. The mono- and diglucoside were, respectively, 71- and 1200-fold more soluble in water than phloretin at room temperature. The a-glucosylation decreased the antioxidant capacity of phloretin, measured by DPPH and ABTS assays; however, this loss was moderate and the activity could be recovered upon deglycosylation in vivo. Since phloretin attracts a great interest in dermocosmetic applications, we analyzed the percutaneous absorption of glucosides and the aglycon employing a pig skin model. Although the three compounds were detected in all skin layers (except the fluid receptor), the diglucoside was present mainly on superficial layers

Engineering a Highly Regioselective Fungal Peroxygenase for the Synthesis of Hydroxy Fatty Acids

The hydroxylation of fatty acids is an appealing reaction in synthetic chemistry, although the lack of selective catalysts hampers its industrial implementation. In this study, we have engineered a highly regioselective fungal peroxygenase for the ω-1 hydroxylation of fatty acids with quenched stepwise over-oxidation. One single mutation near the Phe catalytic tripod narrowed the heme cavity, promoting a dramatic shift toward subterminal hydroxylation with a drop in the over-oxidation activity. While crystallographic soaking experiments and molecular dynamic simulations shed light on this unique oxidation pattern, the selective biocatalyst was produced by Pichia pastoris at 0.4 g L−1 in a fed-batch bioreactor and used in the preparative synthesis of 1.4 g of (ω-1)-hydroxytetradecanoic acid with 95 % regioselectivity and 83 % ee for the S enantiomer.This work was supported by the European Union Project grant H2020-BBI-PPP-2015-2-720297-ENZOX2; the Spanish projects PID2019-106166RB-100-OXYWAVE, PID2020-118968RB-100-LILI, PID2021-123332OB-C21 and PID2019-107098RJ-I00, funded by the Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación (AEI)/doi: 10.13039/501100011033/; the “Comunidad de Madrid” Synergy CAM project Y2018/BIO-4738-EVOCHIMERA-CM; the Generalitat Valenciana projects CIPROM/2021/079-PROMETEO and SEJI/2020/007; and the PIE-CSIC projects PIE-202040E185 and PIE-201580E042. P.G.d.S. thanks the Ministry of Science, Innovation and Universities (Spain) for her FPI scholarship (BES-2017-080040) and the Ministry of Science and Innovation for her contract as part of the PTQ2020-011037 project funded by MCIN/AEI/10.13039/501100011033 within the NextGenerationEU/PRTR. D.G.-P. thanks Juan de la Cierva Incorporación contract Ref. No.: IJC2020-043725-I, funded by MCIN/AEI/10.13039/501100011033, and the EU NextGenerationEU/PRTR program. K.Ś. thanks to Ministerio de Ciencia e Innovación and Fondo Social Europeo for a Ramón y Cajal contract (Ref. RYC2020-030596-I). We thank the Synchrotron Radiation Source at Alba (Barcelona, Spain) for assistance with the BL13-XALOC beamline

Genetically engineered proteins with two active sites for enhanced biocatalysis and synergistic chemo- and biocatalysis

Enzyme engineering has allowed not only the de novo creation of active sites catalysing known biological reactions with rates close to diffusion limits, but also the generation of abiological sites performing new-to-nature reactions. However, the catalytic advantages of engineering multiple active sites into a single protein scaffold are yet to be established. Here, we report on proteins with two active sites of biological and/or abiological origin, for improved natural and non-natural catalysis. The approach increased the catalytic properties, such as enzyme efficiency, substrate scope, stereoselectivity and optimal temperature window, of an esterase containing two biological sites. Then, one of the active sites was metamorphosed into a metal-complex chemocatalytic site for oxidation and Friedel–Crafts alkylation reactions, facilitating synergistic chemo- and biocatalysis in a single protein. The transformations of 1-naphthyl acetate into 1,4-naphthoquinone (conversion approx. 100%) and vinyl crotonate and benzene into 3-phenylbutyric acid (≥83%; e.e. >99.9%) were achieved in one pot with this artificial multifunctional metalloenzyme.This work was funded by grant ‘INMARE’ from the European Union’s Horizon 2020 (grant agreement no. 634486), grants PCIN-2017-078 (within the Marine Biotechnology ERA-NET), CTQ2016-79138-R, BIO2016-76601-C3-1-R, BIO2016-76601-C3-3-R, BIO2017-85522-R, RTI2018-095166-B-I00 and RTI2018-095090-B-100 from the Ministerio de Economía y Competitividad, the Ministerio de Ciencia, Innovación y Universidades (MCIU), the Agencia Estatal de Investigación (AEI), the Fondo Europeo de Desarrollo Regional (FEDER) and the European Union (EU). P.N.G. and R.B. acknowledge the support of the UK Biotechnology and Biological Sciences Research Council (BBSRC; grant No. BB/M029085/1) and the Centre of Environmental Biotechnology Project and the Supercomputing Wales project, which are partly funded by the European Regional Development Fund (ERDF) through the Welsh Government. The authors gratefully acknowledge the financial support provided by the ERDF. C.C. thanks the Ministerio de Economía y Competitividad and FEDER for a Ph.D. fellowship (Grant BES-2015-073829). J.L.G.-A. thanks the support of the Spanish Ministry of Education, Culture and Sport through the National Program FPU (FPU17/00044). I.C.-R. thanks the Regional Government of Madrid for a fellowship (PEJ_BIO_AI_1201). The authors would like to acknowledge S. Ciordia and M. C. Mena for MALDI-TOF/TOF analysis. We thank the staff of both the European Synchrotron Radiation Facility (ESRF, Grenoble, France), for providing access and technical assistance at beamline ID30A-1/MASSIf-1, and the Synchrotron Radiation Source at Alba (Barcelona, Spain), for assistance at BL13-XALOC beamline. The authors would also like to acknowledge M. J. Vicente and M. A. Pascual at the Servicio Interdepartamental de Investigación (SIDI) of the Autonomous University of Madrid for the ESI-MS analyses

Transforming an esterase into an enantioselective catecholase through bioconjugation of a versatile metal-chelating inhibitor

4 pags., 3 figs.Metal complexes introduced into protein scaffolds can generate versatile biomimetic catalysts endowed with a variety of catalytic properties. Here, we synthesized and covalently bound a bipyridinyl derivative to the active centre of an esterase to generate a biomimetic catalyst that shows catecholase activity and enantioselective catalytic oxidation of (+)-catechin.We acknowledge the financial support of the European Union’s Horizon 2020 (GA 101000327), and the Ministerio de Ciencia e Innovacio´n and Agencia Estatal de Investigacio´n (AEI) (DOI 10.13039/501100011033), and the ‘‘NextGenerationEU/PRTR’’ (PID2020-112758RB-I00, PDC2021-121534-I00, TED2021-130544B-I00, PID2019-105838RB-C31, PID2019-105838RB-C33).Peer reviewe

Enzymes for consumer products to achieve climate neutrality

29 pags., 4 figs., 3 tabs., 1 graf.Accumulated greenhouse gas emissions are expected to increase from 36.2 Giga-tons (Gt) to 60 Gt over the next three decades. The global surface temperature has increased by¿+¿1.09¿°C since 2001, and might increase by¿+¿2.2¿°C in 2100, +3.6¿°C in 2200 and +4.6¿°C in 2500. These emissions and temperature rises cannot be reduced in their entirety, but they can be lowered by using enzymes. Enzymes are proteins that catalyze biochemical reactions that make life possible since 3.8 billion years ago. Scientists have been able to "domesticate" them in such a way that enzymes, and their engineered variants, are now key players of the circular economy. With a world production of 117 Kilo-tons and a trade of 14.5 Billion-dollars, they have the potential to annually decrease CO2 emissions by 1 to 2.5 Billion-tons (Bt), the carbon demand to synthesise chemicals by 200 Million tons (Mt), the amount of chemicals by 90¿Mt, and the economic losses derived from global warming by 0.5%, while promoting biodiversity and our planet¿s health. Our success to increase these benefits will depend on better integration of enzymatic solutions in different sectors.This study was conducted under the auspices of the FuturEnzyme Project funded by the European Union’s Horizon 2020 Research and Innovation Programme under Grant Agreement No. 101000327. MF also acknowledges Grants PID2020-112758RB-I00, PDC2021-121534-I00, and TED2021-130544B-I00 from the MCIN/AEI/10.13039/501100011033 and the European Union (“NextGenerationEU/PRTR”)

Choice of the initial antiretroviral treatment for HIV-positive individuals in the era of integrase inhibitors

BACKGROUND: We aimed to describe the most frequently prescribed initial antiretroviral therapy (ART) regimens in recent years in HIV-positive persons in the Cohort of the Spanish HIV/AIDS Research Network (CoRIS) and to investigate factors associated with the choice of each regimen. METHODS: We analyzed initial ART regimens prescribed in adults participating in CoRIS from 2014 to 2017. Only regimens prescribed in >5% of patients were considered. We used multivariable multinomial regression to estimate Relative Risk Ratios (RRRs) for the association between sociodemographic and clinical characteristics and the choice of the initial regimen. RESULTS: Among 2874 participants, abacavir(ABC)/lamivudine(3TC)/dolutegavir(DTG) was the most frequently prescribed regimen (32.1%), followed by tenofovir disoproxil fumarate (TDF)/emtricitabine (FTC)/elvitegravir(EVG)/cobicistat(COBI) (14.9%), TDF/FTC/rilpivirine (RPV) (14.0%), tenofovir alafenamide (TAF)/FTC/EVG/COBI (13.7%), TDF/FTC+DTG (10.0%), TDF/FTC+darunavir/ritonavir or darunavir/cobicistat (bDRV) (9.8%) and TDF/FTC+raltegravir (RAL) (5.6%). Compared with ABC/3TC/DTG, starting TDF/FTC/RPV was less likely in patients with CD4100.000 copies/mL. TDF/FTC+DTG was more frequent in those with CD4100.000 copies/mL. TDF/FTC+RAL and TDF/FTC+bDRV were also more frequent among patients with CD4<200 cells//muL and with transmission categories other than men who have sex with men. Compared with ABC/3TC/DTG, the prescription of other initial ART regimens decreased from 2014-2015 to 2016-2017 with the exception of TDF/FTC+DTG. Differences in the choice of the initial ART regimen were observed by hospitals' location. CONCLUSIONS: The choice of initial ART regimens is consistent with Spanish guidelines' recommendations, but is also clearly influenced by physician's perception based on patient's clinical and sociodemographic variables and by the prescribing hospital location