Assembly in vitro of Rhodococcus jostii RHA1 encapsulin and peroxidase DypB to form a nanocompartment.
TL;DR: Recombinant DypB could be assembled in vitro with monomeric encapsulin to form an assembly of similar size to the encapsulin‐only nanocompartment, as assessed by gel filtration and stoichiometry.
Abstract: Rhodococcus jostii RHA1 peroxidase DypB has been recently identified as a bacterial lignin peroxidase. The dypB gene is co-transcribed with a gene encoding an encapsulin protein, shown in Thermotoga maritima to assemble to form a 60-subunit nano-compartment, and DypB contains a C-terminal sequence motif thought to target the protein to the encapsulin nanocompartment. R. jostii RHA1 encapsulin protein has been overexpressed in R. jostii RHA1, and purified as a high molecular weight assembly (Mr >106). The purified nanocompartment can be denatured to form a low molecular weight species by treatment at pH 3.0, and can be re-assembled to form the nanocompartment at pH 7.0. Recombinant DypB can be assembled in vitro with monomeric encapsulin to form an assembly of similar size and shape to the encapsulin-only nanocompartment, assessed by dynamic light scattering. The assembled complex shows enhanced lignin degradation activity per mg DypB present, compared with native DypB, using a nitrated lignin UV-vis assay method. The measured stoichiometry of 8.6 µmoles encapsulin/µmol DypB in the complex is comparable to the value of 10 predicted from the crystal structure.
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TL;DR: An overview of recent advances in the identification and use of bacterial enzymes acting on lignin or lignIn-derived products is provided, including DyP-type peroxidases and laccases.
393 citations
TL;DR: Recent advances in studying bacterial lignin degradation as an approach to exploring greater diversity in the environment are discussed.
363 citations
TL;DR: The review describes recent developments in the understanding of bacterial enzymes for lignin breakdown, such as DyP peroxidases, bacterial laccases, and beta-etherase enzymes.
207 citations
TL;DR: Physiological data reveal that few nanocompartments are assembled during vegetative growth, but they increase fivefold upon starvation, protecting cells from oxidative stress through iron sequestration.
Abstract: Living cells compartmentalize materials and enzymatic reactions to increase metabolic efficiency. While eukaryotes use membrane-bound organelles, bacteria and archaea rely primarily on protein-bound nanocompartments. Encapsulins constitute a class of nanocompartments widespread in bacteria and archaea whose functions have hitherto been unclear. Here, we characterize the encapsulin nanocompartment from Myxococcus xanthus, which consists of a shell protein (EncA, 32.5 kDa) and three internal proteins (EncB, 17 kDa; EncC, 13 kDa; EncD, 11 kDa). Using cryo-electron microscopy, we determined that EncA self-assembles into an icosahedral shell 32 nm in diameter (26 nm internal diameter), built from 180 subunits with the fold first observed in bacteriophage HK97 capsid. The internal proteins, of which EncB and EncC have ferritin-like domains, attach to its inner surface. Native nanocompartments have dense iron-rich cores. Functionally, they resemble ferritins, cage-like iron storage proteins, but with a massively greater capacity (~30,000 iron atoms versus ~3,000 in ferritin). Physiological data reveal that few nanocompartments are assembled during vegetative growth, but they increase fivefold upon starvation, protecting cells from oxidative stress through iron sequestration.
147 citations
Cites background from "Assembly in vitro of Rhodococcus jo..."
...…have been suggested to perform different functions, including antibacterial activity in Brevibacterium linens (Valdes-Stauber & Scherer, 1994), proteolytic activity in T. maritima (Hicks et al, 1998), and lignin degradation activity in Rhodococcus jostii RHA1 (Rahmanpour & Bugg, 2013)....
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...However, other encapsulin nanocompartments have been suggested to perform different functions, including antibacterial activity in Brevibacterium linens (Valdes-Stauber & Scherer, 1994), proteolytic activity in T. maritima (Hicks et al, 1998), and lignin degradation activity in Rhodococcus jostii RHA1 (Rahmanpour & Bugg, 2013)....
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TL;DR: Treatment of powdered lignocellulose with Dyp1B in the presence of Mn(II) and hydrogen peroxide leads to the release of a low molecular weight lignin fragment, which has been identified by mass spectrometry as a β-aryl ether lign in dimer containing one G unit and one H unit bearing a benzylic ketone.
123 citations
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TL;DR: In this article, a number of surfactants were screened for their ability to improve enzymatic hydrolysis of steam-pretreated spruce (SPS), and non-ionic surfactant was found to be the most effective.
875 citations
TL;DR: Overall, RHA1 appears to have evolved to simultaneously catabolize a diverse range of plant-derived compounds in an O2-rich environment and is established as an important model for studying actinomycete physiology.
Abstract: Rhodococcus sp. RHA1 (RHA1) is a potent polychlorinated biphenyl-degrading soil actinomycete that catabolizes a wide range of compounds and represents a genus of considerable industrial interest. RHA1 has one of the largest bacterial genomes sequenced to date, comprising 9,702,737 bp (67% G+C) arranged in a linear chromosome and three linear plasmids. A targeted insertion methodology was developed to determine the telomeric sequences. RHA1's 9,145 predicted protein-encoding genes are exceptionally rich in oxygenases (203) and ligases (192). Many of the oxygenases occur in the numerous pathways predicted to degrade aromatic compounds (30) or steroids (4). RHA1 also contains 24 nonribosomal peptide synthase genes, six of which exceed 25 kbp, and seven polyketide synthase genes, providing evidence that rhodococci harbor an extensive secondary metabolism. Among sequenced genomes, RHA1 is most similar to those of nocardial and mycobacterial strains. The genome contains few recent gene duplications. Moreover, three different analyses indicate that RHA1 has acquired fewer genes by recent horizontal transfer than most bacteria characterized to date and far fewer than Burkholderia xenovorans LB400, whose genome size and catabolic versatility rival those of RHA1. RHA1 and LB400 thus appear to demonstrate that ecologically similar bacteria can evolve large genomes by different means. Overall, RHA1 appears to have evolved to simultaneously catabolize a diverse range of plant-derived compounds in an O(2)-rich environment. In addition to establishing RHA1 as an important model for studying actinomycete physiology, this study provides critical insights that facilitate the exploitation of these industrially important microorganisms.
625 citations
TL;DR: The results indicate that the cellulose binding domain has a significant role in the unspecific binding of cellulases to lignin.
508 citations
"Assembly in vitro of Rhodococcus jo..." refers background in this paper
...The non-specific binding of cellulase enzymes to the hydrophobic surface of lignin is thought to slow down the rate of lignocellulose breakdown by cellulases [12], which can be alleviated by addition of non-ionic detergents that preferentially bind to lignin [13]....
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TL;DR: Two proteins of previously unknown function assemble to form pentameric structures whose size and shape are compatible with formation of vertices in an icosahedral shell, and combining these pentamers with the hexamers previously elucidated gives two plausible, preliminary atomic models for the carboxysome shell.
Abstract: The carboxysome is a bacterial microcompartment that functions as a simple organelle by sequestering enzymes involved in carbon fixation. The carboxysome shell is roughly 800 to 1400 angstroms in diameter and is assembled from several thousand protein subunits. Previous studies have revealed the three-dimensional structures of hexameric carboxysome shell proteins, which self-assemble into molecular layers that most likely constitute the facets of the polyhedral shell. Here, we report the three-dimensional structures of two proteins of previously unknown function, CcmL and OrfA (or CsoS4A), from the two known classes of carboxysomes, at resolutions of 2.4 and 2.15 angstroms. Both proteins assemble to form pentameric structures whose size and shape are compatible with formation of vertices in an icosahedral shell. Combining these pentamers with the hexamers previously elucidated gives two plausible, preliminary atomic models for the carboxysome shell.
362 citations
TL;DR: Results indicate that DypB has a significant role in lignin degradation in R. jostii RHA1, is able to oxidize both polymericLignin and a lignIn model compound, and appears to have both Mn(II) and lign in oxidation sites, which is the first detailed characterization of a recombinant bacterial lignine peroxidase.
Abstract: Rhodococcus jostii RHA1, a polychlorinated biphenyl-degrading soil bacterium whose genome has been sequenced, shows lignin degrading activity in two recently developed spectrophotometric assays. Bioinformatic analysis reveals two unannotated peroxidase genes present in the genome of R. jostii RHA1 with sequence similarity to open reading frames in other lignin-degrading microbes. They are members of the Dyp peroxidase family and were annotated as DypA and DypB, on the basis of bioinformatic analysis. Assay of gene deletion mutants using a colorimetric lignin degradation assay reveals that a ΔdypB mutant shows greatly reduced lignin degradation activity, consistent with a role in lignin breakdown. Recombinant DypB protein shows activity in the colorimetric assay and shows Michaelis–Menten kinetic behavior using Kraft lignin as a substrate. DypB is activated by Mn2+ by 5–23-fold using a range of assay substrates, and breakdown of wheat straw lignocellulose by recombinant DypB is observed over 24–48 h in the...
352 citations