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1.
Carbohydr Polym ; 262: 117968, 2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-33838833

RESUMO

Enzymatically rearranging α-1,4 and α-1,6 glycosidic bonds in starch is a green approach to regulating its digestibility. A two-step modification process successively catalyzed by 1,4-α-glucan branching enzymes (GBEs) from Rhodothermus obamensi STB05 (Ro-GBE) and Geobacillus thermoglucosidans STB02 (Gt-GBE) was investigated as a strategy to reduce the digestibility of corn starch. This dual GBE modification process caused a reduction of 25.8 % in rapidly digestible starch fraction in corn starch, which were more effective than single GBE-catalyzed modification with the same duration. Structural analysis indicated that the dual GBE modified product contained higher branching density, more abundant short branches, and shorter external chains than those in single GBE-modified product. These results demonstrated that a moderate Ro-GBE treatment prior to starch gelatinization caused several suitable alterations in starch molecules, which promoted the transglycosylation efficiency of the following Gt-GBE treatment. This dual GBE-catalyzed modification process offered an efficient strategy for regulating starch digestibility.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/química , Glicosídeos/química , Amido/química , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Amilose/química , Amilose/metabolismo , Bacillaceae/enzimologia , Digestão , Glicosídeos/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Estrutura Molecular , Rhodothermus/enzimologia , Amido/metabolismo
2.
Sci Rep ; 10(1): 1329, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992772

RESUMO

The genome of Rhodothermus marinus DSM 4253 encodes six glycoside hydrolases (GH) classified under GH family 3 (GH3): RmBgl3A, RmBgl3B, RmBgl3C, RmXyl3A, RmXyl3B and RmNag3. The biochemical function, modelled 3D-structure, gene cluster and evolutionary relationships of each of these enzymes were studied. The six enzymes were clustered into three major evolutionary lineages of GH3: ß-N-acetyl-glucosaminidases, ß-1,4-glucosidases/ß-xylosidases and macrolide ß-glucosidases. The RmNag3 with additional ß-lactamase domain clustered with the deepest rooted GH3-lineage of ß-N-acetyl-glucosaminidases and was active on acetyl-chitooligosaccharides. RmBgl3B displayed ß-1,4-glucosidase activity and was the only representative of the lineage clustered with macrolide ß-glucosidases from Actinomycetes. The ß-xylosidases, RmXyl3A and RmXyl3B, and the ß-glucosidases RmBgl3A and RmBgl3C clustered within the major ß-glucosidases/ß-xylosidases evolutionary lineage. RmXyl3A and RmXyl3B showed ß-xylosidase activity with different specificities for para-nitrophenyl (pNP)-linked substrates and xylooligosaccharides. RmBgl3A displayed ß-1,4-glucosidase/ß-xylosidase activity while RmBgl3C was active on pNP-ß-Glc and ß-1,3-1,4-linked glucosyl disaccharides. Putative polysaccharide utilization gene clusters were also investigated for both R. marinus DSM 4253 and DSM 4252T (homolog strain). The analysis showed that in the homolog strain DSM 4252T Rmar_1080 (RmXyl3A) and Rmar_1081 (RmXyl3B) are parts of a putative polysaccharide utilization locus (PUL) for xylan utilization.


Assuntos
Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Família Multigênica , Rhodothermus/enzimologia , Rhodothermus/genética , Ativação Enzimática , Ordem dos Genes , Genes Bacterianos , Loci Gênicos , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/classificação , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Modelos Moleculares , Conformação Proteica , Relação Estrutura-Atividade , Temperatura
3.
J Am Chem Soc ; 141(25): 9798-9802, 2019 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-31187993

RESUMO

The introduction of fluoroalkyl groups into organic compounds can significantly alter pharmacological characteristics. One enabling but underexplored approach for the installation of fluoroalkyl groups is selective C( sp3)-H functionalization due to the ubiquity of C-H bonds in organic molecules. We have engineered heme enzymes that can insert fluoroalkyl carbene intermediates into α-amino C( sp3)-H bonds and enable enantiodivergent synthesis of fluoroalkyl-containing molecules. Using directed evolution, we engineered cytochrome P450 enzymes to catalyze this abiological reaction under mild conditions with total turnovers (TTN) up to 4070 and enantiomeric excess (ee) up to 99%. The iron-heme catalyst is fully genetically encoded and configurable by directed evolution so that just a few mutations to the enzyme completely inverted product enantioselectivity. These catalysts provide a powerful method for synthesis of chiral organofluorine molecules that is currently not possible with small-molecule catalysts.


Assuntos
Aminas/síntese química , Proteínas de Bactérias/química , Sistema Enzimático do Citocromo P-450/química , Fluorcarbonetos/síntese química , Alquilação , Bacillus megaterium/enzimologia , Proteínas de Bactérias/genética , Biocatálise , Carbono/química , Sistema Enzimático do Citocromo P-450/genética , Evolução Molecular Direcionada , Escherichia coli/enzimologia , Hidrogênio/química , Estudo de Prova de Conceito , Engenharia de Proteínas , Rhodothermus/enzimologia , Estereoisomerismo
4.
Int J Biol Macromol ; 132: 759-765, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30953720

RESUMO

1,4-α-Glucan branching enzyme (GBE, EC. 2.4.1.18), which plays a key role in the synthesis of starch and glycogen, has been overexpressed in E. coli as an intracellular enzyme by many researchers. In this study, it was found that the GBEs from Geobacillus thermoglucosidans and Rhodothermus obamensis were secreted into the culture medium when they were expressed separately, in E. coli. This occurred despite the absence of any signal peptide. In fact, although bioinformatics tools predicted that both of these proteins would localize to the cytoplasm, a high level of expression and non-classical secretion was found to achieve without addition of the inducer isopropyl ß-d-thiogalactopyranoside. Further experiments revealed that secretion was a two-step process that occurred via the periplasmic space. Results excluded the involvement of the Sec pathway or the TAT pathway. Instead, the findings indicated a relationship between cell membrane integrity and the secretion of the two GBEs, and suggested that their N-termini play an essential role in their expression and secretion.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Escherichia coli/enzimologia , Enzima Ramificadora de 1,4-alfa-Glucana/química , Permeabilidade da Membrana Celular , Estabilidade Enzimática , Escherichia coli/citologia , Geobacillus/enzimologia , Rhodothermus/enzimologia
5.
Proc Natl Acad Sci U S A ; 116(18): 8852-8858, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-30979809

RESUMO

To reduce experimental effort associated with directed protein evolution and to explore the sequence space encoded by mutating multiple positions simultaneously, we incorporate machine learning into the directed evolution workflow. Combinatorial sequence space can be quite expensive to sample experimentally, but machine-learning models trained on tested variants provide a fast method for testing sequence space computationally. We validated this approach on a large published empirical fitness landscape for human GB1 binding protein, demonstrating that machine learning-guided directed evolution finds variants with higher fitness than those found by other directed evolution approaches. We then provide an example application in evolving an enzyme to produce each of the two possible product enantiomers (i.e., stereodivergence) of a new-to-nature carbene Si-H insertion reaction. The approach predicted libraries enriched in functional enzymes and fixed seven mutations in two rounds of evolution to identify variants for selective catalysis with 93% and 79% ee (enantiomeric excess). By greatly increasing throughput with in silico modeling, machine learning enhances the quality and diversity of sequence solutions for a protein engineering problem.


Assuntos
Técnicas de Química Combinatória/métodos , Evolução Molecular Direcionada , Aprendizado de Máquina , Oxigenases/genética , Rhodothermus/enzimologia , Bibliotecas de Moléculas Pequenas , Sequência de Aminoácidos , Humanos , Modelos Moleculares , Oxigenases/metabolismo , Conformação Proteica
6.
Chembiochem ; 20(18): 2360-2372, 2019 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-31017712

RESUMO

Lanthanide (Ln)-dependent methanol dehydrogenases (MDHs) have recently been shown to be widespread in methylotrophic bacteria. Along with the core MDH protein, XoxF, these systems contain two other proteins, XoxG (a c-type cytochrome) and XoxJ (a periplasmic binding protein of unknown function), about which little is known. In this work, we have biochemically and structurally characterized these proteins from the methyltroph Methylobacterium extorquens AM1. In contrast to results obtained in an artificial assay system, assays of XoxFs metallated with LaIII , CeIII , and NdIII using their physiological electron acceptor, XoxG, display Ln-independent activities, but the Km for XoxG markedly increases from La to Nd. This result suggests that XoxG's redox properties are tuned specifically for lighter Lns in XoxF, an interpretation supported by the unusually low reduction potential of XoxG (+172 mV). The X-ray crystal structure of XoxG provides a structural basis for this reduction potential and insight into the XoxG-XoxF interaction. Finally, the X-ray crystal structure of XoxJ reveals a large hydrophobic cleft and suggests a role in the activation of XoxF. These studies enrich our understanding of the underlying chemical principles that enable the activity of XoxF with multiple lanthanides in vitro and in vivo.


Assuntos
Oxirredutases do Álcool/química , Proteínas de Bactérias/química , Grupo dos Citocromos c/química , Elementos da Série dos Lantanídeos/química , Proteínas Periplásmicas de Ligação/química , Ensaios Enzimáticos , Cinética , Metanol/química , Methylobacterium extorquens/enzimologia , Oxirredução , Rhodothermus/enzimologia , Saccharomyces cerevisiae/enzimologia
7.
Angew Chem Int Ed Engl ; 58(10): 3138-3142, 2019 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-30600873

RESUMO

Chiral 1,2-amino alcohols are widely represented in biologically active compounds from neurotransmitters to antivirals. While many synthetic methods have been developed for accessing amino alcohols, the direct aminohydroxylation of alkenes to unprotected, enantioenriched amino alcohols remains a challenge. Using directed evolution, we have engineered a hemoprotein biocatalyst based on a thermostable cytochrome c that directly transforms alkenes to amino alcohols with high enantioselectivity (up to 2500 TTN and 90 % ee) under anaerobic conditions with O-pivaloylhydroxylamine as an aminating reagent. The reaction is proposed to proceed via a reactive iron-nitrogen species generated in the enzyme active site, enabling tuning of the catalyst's activity and selectivity by protein engineering.


Assuntos
Alcenos/química , Amino Álcoois/química , Citocromos c/química , Rhodothermus/enzimologia , Aminação , Biocatálise , Hidroxilação , Modelos Moleculares , Estereoisomerismo
8.
Molecules ; 23(10)2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-30275414

RESUMO

Cellobiose 2-epimerase from Rhodothermus marinus (RmCE) reversibly converts a glucose residue to a mannose residue at the reducing end of ß-1,4-linked oligosaccharides. In this study, the monosaccharide specificity of RmCE has been mapped and the synthesis of d-talose from d-galactose was discovered, a reaction not yet known to occur in nature. Moreover, the conversion is industrially relevant, as talose and its derivatives have been reported to possess important antimicrobial and anti-inflammatory properties. As the enzyme also catalyzes the keto-aldo isomerization of galactose to tagatose as a minor side reaction, the purity of talose was found to decrease over time. After process optimization, 23 g/L of talose could be obtained with a product purity of 86% and a yield of 8.5% (starting from 4 g (24 mmol) of galactose). However, higher purities and concentrations can be reached by decreasing and increasing the reaction time, respectively. In addition, two engineering attempts have also been performed. First, a mutant library of RmCE was created to try and increase the activity on monosaccharide substrates. Next, two residues from RmCE were introduced in the cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus (CsCE) (S99M/Q371F), increasing the kcat twofold.


Assuntos
Carboidratos Epimerases/química , Galactose/química , Lactonas/química , Rhodothermus/enzimologia , Carboidratos Epimerases/genética , Catálise , Celobiose/química , Simulação por Computador , Biblioteca Gênica , Hexoses/química , Isomerismo , Cinética , Monossacarídeos/química , Mutação , Oligossacarídeos/química , Especificidade por Substrato
9.
Proc Natl Acad Sci U S A ; 115(28): 7308-7313, 2018 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-29946033

RESUMO

Recently, heme proteins have been discovered and engineered by directed evolution to catalyze chemical transformations that are biochemically unprecedented. Many of these nonnatural enzyme-catalyzed reactions are assumed to proceed through a catalytic iron porphyrin carbene (IPC) intermediate, although this intermediate has never been observed in a protein. Using crystallographic, spectroscopic, and computational methods, we have captured and studied a catalytic IPC intermediate in the active site of an enzyme derived from thermostable Rhodothermus marinus (Rma) cytochrome c High-resolution crystal structures and computational methods reveal how directed evolution created an active site for carbene transfer in an electron transfer protein and how the laboratory-evolved enzyme achieves perfect carbene transfer stereoselectivity by holding the catalytic IPC in a single orientation. We also discovered that the IPC in Rma cytochrome c has a singlet ground electronic state and that the protein environment uses geometrical constraints and noncovalent interactions to influence different IPC electronic states. This information helps us to understand the impressive reactivity and selectivity of carbene transfer enzymes and offers insights that will guide and inspire future engineering efforts.


Assuntos
Proteínas de Bactérias/química , Evolução Molecular Direcionada , Metano/análogos & derivados , Porfirinas/química , Rhodothermus/enzimologia , Transferases/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Metano/química , Metano/metabolismo , Porfirinas/genética , Porfirinas/metabolismo , Rhodothermus/genética , Transferases/genética , Transferases/metabolismo
10.
Nat Commun ; 9(1): 1728, 2018 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-29712914

RESUMO

Electron transfer in respiratory chains generates the electrochemical potential that serves as energy source for the cell. Prokaryotes can use a wide range of electron donors and acceptors and may have alternative complexes performing the same catalytic reactions as the mitochondrial complexes. This is the case for the alternative complex III (ACIII), a quinol:cytochrome c/HiPIP oxidoreductase. In order to understand the catalytic mechanism of this respiratory enzyme, we determined the structure of ACIII from Rhodothermus marinus at 3.9 Å resolution by single-particle cryo-electron microscopy. ACIII presents a so-far unique structure, for which we establish the arrangement of the cofactors (four iron-sulfur clusters and six c-type hemes) and propose the location of the quinol-binding site and the presence of two putative proton pathways in the membrane. Altogether, this structure provides insights into a mechanism for energy transduction and introduces ACIII as a redox-driven proton pump.


Assuntos
Proteínas de Bactérias/química , Complexo III da Cadeia de Transporte de Elétrons/química , Heme/química , Hidroquinonas/química , Subunidades Proteicas/química , Prótons , Rhodothermus/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Expressão Gênica , Heme/metabolismo , Hidroquinonas/metabolismo , Cinética , Modelos Moleculares , Oxirredução , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Rhodothermus/genética , Termodinâmica
11.
Extremophiles ; 22(3): 553-562, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29523972

RESUMO

YcfD from Escherichia coli is a homologue of the human ribosomal oxygenases NO66 and MINA53, which catalyse histidyl-hydroxylation of the 60S subunit and affect cellular proliferation (Ge et al., Nat Chem Biol 12:960-962, 2012). Bioinformatic analysis identified a potential homologue of ycfD in the thermophilic bacterium Rhodothermus marinus (ycfDRM). We describe studies on the characterization of ycfDRM, which is a functional 2OG oxygenase catalysing (2S,3R)-hydroxylation of the ribosomal protein uL16 at R82, and which is active at significantly higher temperatures than previously reported for any other 2OG oxygenase. Recombinant ycfDRM manifests high thermostability (Tm 84 °C) and activity at higher temperatures (Topt 55 °C) than ycfDEC (Tm 50.6 °C, Topt 40 °C). Mass spectrometric studies on purified R. marinus ribosomal proteins demonstrate a temperature-dependent variation in uL16 hydroxylation. Kinetic studies of oxygen dependence suggest that dioxygen availability can be a limiting factor for ycfDRM catalysis at high temperatures, consistent with incomplete uL16 hydroxylation observed in R. marinus cells. Overall, the results that extend the known range of ribosomal hydroxylation, reveal the potential for ycfD-catalysed hydroxylation to be regulated by temperature/dioxygen availability, and that thermophilic 2OG oxygenases are of interest from a biocatalytic perspective.


Assuntos
Proteínas de Escherichia coli/metabolismo , Oxigenases de Função Mista/metabolismo , Rhodothermus/enzimologia , Proteínas Ribossômicas/metabolismo , Estabilidade Enzimática , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Hidroxilação , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rhodothermus/genética , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Homologia de Sequência
12.
Biochim Biophys Acta Proteins Proteom ; 1866(2): 292-306, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29155107

RESUMO

Hydrolysis of arabinoxylan (AX) by glycoside hydrolase family 10 (GH10) xylanases produces xylo- and arabinoxylo-oligosaccharides ((A)XOS) which have shown prebiotic effects. The thermostable GH10 xylanase RmXyn10A has shown great potential to produce (A)XOS. In this study, the structure of RmXyn10A was investigated, the catalytic module by homology modelling and site-directed mutagenesis and the arrangement of its five domains by small-angle X-ray scattering (SAXS). Substrate specificity was explored in silico by manual docking and molecular dynamic simulations. It has been shown in the literature that the glycone subsites of GH10 xylanases are well conserved and our results suggest that RmXyn10A is no exception. The aglycone subsites are less investigated, and the modelled structure of RmXyn10A suggests that loop ß6α6 in the aglycone part of the active site contains a non-conserved α-helix, which blocks the otherwise conserved space of subsite +2. This structural feature has only been observed for one other GH10 xylanase. In RmXyn10A, docking revealed two alternative binding regions, one on either side of the α-helix. However, only one was able to accommodate arabinose-substitutions and the mutation study suggests that the same region is responsible for binding XOS. Several non-conserved structural features are most likely to be responsible for providing affinity for arabinose-substitutions in subsites +1 and +2. The SAXS rigid model of the modular arrangement of RmXyn10A displays the catalytic module close to the cell-anchoring domain while the carbohydrate binding modules are further away, likely explaining the observed lack of contribution of the CBMs to activity.


Assuntos
Proteínas de Bactérias/genética , Endo-1,4-beta-Xilanases/química , Rhodothermus/enzimologia , Proteínas de Bactérias/química , Endo-1,4-beta-Xilanases/genética , Domínios Proteicos , Estrutura Secundária de Proteína , Rhodothermus/genética
13.
Angew Chem Int Ed Engl ; 57(16): 4143-4148, 2018 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-29064156

RESUMO

Tailor-made: Discussed herein is the ability to adapt biology's mechanisms for innovation and optimization to solving problems in chemistry and engineering. The evolution of nature's enzymes can lead to the discovery of new reactivity, transformations not known in biology, and reactivity inaccessible by small-molecule catalysts.


Assuntos
Boranos/metabolismo , Citocromos c/metabolismo , Evolução Molecular Direcionada , Engenharia de Proteínas , Silanos/metabolismo , Biocatálise , Boranos/química , Citocromos c/química , Ensaios de Triagem em Larga Escala , Rhodothermus/enzimologia , Silanos/química
14.
Microb Cell Fact ; 16(1): 232, 2017 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-29274637

RESUMO

BACKGROUND: The gene encoding a thermostable cellulase of family 12 was previously isolated from a Rhodothermus marinus through functional screening. CelA is a protein of 260 aminoacyl residues with a 28-residue amino-terminal signal peptide. Mature CelA was poorly synthesized in some Escherichia coli strains and not at all in others. Here we present an alternative approach for its heterologous production as a secreted polypeptide in Streptomyces. RESULTS: CelA was successfully over-expressed as a secreted polypeptide in Streptomyces lividans TK24. To this end, CelA was fused C-terminally to the secretory signal peptide of the subtilisin inhibitor protein (Sianidis et al. in J Biotechnol. 121: 498-507, 2006) from Streptomyces venezuelae and a new cloning strategy developed. Optimal growth media and conditions that stall biomass production promote excessive CelA secretion. Under optimal growth conditions in nutrient broth medium, significant amounts of mature CelA (50-90 mg/L or 100-120 mg/g of dry cell weight) are secreted in the spent growth media after 7 days. A protocol to rapidly purify CelA to homogeneity from culture supernatants was developed and specific anti-sera raised against it. Biophysical, biochemical and immmuno-detection analyses indicate that the enzyme is intact, stable and fully functional. CelA is the most thermostable heterologous polypeptide shown to be secreted from S. lividans. CONCLUSION: This study further validates and extends the use of the S. lividans platform for production of heterologous enzymes of industrial importance and extends it to active thermostable enzymes. This study contributes to developing a platform for poly-omics analysis of protein secretion in S. lividans.


Assuntos
Proteínas de Bactérias/metabolismo , Celulase/metabolismo , Expressão Gênica , Rhodothermus/enzimologia , Streptomyces lividans/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Celulase/química , Celulase/genética , Estabilidade Enzimática , Temperatura Alta , Transporte Proteico , Rhodothermus/genética , Streptomyces lividans/metabolismo
15.
Nature ; 552(7683): 132-136, 2017 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-29186119

RESUMO

Recent advances in enzyme engineering and design have expanded nature's catalytic repertoire to functions that are new to biology. However, only a subset of these engineered enzymes can function in living systems. Finding enzymatic pathways that form chemical bonds that are not found in biology is particularly difficult in the cellular environment, as this depends on the discovery not only of new enzyme activities, but also of reagents that are both sufficiently reactive for the desired transformation and stable in vivo. Here we report the discovery, evolution and generalization of a fully genetically encoded platform for producing chiral organoboranes in bacteria. Escherichia coli cells harbouring wild-type cytochrome c from Rhodothermus marinus (Rma cyt c) were found to form carbon-boron bonds in the presence of borane-Lewis base complexes, through carbene insertion into boron-hydrogen bonds. Directed evolution of Rma cyt c in the bacterial catalyst provided access to 16 novel chiral organoboranes. The catalyst is suitable for gram-scale biosynthesis, providing up to 15,300 turnovers, a turnover frequency of 6,100 h-1, a 99:1 enantiomeric ratio and 100% chemoselectivity. The enantiopreference of the biocatalyst could also be tuned to provide either enantiomer of the organoborane products. Evolved in the context of whole-cell catalysts, the proteins were more active in the whole-cell system than in purified forms. This study establishes a DNA-encoded and readily engineered bacterial platform for borylation; engineering can be accomplished at a pace that rivals the development of chemical synthetic methods, with the ability to achieve turnovers that are two orders of magnitude (over 400-fold) greater than those of known chiral catalysts for the same class of transformation. This tunable method for manipulating boron in cells could expand the scope of boron chemistry in living systems.


Assuntos
Boro/química , Citocromos c/genética , Citocromos c/metabolismo , Evolução Molecular Direcionada , Escherichia coli/metabolismo , Hidrogênio/química , Engenharia Metabólica , Rhodothermus/enzimologia , Biocatálise , Boro/metabolismo , Escherichia coli/genética , Hidrogênio/metabolismo , Ligação de Hidrogênio , Redes e Vias Metabólicas/genética , Estrutura Molecular , Rhodothermus/genética , Estereoisomerismo
16.
Appl Biochem Biotechnol ; 183(4): 1503-1515, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28555295

RESUMO

The gene RmGH28 from the organism Rhodothermus marinus, a putative glycosyl hydrolase family 28 polygalacturonase, was expressed in Escherichia coli and biochemically characterized. The gene was found to encode an exopolygalacturonase termed RmGH28, with galacturonic acid monomer and the polymer substrate (n-1) as the products released when acting on de-esterified polygalacturonic acid from citrus pectin. The enzyme at 25 °C had k cat ∼6 s-1 when acting on polygalacturonic acid, with K m ∼0.7 µM and a substrate inhibition constant K si ∼70 µM. The enzyme was hyperthermophilic, with one half initial enzyme activity remaining after 1-h incubation at 93.9 °C. Since the enzyme can function at high temperatures where reaction rates are increased and the risk of bacterial contamination is decreased, this indicates that RmGH28 can be useful in industry for generating galacturonic acid from pectin. The amino acid sequence of RmGH28 is highly homologous to the known hyperthermophilic exopolygalacturonases TtGH28 and Tm0437, which together can serve as starting points for structure-function studies and molecular breeding enzyme engineering approaches.


Assuntos
Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Expressão Gênica , Glicosídeo Hidrolases/biossíntese , Glicosídeo Hidrolases/química , Rhodothermus/enzimologia , Proteínas de Bactérias/genética , Estabilidade Enzimática , Glicosídeo Hidrolases/genética , Temperatura Alta , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Rhodothermus/genética
17.
Biol Chem ; 398(9): 1037-1044, 2017 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-28141544

RESUMO

Alternative Complex III (ACIII) is an example of the robustness and flexibility of prokaryotic respiratory chains. It performs quinol:cytochrome c oxidoreductase activity, being functionally equivalent to the bc1 complex but structurally unrelated. In this work we further explored ACIII investigating the role of its monoheme cytochrome c subunit (ActE). We expressed and characterized the individually isolated ActE, which allowed us to suggest that ActE is a lipoprotein and to show its function as a direct electron donor to the caa3 oxygen reductase.


Assuntos
Grupo dos Citocromos c/metabolismo , Citocromos a3/metabolismo , Citocromos a/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Oxirredutases/metabolismo , Subunidades Proteicas/metabolismo , Rhodothermus/enzimologia , Transporte de Elétrons , Metabolismo dos Lipídeos , Modelos Moleculares , Conformação Proteica , Subunidades Proteicas/química
18.
Science ; 354(6315): 1048-1051, 2016 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-27885032

RESUMO

Enzymes that catalyze carbon-silicon bond formation are unknown in nature, despite the natural abundance of both elements. Such enzymes would expand the catalytic repertoire of biology, enabling living systems to access chemical space previously only open to synthetic chemistry. We have discovered that heme proteins catalyze the formation of organosilicon compounds under physiological conditions via carbene insertion into silicon-hydrogen bonds. The reaction proceeds both in vitro and in vivo, accommodating a broad range of substrates with high chemo- and enantioselectivity. Using directed evolution, we enhanced the catalytic function of cytochrome c from Rhodothermus marinus to achieve more than 15-fold higher turnover than state-of-the-art synthetic catalysts. This carbon-silicon bond-forming biocatalyst offers an environmentally friendly and highly efficient route to producing enantiopure organosilicon molecules.


Assuntos
Proteínas de Bactérias/química , Biocatálise , Carbono/química , Citocromos c/química , Evolução Molecular Direcionada/métodos , Compostos de Organossilício/síntese química , Rhodothermus/enzimologia , Silício/química , Proteínas de Bactérias/genética , Citocromos c/genética , Ligação de Hidrogênio , Metano/análogos & derivados , Metano/química , Especificidade por Substrato
19.
Carbohydr Polym ; 152: 51-61, 2016 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-27516249

RESUMO

Thermostable branching enzyme (BE, EC 2.4.1.18) from Rhodothermus obamensis in combination with amylomaltase (AM, EC 2.4.1.25) from Thermus thermophilus was used to modify starch structure exploring potentials to extensively increase the number of branch points in starch. Amylose is an important constituent in starch and the effect of amylose on enzyme catalysis was investigated using amylose-only barley starch (AO) and waxy maize starch (WX) in well-defined ratios. All products were analysed for amylopectin chain length distribution, α-1,6 glucosidic linkages content, molar mass distribution and digestibility by using rat intestinal α-glucosidases. For each enzyme treatment series, increased AO content resulted in a higher rate of α-1,6 glucosidic linkage formation but as an effect of the very low initial branching of the AO, the final content of α-1,6 glucosidic linkages was slightly lower as compared to the high amylopectin substrates. However, an increase specifically in short chains was produced at high AO levels. The molar mass distribution for the enzyme treated samples was lower as compared with substrate WX and AO, indicating the presence of hydrolytic activity as well as cyclisation of the substrate. For all samples, increased amylose substrate showed decreased α- and ß-amylolysis. Surprisingly, hydrolysis with rat intestinal α-glucosidases was higher with increasing α-1,6 glucosidic linkage content and decreasing M¯w indicating that steric hindrance towards the α-glucosidases was directed by the molar mass rather that the branching density of the glucan per se. Our data demonstrate that a higher amylose content in the substrate starch efficiently produces α-1,6 glucosidic linkages and that the present of amylose generates a higher M¯w and more resistant product than amylopectin. The combination of BE→AM→BE provided somewhat more resistant α-glucan products as compared to BE alone.


Assuntos
Amilopectina/química , Amilose/química , Proteínas de Bactérias/química , Sistema da Enzima Desramificadora do Glicogênio/química , Rhodothermus/enzimologia , Thermus thermophilus/enzimologia
20.
J Am Chem Soc ; 138(28): 8678-81, 2016 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-27336299

RESUMO

We describe palladium-mediated S-arylation that exploits natural metal-binding motifs to ensure high site selectivity for a proximal reactive residue. This allows the chemical identification not only of proteins that bind metals but also the environment of the metal-binding site itself through proteomic analysis of arylation sites. The transformation is easy to perform under standard conditions, does not require the isolation of a reactive Ar-Pd complex, is broad in scope, and is applicable in cell lysates as well as to covalent inhibition/modulation of metal-dependent enzymatic activity.


Assuntos
Manosiltransferases/metabolismo , Paládio/química , Sítios de Ligação , Catálise , Hidrocarbonetos Aromáticos/química , Manosiltransferases/química , Modelos Moleculares , Conformação Proteica , Rhodothermus/enzimologia
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