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1.
BMC Genomics ; 22(1): 652, 2021 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-34507539

RESUMO

BACKGROUND: Composting is an important technique for environment-friendly degradation of organic material, and is a microbe-driven process. Previous metagenomic studies of composting have presented a general description of the taxonomic and functional diversity of its microbial populations, but they have lacked more specific information on the key organisms that are active during the process. RESULTS: Here we present and analyze 60 mostly high-quality metagenome-assembled genomes (MAGs) recovered from time-series samples of two thermophilic composting cells, of which 47 are potentially new bacterial species; 24 of those did not have any hits in two public MAG datasets at the 95% average nucleotide identity level. Analyses of gene content and expressed functions based on metatranscriptome data for one of the cells grouped the MAGs in three clusters along the 99-day composting process. By applying metabolic modeling methods, we were able to predict metabolic dependencies between MAGs. These models indicate the importance of coadjuvant bacteria that do not carry out lignocellulose degradation but may contribute to the management of reactive oxygen species and with enzymes that increase bioenergetic efficiency in composting, such as hydrogenases and N2O reductase. Strong metabolic dependencies predicted between MAGs revealed key interactions relying on exchange of H+, NH3, O2 and CO2, as well as glucose, glutamate, succinate, fumarate and others, highlighting the importance of functional stratification and syntrophic interactions during biomass conversion. Our model includes 22 out of 49 MAGs recovered from one composting cell data. Based on this model we highlight that Rhodothermus marinus, Thermobispora bispora and a novel Gammaproteobacterium are dominant players in chemolithotrophic metabolism and cross-feeding interactions. CONCLUSIONS: The results obtained expand our knowledge of the taxonomic and functional diversity of composting bacteria and provide a model of their dynamic metabolic interactions.


Assuntos
Compostagem , Metagenoma , Actinobacteria , Bactérias/genética , Rhodothermus
2.
Chemistry ; 27(56): 13998-14006, 2021 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-34355437

RESUMO

To protect their intracellular proteins, extremophile microorganisms synthesize molecules called compatible solutes. These molecules are the result of the attachment of a small negatively charged molecule to a sugar molecule. It has been found that these molecules, not only protect the microorganism against osmotic stress but also against other extreme conditions. They can also confer protection against extreme conditions to isolated enzymes from different organisms making them an exciting prospect for potential biotechnological applications. One of the most widespread compatible solute in hyperthermophile organisms is the molecule 2-O-α-D-mannosyl-D-glycerate (MG). In addition to confer protection to proteins against extreme conditions, MG was found to prevent Alzheimer's ß-amyloid aggregation and reduce α-synuclein fibril formation in Parkinson's disease. In this work we studied, using computational methods, the catalytic mechanism of the synthesis of MG by the enzyme mannosylglycerate synthase (MGS) from the thermophilic bacteria Rhodothermus marinus.


Assuntos
Biotecnologia , Glicosiltransferases , Manosiltransferases , Rhodothermus
3.
J Agric Food Chem ; 69(20): 5755-5763, 2021 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-33988022

RESUMO

The 1,4-α-glucan branching enzyme (GBE, EC 2.4.1.18) catalyzes the formation of α-1,6 branching points in starch and plays a key role in synthesis. To obtain mechanistic insights into the catalytic action of the enzyme, we first determined the crystal structure of GBE from Rhodothermus obamensis STB05 (RoGBE) to a resolution of 2.39 Å (PDB ID: 6JOY). The structure consists of three domains: domain A, domain C, and the carbohydrate-binding module 48 (CBM48). An engineered truncated mutant lacking the CBM48 domain (ΔCBM48) showed significantly reduced ligand binding affinity and enzyme activity. Comparison of the structures of RoGBE with other GBEs showed that CBM48 of RoGBE had a longer flexible loop. Truncation of the flexible loops resulted in reduced binding affinity and activity, thereby substantiating the importance of the optimum loop structure for catalysis. In essence, our study shows that CBM48, especially the flexible loop, plays an important role in substrate binding and enzymatic activity of RoGBE. Further, based on the structural analysis, kinetics, and activity assays on wild type and mutants, as well as homology modeling, we proposed a mechanistic model (called the "lid model") to illustrate how the flexible loop triggers substrate binding, ultimately leading to catalysis.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana , Rhodothermus , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Glucanos , Rhodothermus/metabolismo , Especificidade por Substrato
4.
Methods Mol Biol ; 2290: 187-201, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34009591

RESUMO

Polymerase chain reaction (PCR) is a popular molecular tool for detection of bacteria. PCR allows millions of copies of a target segment of DNA to be produced. The DNA is extracted from overnight grown cultures of pure bacterial isolates using either the organo-solvent method or a commercial DNA extraction kit. The quality and purity of the DNA is determined by performing gel electrophoresis on 0.8% agarose gel. The DNA is amplified by performing PCR assay. Bands of approximately 1.5 kb in size are obtained from the amplified products of DNA. The PCR products run on 1.5% agarose gel are visualized with UV light and imaged by gel documentation system. This chapter outlines the protocol for isolation and amplification of DNA from cellulolytic bacteria. Cellulolytic bacteria are considered a potential source of cellulases for pretreatment of crop residues during biogas production. PCR is considered a very powerful, sensitive, specific, fast, and reliable tool in molecular detection and diagnostics.


Assuntos
Biocombustíveis/microbiologia , DNA Bacteriano/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Bacillus/genética , Bactérias/classificação , Bactérias/genética , Cellulomonas/genética , Clostridium/genética , DNA Bacteriano/genética , Eletroforese/métodos , Pseudomonas/genética , Rhodothermus/genética
5.
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
6.
Protein J ; 40(2): 184-191, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33651244

RESUMO

Substrate binding proteins (SBPs) bind to specific ligands in the periplasmic regions of cells and then bind to membrane proteins to participate in transport or signal transduction. Typically, SBPs consist of two α/ß domains and recognize the substrate by a flexible hinge region between the two domains. Conversely, the short-length SBPs are often observed in protein databases, which are located around methyl-accepting chemotaxis protein genes. We previously determined the crystal structure of Rhodothermus marinus SBP (named as RmSBP), consisting of a single α/ß domain; however, the substrate recognition mechanism is still unclear. To better understand the functions of short length RmSBP, we performed a comprehensive study, involving comparative structure analysis, computational substrate docking, and X-ray crystallographic data. RmSBP shares a high level of similarity in the α/ß domain region with other SBPs, but it has a distinct topology in the C-terminal domain. The substrate binding model suggested that conformational changes in the peripheral region of RmSBP was required to recognize the substrate. We determined the crystal structures of RmSBP at pH 5.5, 6.0, and 7.5. RmSBP showed structural flexibility in the ß1-α2 loop, ß5-ß6 loop, and extended C-terminal domains, based on the electron density map and temperature B-factor analysis. These results provide information that will further our understanding on the functions of the short length SBP.


Assuntos
Proteínas de Bactérias/metabolismo , Rhodothermus/metabolismo , Cristalografia por Raios X , Ligação Proteica , Conformação Proteica
7.
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
8.
Extremophiles ; 23(6): 735-745, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31522265

RESUMO

This work presents an evaluation of batch, fed-batch, and sequential batch cultivation techniques for production of R. marinus DSM 16675 and its exopolysaccharides (EPSs) and carotenoids in a bioreactor, using lysogeny broth (LB) and marine broth (MB), respectively, in both cases supplemented with 10 g/L maltose. Batch cultivation using LB supplemented with maltose (LBmalt) resulted in higher cell density (OD620 = 6.6) than use of MBmalt (OD620 = 1.7). Sequential batch cultivation increased the cell density threefold (OD620 = 20) in LBmalt and eightfold (OD620 = 14) in MBmalt. In both single and sequential batches, the production of carotenoids and EPSs using LBmalt was detected in the exponential phase and stationary phase, respectively, while in MBmalt formation of both products was detectable in both the exponential and stationary phases of the culture. Heteropolymeric EPSs were produced with an overall volumetric productivity (QE) of 0.67 (mg/L h) in MBmalt and the polymer contained xylose. In LB, QE was lower (0.1 mg/L h) and xylose could not be detected in the composition of the produced EPSs. In conclusion, this study showed the importance of a process design and medium source for production of R. marinus DSM 16675 and its metabolites.


Assuntos
Reatores Biológicos , Rhodothermus/crescimento & desenvolvimento , Carotenoides/metabolismo , Meios de Cultura/química
9.
Protein Expr Purif ; 164: 105464, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31376486

RESUMO

Xylanases (EC 3.2.1.8) are essential enzymes due to their applications in various industries such as textile, animal feed, paper and pulp, and biofuel industries. Halo-thermophilic Rhodothermaceae bacterium RA was previously isolated from a hot spring in Malaysia. Genomic analysis revealed that this bacterium is likely to be a new genus of the family Rhodothermaceae. In this study, a xylanase gene (1140 bp) that encoded 379 amino acids from the bacterium was cloned and expressed in Escherichia coli BL21(DE3). Based on InterProScan, this enzyme XynRA1 contained a GH10 domain and a signal peptide sequence. XynRA1 shared low similarity with the currently known xylanases (the closest is 57.2-65.4% to Gemmatimonadetes spp.). The purified XynRA1 achieved maximum activity at pH 8 and 60 °C. The protein molecular weight was 43.1 kDa XynRA1 exhibited an activity half-life (t1/2) of 1 h at 60 °C and remained stable at 50 °C throughout the experiment. However, it was NaCl intolerant, and various types of salt reduced the activity. This enzyme effectively hydrolyzed xylan (beechwood, oat spelt, and Palmaria palmata) and xylodextrin (xylotriose, xylotetraose, xylopentaose, and xylohexaose) to produce predominantly xylobiose. This xylanase is the first functionally characterized enzyme from the bacterium, and this work broadens the knowledge of GH10 xylanases.


Assuntos
Proteínas de Bactérias/genética , Endo-1,4-beta-Xilanases/genética , Rhodothermus/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/isolamento & purificação , Endo-1,4-beta-Xilanases/metabolismo , Escherichia coli/genética , Expressão Gênica , Vetores Genéticos/genética , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Rhodothermus/química , Rhodothermus/isolamento & purificação , Rhodothermus/metabolismo , Alinhamento de Sequência , Especificidade por Substrato
10.
Protein Expr Purif ; 164: 105478, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31421223

RESUMO

A gene encoding 1,4-α-glucan branching enzyme (GBE, EC 2.4.1.18) from the extremely thermophilic bacterium Rhodothermus obamensis STB05 was successfully cloned and expressed in Escherichia coli. Extracellular expression of the recombinant enzyme (R.o-GBE) was achieved with a yield of 1080 mg/L. Then it was purified and further characterized biochemically. R.o-GBE was optimally active at pH 7.0 and 65 °C. It remained stable at temperatures up to 80 °C and had a half-life at 85 °C of approximately 31 min. Far-UV circular dichroism and intrinsic fluorescence analyses revealed that high temperatures reduced its activity by changing the secondary and tertiary structure of R.o-GBE. The enzyme had broad pH stability between pH 3.0 and 11.0 at 4 °C, and preferred weakly acidic conditions at high temperatures. None of the metal ions enhanced the activity of R.o-GBE, but Ca2+ may be required for its activity. Its specific activity with amylopectin was 6651 U/mg, which is much higher than that reported for other GBEs. Its excellent thermostability, broad pH stability, and high specific activity make R.o-GBE highly suitable for industrial applications.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/genética , Proteínas de Bactérias/genética , Rhodothermus/genética , Enzima Ramificadora de 1,4-alfa-Glucana/química , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cálcio/metabolismo , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Expressão Gênica , Concentração de Íons de Hidrogênio , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rhodothermus/química , Rhodothermus/metabolismo
11.
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
12.
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
13.
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
14.
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
15.
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
16.
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
17.
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
18.
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
19.
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
20.
Biochem Biophys Res Commun ; 497(1): 368-373, 2018 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-29432740

RESUMO

Substrate-binding proteins (SBPs) bind to specific ligands and are associated with membrane protein complexes for transport or signal transduction. Most SBPs recognize substrates by the hinge motion between two distinct α/ß domains. However, short SBP motifs are often observed in protein databases, which are located around methyl-accepting chemotaxis protein genes, but structural and functional studies have yet to be performed. Here, we report the crystal structure of an unusually small SBP from Rhodothermus marinus (named as RmSBP) at 1.9 Å. This protein is composed of a single α/ß-domain, unlike general SBPs that have two distinct domains. RmSBP exhibits a high structural similarity to the C-terminal domain of the previously reported amino acid bound SBPs, while it does not contain an N-terminal domain for substrate recognition. As a result of the structural comparison analysis, RmSBP has a putative SBP that is different from the previously reported SBP. Our results provide insight into a new class of substrate recognition mechanism by the mini SBP protein.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Proteínas de Transporte/química , Proteínas de Transporte/ultraestrutura , Modelos Químicos , Modelos Moleculares , Rhodothermus/química , Sequência de Aminoácidos , Sítios de Ligação , Simulação por Computador , Cristalização , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Análise de Sequência de Proteína , Especificidade por Substrato
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