56 research outputs found
Oxidation of Monolignols by Members of the Berberine Bridge Enzyme Family Suggests a Role in Plant Cell Wall Metabolism
Plant genomes contain a large number of genes encoding for berberine bridge enzyme (BBE)-like enzymes. Despite the widespread occurrence and abundance of this protein family in the plant kingdom, the biochemical function remains largely unexplored. In this study, we have expressed two members of the BBE-like enzyme family from Arabidopsis thaliana in the host organism Komagataella pastoris. The two proteins, termed AtBBE-like 13 and AtBBE-like 15, were purified, and their catalytic properties were determined. In addition, AtBBE-like 15 was crystallized and structurally characterized by x-ray crystallography. Here, we show that the enzymes catalyze the oxidation of aromatic allylic alcohols, such as coumaryl, sinapyl, and coniferyl alcohol, to the corresponding aldehydes and that AtBBE-like 15 adopts the same fold as vanillyl alcohol oxidase as reported previously for berberine bridge enzyme and other FAD-dependent oxidoreductases. Further analysis of the substrate range identified coniferin, the glycosylated storage form of coniferyl alcohol, as a substrate of the enzymes, whereas other glycosylated monolignols were rather poor substrates. A detailed analysis of the motifs present in the active sites of the BBE-like enzymes in A. thaliana suggested that 14 out of 28 members of the family might catalyze similar reactions. Based on these findings, we propose a novel role of BBE-like enzymes in monolignol metabolism that was previously not recognized for this enzyme family
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Rationally engineered flavin-dependent oxidase reveals steric control of dioxygen reduction
The ability of flavoenzymes to reduce dioxygen varies greatly and is controlled by the protein environment that can cause either a rapid (oxidases) or sluggish (dehydrogenases) reaction. Previously, a “gatekeeper” amino acid residue was identified that controls the reactivity to dioxygen in proteins from the vanillyl alcohol oxidase superfamily of flavoenzymes. We have identified an alternate gatekeeper residue that similarly controls dioxygen reactivity in the grass pollen allergen Phl p 4, a member of this superfamily, which has glucose dehydrogenase activity and the highest redox potential measured in a flavoenzyme. A substitution at the alternate gatekeeper site (I153V) transformed the enzyme into an efficient oxidase by increasing dioxygen reactivity by a factor of 60,000. An inverse exchange (V169I) in the structurally related berberine bridge enzyme (BBE) lowered its dioxygen reactivity by a factor of 500. Structural and biochemical characterization of these and additional variants showed that our model enzymes have a cavity binding an anion and resembling the “oxyanion hole” in the proximity of the flavin ring. We showed also that steric control of access to this site is the most important parameter affecting dioxygen reactivity in BBE-like enzymes. Analysis of flavin-dependent oxidases from other superfamilies revealed similar structural features suggesting that dioxygen reactivity might be governed by a common mechanistic principle.This is an author's peer-reviewed final manuscript, as accepted by the publisher. The published article is copyrighted by FEBS (Federation of European Biochemical Societies) and published by John Wiley & Sons, Inc. It can be found at: http://febs.onlinelibrary.wiley.com/hub/journal/10.1111/%28ISSN%291742-4658
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An analysis and evaluation of the WeFold collaborative for protein structure prediction and its pipelines in CASP11 and CASP12
Every two years groups worldwide participate in the Critical Assessment of Protein Structure Prediction (CASP) experiment to blindly test the strengths and weaknesses of their computational methods. CASP has significantly advanced the field but many hurdles still remain, which may require new ideas and collaborations. In 2012 a web-based effort called WeFold, was initiated to promote collaboration within the CASP community and attract researchers from other fields to contribute new ideas to CASP. Members of the WeFold coopetition (cooperation and competition) participated in CASP as individual teams, but also shared components of their methods to create hybrid pipelines and actively contributed to this effort. We assert that the scale and diversity of integrative prediction pipelines could not have been achieved by any individual lab or even by any collaboration among a few partners. The models contributed by the participating groups and generated by the pipelines are publicly available at the WeFold website providing a wealth of data that remains to be tapped. Here, we analyze the results of the 2014 and 2016 pipelines showing improvements according to the CASP assessment as well as areas that require further adjustments and research
X-ray crystal structure of Saccharomyces cerevisiae Pdx1 provides insights into the oligomeric nature of PLP synthases
The universal enzymatic cofactor vitamin B6 can be synthesized as pyridoxal 5-phosphate (PLP) by the glutamine amidotransferase Pdx1. We show that Saccharomyces cerevisiae Pdx1 is hexameric by analytical ultracentrifugation and by crystallographic 3D structure determination. Bacterial homologues were previously reported to exist in hexamer:dodecamer equilibrium. A small sequence insertion found in yeast Pdx1 elevates the dodecamer dissociation constant when introduced into Bacillus subtilis Pdx1. Further, we demonstrate that the yeast Pdx1 C-terminus contacts an adjacent subunit, and deletion of this segment decreases enzymatic activity 3.5-fold, suggesting a role in catalysis
The origins and spread of domestic horses from the Western Eurasian steppes
This is the final version. Available on open access from Nature Research via the DOI in this recordData availability: All collapsed and paired-end sequence data for samples sequenced in this study are available in compressed fastq format through the European Nucleotide Archive under accession number PRJEB44430, together with rescaled and trimmed bam sequence alignments against both the nuclear and mitochondrial horse reference genomes. Previously published ancient data used in this study are available under accession numbers PRJEB7537, PRJEB10098, PRJEB10854, PRJEB22390 and PRJEB31613, and detailed in Supplementary Table 1. The genomes of ten modern horses, publicly available, were also accessed as indicated in their corresponding original publications57,61,85-87.NOTE: see the published version available via the DOI in this record for the full list of authorsDomestication of horses fundamentally transformed long-range mobility and warfare. However, modern domesticated breeds do not descend from the earliest domestic horse lineage associated with archaeological evidence of bridling, milking and corralling at Botai, Central Asia around 3500 BC. Other longstanding candidate regions for horse domestication, such as Iberia and Anatolia, have also recently been challenged. Thus, the genetic, geographic and temporal origins of modern domestic horses have remained unknown. Here we pinpoint the Western Eurasian steppes, especially the lower Volga-Don region, as the homeland of modern domestic horses. Furthermore, we map the population changes accompanying domestication from 273 ancient horse genomes. This reveals that modern domestic horses ultimately replaced almost all other local populations as they expanded rapidly across Eurasia from about 2000 BC, synchronously with equestrian material culture, including Sintashta spoke-wheeled chariots. We find that equestrianism involved strong selection for critical locomotor and behavioural adaptations at the GSDMC and ZFPM1 genes. Our results reject the commonly held association between horseback riding and the massive expansion of Yamnaya steppe pastoralists into Europe around 3000 BC driving the spread of Indo-European languages. This contrasts with the scenario in Asia where Indo-Iranian languages, chariots and horses spread together, following the early second millennium BC Sintashta culture
Buchbesprechungen
Frank Daubner, Makedonien nach den Königen (168 v. Chr.–14 n. Chr.) (Historia. Einzelschriften 251), Stuttgart: Steiner 2018
Luis Ángel Hidalgo Martín, Jonathan Edmondsen, Juana Márquez Pérez, José Luis Ramírez Sádaba, Nueva epigrafía funeraria de Augusta Emerita. Tituli sepulcrales urbanos (ss. I–VII) y su contexto arqueológico (NEFAE) (Memoria 1. Monografías arqueológicas de Mérida), Mérida 2019
Stephen Mitchell, David French, The Greek and Latin Inscriptions of Ankara (Ancyra). Vol. II: Late Roman, Byzantine and other Texts (Vestigia 72), München 2019Frank Daubner, Makedonien nach den Königen (168 v. Chr.–14 n. Chr.) (Historia. Einzelschriften 251), Stuttgart: Steiner 2018
Luis Ángel Hidalgo Martín, Jonathan Edmondsen, Juana Márquez Pérez, José Luis Ramírez Sádaba, Nueva epigrafía funeraria de Augusta Emerita. Tituli sepulcrales urbanos (ss. I–VII) y su contexto arqueológico (NEFAE) (Memoria 1. Monografías arqueológicas de Mérida), Mérida 2019
Stephen Mitchell, David French, The Greek and Latin Inscriptions of Ankara (Ancyra). Vol. II: Late Roman, Byzantine and other Texts (Vestigia 72), München 201
Primary Care and Physical Literacy: A Non-Randomized Controlled Pilot Study to Combat the High Prevalence of Physically Inactive Adults in Austria
The multidimensional concept of physical literacy is fundamental for lifelong physical activity engagement. However, physical literacy-based interventions are in their infancy, especially among adults. Therefore, the purpose of this pilot study was to assess the association of a physical literacy-based intervention with changes in self-reported physical literacy among inactive adults. A non-randomized controlled study (2 × 2 design) was conducted, comparing pre- vs. postintervention. Twenty-eight inactive healthy participants in the intervention group (89% female, 53 ± 10 years) entered a physical literacy-based intervention once weekly for 14 weeks. The non-treated control group consisted of 22 inactive adults (96% female, 50 ± 11 years). Physical literacy was evaluated with a questionnaire encompassing five domains: physical activity behavior, attitude/understanding, motivation, knowledge, and self-efficacy/confidence. ANOVA models were applied to evaluate changes by time and condition. Following the intervention, significant improvements were seen for overall physical literacy and in four out of five physical literacy domains, including physical activity behavior, attitude/understanding, knowledge, and self-efficacy/confidence (all p < 0.01, Cohen’s d = 0.38–0.83). No changes by time x condition were found for motivation. The physical literacy-based intervention applied in this study may be a promising approach to help inactive adults to adopt an active lifestyle
Rationally engineered flavin-dependent oxidase reveals steric control of dioxygen reduction
The ability of flavoenzymes to reduce dioxygen varies greatly, and is controlled by the protein environment, which may cause either a rapid reaction (oxidases) or a sluggish reaction (dehydrogenases). Previously, a ‘gatekeeper’ amino acid residue was identified that controls the reactivity to dioxygen in proteins from the vanillyl alcohol oxidase superfamily of flavoenzymes. We have identified an alternative gatekeeper residue that similarly controls dioxygen reactivity in the grass pollen allergen Phl p 4, a member of this superfamily that has glucose dehydrogenase activity and the highest redox potential measured in a flavoenzyme. A substitution at the alternative gatekeeper site (I153V) transformed the enzyme into an efficient oxidase by increasing dioxygen reactivity by a factor of 60 000. An inverse exchange (V169I) in the structurally related berberine bridge enzyme (BBE) decreased its dioxygen reactivity by a factor of 500. Structural and biochemical characterization of these and additional variants showed that our model enzymes possess a cavity that binds an anion and resembles the ‘oxyanion hole’ in the proximity of the flavin ring. We showed also that steric control of access to this site is the most important parameter affecting dioxygen reactivity in BBE-like enzymes. Analysis of flavin-dependent oxidases from other superfamilies revealed similar structural features, suggesting that dioxygen reactivity may be governed by a common mechanistic principle
Biocatalytic Organic Synthesis of Optically Pure (S)-Scoulerine and Berbine and Benzylisoquinoline Alkaloids
A chemoenzymatic approach for the asymmetric total synthesis of the title compounds is described that employs an enantioselective oxidative CC bond formation catalyzed by berberine bridge enzyme (BBE) in the asymmetric key step. This unique reaction yielded enantiomerically pure (R)-benzylisoquinoline derivatives and (S)-berbines such as the natural product (S)-scoulerine, a sedative and muscle relaxing agent. The racemic substrates rac-1 required for the biotransformation were prepared in 48 linear steps using either a BischlerNapieralski cyclization or a C1C alkylation approach. The chemoenzymatic synthesis was applied to the preparation of fourteen enantiomerically pure alkaloids, including the natural products (S)-scoulerine and (R)-reticuline, and gave overall yields of up to 20% over 59 linear steps
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