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Loosenin, a novel protein with cellulose-disrupting activity from Bjerkandera adusta.

Quiroz-Castañeda RE, Martínez-Anaya C, Cuervo-Soto LI, Segovia L, Folch-Mallol JL - Microb. Cell Fact. (2011)

Bottom Line: LOOS1 is distantly related to plant expansins through the shared presence of a DPBB domain, however domain II found in plant expansins is absent.LOOS1 is a new type of protein with disrupting activity on cellulose.LOOS1 binds polysaccharides, and given its enhancing properties on the action of hydrolytic enzymes, LOOS1 represents a potential additive in the production of fermentable sugars from lignocellulose.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratorio de Biología Molecular de Hongos, Centro de Investigación en Biotecnología, Universidad Autónoma del Estado de Morelos, Avenida Universidad 1001 Col, Chamilpa, Cuernavaca 62209, Morelos, Mexico.

ABSTRACT

Background: Expansins and expansin-like proteins loosen cellulose microfibrils, possibly through the rupture of intramolecular hydrogen bonds. Together with the use of lignocellulolytic enzymes, these proteins are potential molecular tools to treat plant biomass to improve saccharification yields.

Results: Here we describe a new type of expansin-related fungal protein that we have called loosenin. Its corresponding gene, loos1, from the basidiomycete Bjerkandera adusta, was cloned and heterologously expressed in Saccharomyces cerevisiae. LOOS1 is distantly related to plant expansins through the shared presence of a DPBB domain, however domain II found in plant expansins is absent. LOOS1 binds tightly to cellulose and chitin, and we demonstrate that cotton fibers become susceptible to the action of a commercial cellulase following treatment with LOOS1. Natural fibers of Agave tequilana also become susceptible to hydrolysis by cellulases after loosenin treatment.

Conclusions: LOOS1 is a new type of protein with disrupting activity on cellulose. LOOS1 binds polysaccharides, and given its enhancing properties on the action of hydrolytic enzymes, LOOS1 represents a potential additive in the production of fermentable sugars from lignocellulose.

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Related in: MedlinePlus

Structural superposition of the Loosenin model (green) with 2hcz, an EXPB (dark grey). The residues identified by Kerff et al as belonging to the polysaccharide binding groove in BsEXLX are indicated as sticks. Strictly conserved residues in Kerff's analysis are indicated in cyan and conserved residues in yellow. The structural alignment and figure were produced with Pymol.
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Figure 2: Structural superposition of the Loosenin model (green) with 2hcz, an EXPB (dark grey). The residues identified by Kerff et al as belonging to the polysaccharide binding groove in BsEXLX are indicated as sticks. Strictly conserved residues in Kerff's analysis are indicated in cyan and conserved residues in yellow. The structural alignment and figure were produced with Pymol.

Mentions: The loosenin amino acid sequence was used for fold recognition using the PHYRE web server version 0.1. The top three results were EXLX1 from Bacillus subtilis [PDB:2BH0], the homologue pollen allergen PHL P1 N-terminal domain from Phleum pratense [PDB:1N10] and an EXPB and group-1 allergen from maize [PDB:2HCZ]. All had an estimated precision of 100% indicating a successful fold assignment to the DPBB fold family. Primary sequences identities were 19, 19 and 20% respectively confirming that loosenin is indeed a remote homologue of the expansins. We used the alignments provided by PHYRE to construct five models of the complete loosenin amino acid sequence with 2BH0, 1N10 and 2HCZ as templates using maximum MD-refinement. All five models were essentially identical with an average RMSD of 0.4 Å. Structures 2BH0 and 1N10 had an RMSD (of the equivalent superimposed alpha-carbons) of around 1.4 Å to the models when 2HCZ had an RMSD of 0.5 Å, although amino acid identity to loosenin was very marginally greater (Figure 2). This measure would reflect the similarity of the proteins cores. Unlike typical expansins, loosenin is composed of a single domain, albeit one highly similar to domain I of expansins, as evidenced by fold recognition. Kerff et al [27] performed a sequence analysis of EXLX1 (structure 2BH0) together with other polysaccharide recognizing proteins through which they identified several conserved residues. Similarly, we superimposed the loosenin model with the above-mentioned structures and identified the equivalent residues. In loosenin, T31 and D105 (highlighted in cyan in Additional File 1, Figure S2) correspond to the two strictly conserved residues, equivalent to T12 and D82 in EXLX1, and known to form a conserved hydrogen bond between the OH group of Thr and the carboxylic group of Asp. Other residues that show perfect sequence conservation between loosenin and the other three structures are G38, A39, G75, T92, and D93. Four more positions identified as important by Kerff et al correspond to loosenin Y33 (also conserved in 2HCZ and 1N10, however it is substituted by a T in 2BH0); A52 (conserved in 2BH0, but found as C in the other two structures); D103 (is substituted by A in 2BH0, or H in 1N10 and 2HCZ); and finally F109 (found conserved in the rest of the structures as an L). Except for G75, all these residues were identified by Kerff et al. as part of the groove which is thought to serve as the polysaccharide binding site by means of hydrogen bonding. The model thus suggests that loosenin is also a polysaccharide binding protein.


Loosenin, a novel protein with cellulose-disrupting activity from Bjerkandera adusta.

Quiroz-Castañeda RE, Martínez-Anaya C, Cuervo-Soto LI, Segovia L, Folch-Mallol JL - Microb. Cell Fact. (2011)

Structural superposition of the Loosenin model (green) with 2hcz, an EXPB (dark grey). The residues identified by Kerff et al as belonging to the polysaccharide binding groove in BsEXLX are indicated as sticks. Strictly conserved residues in Kerff's analysis are indicated in cyan and conserved residues in yellow. The structural alignment and figure were produced with Pymol.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3050684&req=5

Figure 2: Structural superposition of the Loosenin model (green) with 2hcz, an EXPB (dark grey). The residues identified by Kerff et al as belonging to the polysaccharide binding groove in BsEXLX are indicated as sticks. Strictly conserved residues in Kerff's analysis are indicated in cyan and conserved residues in yellow. The structural alignment and figure were produced with Pymol.
Mentions: The loosenin amino acid sequence was used for fold recognition using the PHYRE web server version 0.1. The top three results were EXLX1 from Bacillus subtilis [PDB:2BH0], the homologue pollen allergen PHL P1 N-terminal domain from Phleum pratense [PDB:1N10] and an EXPB and group-1 allergen from maize [PDB:2HCZ]. All had an estimated precision of 100% indicating a successful fold assignment to the DPBB fold family. Primary sequences identities were 19, 19 and 20% respectively confirming that loosenin is indeed a remote homologue of the expansins. We used the alignments provided by PHYRE to construct five models of the complete loosenin amino acid sequence with 2BH0, 1N10 and 2HCZ as templates using maximum MD-refinement. All five models were essentially identical with an average RMSD of 0.4 Å. Structures 2BH0 and 1N10 had an RMSD (of the equivalent superimposed alpha-carbons) of around 1.4 Å to the models when 2HCZ had an RMSD of 0.5 Å, although amino acid identity to loosenin was very marginally greater (Figure 2). This measure would reflect the similarity of the proteins cores. Unlike typical expansins, loosenin is composed of a single domain, albeit one highly similar to domain I of expansins, as evidenced by fold recognition. Kerff et al [27] performed a sequence analysis of EXLX1 (structure 2BH0) together with other polysaccharide recognizing proteins through which they identified several conserved residues. Similarly, we superimposed the loosenin model with the above-mentioned structures and identified the equivalent residues. In loosenin, T31 and D105 (highlighted in cyan in Additional File 1, Figure S2) correspond to the two strictly conserved residues, equivalent to T12 and D82 in EXLX1, and known to form a conserved hydrogen bond between the OH group of Thr and the carboxylic group of Asp. Other residues that show perfect sequence conservation between loosenin and the other three structures are G38, A39, G75, T92, and D93. Four more positions identified as important by Kerff et al correspond to loosenin Y33 (also conserved in 2HCZ and 1N10, however it is substituted by a T in 2BH0); A52 (conserved in 2BH0, but found as C in the other two structures); D103 (is substituted by A in 2BH0, or H in 1N10 and 2HCZ); and finally F109 (found conserved in the rest of the structures as an L). Except for G75, all these residues were identified by Kerff et al. as part of the groove which is thought to serve as the polysaccharide binding site by means of hydrogen bonding. The model thus suggests that loosenin is also a polysaccharide binding protein.

Bottom Line: LOOS1 is distantly related to plant expansins through the shared presence of a DPBB domain, however domain II found in plant expansins is absent.LOOS1 is a new type of protein with disrupting activity on cellulose.LOOS1 binds polysaccharides, and given its enhancing properties on the action of hydrolytic enzymes, LOOS1 represents a potential additive in the production of fermentable sugars from lignocellulose.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratorio de Biología Molecular de Hongos, Centro de Investigación en Biotecnología, Universidad Autónoma del Estado de Morelos, Avenida Universidad 1001 Col, Chamilpa, Cuernavaca 62209, Morelos, Mexico.

ABSTRACT

Background: Expansins and expansin-like proteins loosen cellulose microfibrils, possibly through the rupture of intramolecular hydrogen bonds. Together with the use of lignocellulolytic enzymes, these proteins are potential molecular tools to treat plant biomass to improve saccharification yields.

Results: Here we describe a new type of expansin-related fungal protein that we have called loosenin. Its corresponding gene, loos1, from the basidiomycete Bjerkandera adusta, was cloned and heterologously expressed in Saccharomyces cerevisiae. LOOS1 is distantly related to plant expansins through the shared presence of a DPBB domain, however domain II found in plant expansins is absent. LOOS1 binds tightly to cellulose and chitin, and we demonstrate that cotton fibers become susceptible to the action of a commercial cellulase following treatment with LOOS1. Natural fibers of Agave tequilana also become susceptible to hydrolysis by cellulases after loosenin treatment.

Conclusions: LOOS1 is a new type of protein with disrupting activity on cellulose. LOOS1 binds polysaccharides, and given its enhancing properties on the action of hydrolytic enzymes, LOOS1 represents a potential additive in the production of fermentable sugars from lignocellulose.

Show MeSH
Related in: MedlinePlus