AA16 Oxidoreductases Boost Cellulose-Active AA9 Lytic Polysaccharide Monooxygenases from Myceliophthora thermophila.

Copper-dependent lytic polysaccharide monooxygenases (LPMOs) classified in Auxiliary Activity (AA) families are considered indispensable as synergistic partners for cellulolytic enzymes to saccharify recalcitrant lignocellulosic plant biomass. In this study, we characterized two fungal oxidoreductases from the new AA16 family. We found that MtAA16A from Myceliophthora thermophila and AnAA16A from Aspergillus nidulans did not catalyze the oxidative cleavage of oligo- and polysaccharides. Indeed, the MtAA16A crystal structure showed a fairly LPMO-typical histidine brace active site, but the cellulose-acting LPMO-typical flat aromatic surface parallel to the histidine brace region was lacking. Further, we showed that both AA16 proteins are able to oxidize low-molecular-weight reductants to produce H2O2. The oxidase activity of the AA16s substantially boosted cellulose degradation by four AA9 LPMOs from M. thermophila (MtLPMO9s) but not by three AA9 LPMOs from Neurospora crassa (NcLPMO9s). The interplay with MtLPMO9s is explained by the H2O2-producing capability of the AA16s, which, in the presence of cellulose, allows the MtLPMO9s to optimally drive their peroxygenase activity. Replacement of MtAA16A by glucose oxidase (AnGOX) with the same H2O2-producing activity could only achieve less than 50% of the boosting effect achieved by MtAA16A, and earlier MtLPMO9B inactivation (6 h) was observed. To explain these results, we hypothesized that the delivery of AA16-produced H2O2 to the MtLPMO9s is facilitated by protein-protein interaction. Our findings provide new insights into the functions of copper-dependent enzymes and contribute to a further understanding of the interplay of oxidative enzymes within fungal systems to degrade lignocellulose.

Unlocking the structural features for the xylobiohydrolase activity of an unusual GH11 member identified in a compost-derived consortium.

The heteropolysaccharide xylan is a valuable source of sustainable chemicals and materials from renewable biomass sources. A complete hydrolysis of this major hemicellulose component requires a diverse set of enzymes including endo-ß-1,4-xylanases, ß-xylosidases, acetylxylan esterases, α-l-arabinofuranosidases, and α-glucuronidases. Notably, the most studied xylanases from glycoside hydrolase family 11 (GH11) have exclusively been endo-ß-1,4- and ß-1,3-xylanases. However, a recent analysis of a metatranscriptome library from a microbial lignocellulose community revealed GH11 enzymes capable of releasing solely xylobiose from xylan. Although initial biochemical studies clearly indicated their xylobiohydrolase mode of action, the structural features that drive this new activity still remained unclear. It was also not clear whether the enzymes acted on the reducing or nonreducing end of the substrate. Here, we solved the crystal structure of MetXyn11 in the apo and xylobiose-bound forms. The structure of MetXyn11 revealed the molecular features that explain the observed pattern on xylooligosaccharides released by this nonreducing end xylobiohydrolase.

Impact of cellulose properties on enzymatic degradation by bacterial GH48 enzymes: Structural and mechanistic insights from processive Bacillus licheniformis Cel48B cellulase.

Processive cellulases are highly efficient molecular engines involved in the cellulose breakdown process. However, the mechanism that processive bacterial enzymes utilize to recruit and retain cellulose strands in the catalytic site remains poorly understood. Here, integrated enzymatic assays, protein crystallography and computational approaches were combined to study the enzymatic properties of the processive BlCel48B cellulase from Bacillus licheniformis. Hydrolytic efficiency, substrate binding affinity, cleavage patterns, and the apparent processivity of bacterial BlCel48B are significantly impacted by the cellulose size and its surface morphology. BlCel48B crystallographic structure was solved with ligands spanning -5 to -2 and +1 to +2 subsites. Statistical coupling analysis and molecular dynamics show that co-evolved residues on active site are critical for stabilizing ligands in the catalytic tunnel. Our results provide mechanistic insights into BlCel48B molecular-level determinants of activity, substrate binding, and processivity on insoluble cellulose, thus shedding light on structure-activity correlations of GH48 family members in general.

Enzyme-Functionalized Cellulose Beads as a Promising Antimicrobial Material.

The extensive use of antibiotics over the last decades is responsible for the emergence of multidrug-resistant (MDR) microorganisms that are challenging health care systems worldwide. The use of alternative antimicrobial materials could mitigate the selection of new MDR strains by reducing antibiotic overuse. This paper describes the design of enzyme-based antimicrobial cellulose beads containing a covalently coupled glucose oxidase from Aspergillus niger (GOx) able to release antimicrobial concentrations of hydrogen peroxide (H2O2) (≈ 1.8 mM). The material preparation was optimized to obtain the best performance in terms of mechanical resistance, shelf life, and H2O2 production. As a proof of concept, agar inhibition halo assays (Kirby-Bauer test) against model pathogens were performed. The two most relevant factors affecting the bead functionalization process were the degree of oxidation and the pH used for the enzyme binding process. Slightly acidic conditions during the functionalization process (pH 6) showed the best results for the GOx/cellulose system. The functionalized beads inhibited the growth of all the microorganisms assayed, confirming the release of sufficient antimicrobial levels of H2O2. The maximum inhibition efficiency was exhibited toward Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli), although significant inhibitory effects toward methicillin-resistant Staphylococcus aureus (MRSA) and S. aureus were also observed. These enzyme-functionalized cellulose beads represent an inexpensive, sustainable, and biocompatible antimicrobial material with potential use in many applications, including the manufacturing of biomedical products and additives for food preservation.

Multienzyme Cellulose Films as Sustainable and Self-Degradable Hydrogen Peroxide-Producing Material.

The use of hydrogen peroxide-releasing enzymes as a component to produce alternative and sustainable antimicrobial materials has aroused interest in the scientific community. However, the preparation of such materials requires an effective enzyme binding method that often involves the use of expensive and toxic chemicals. Here, we describe the development of an enzyme-based hydrogen peroxide-producing regenerated cellulose film (RCF) in which a cellobiohydrolase (TrCBHI) and a cellobiose dehydrogenase (MtCDHA) were efficiently adsorbed, 90.38 ± 2.2 and 82.40 ± 5.7%, respectively, without making use of cross-linkers. The enzyme adsorption kinetics and binding isotherm experiments showed high affinity of the proteins possessing cellulose-binding modules for RCF, suggesting that binding on regenerated cellulose via specific interactions can be an alternative method for enzyme immobilization. Resistance to compression and porosity at a micrometer scale were found to be tunable by changing cellulose concentration prior to film regeneration. The self-degradation process, triggered by stacking TrCBHI and MtCDHA (previously immobilized onto separate RCF), produced 0.15 nmol/min·cm2 of H2O2. Moreover, the production of H2O2 was sustained for at least 24 h reaching a concentration of ∼2 mM. The activity of MtCDHA immobilized on RCF was not affected by reuse for at least 3 days (1 cycle/day), suggesting that no significant enzyme leakage occurred in that timeframe. In the material herein designed, cellulose (regenerated from a 1-ethyl-3-methylimidazolium acetate/dimethyl sulfoxide (DMSO) solution) serves both as support and substrate for the immobilized enzymes. The sequential reaction led to the production of H2O2 at a micromolar-millimolar level revealing the potential use of the material as a self-degradable antimicrobial agent.

Structure and dynamics of Trichoderma harzianum Cel7B suggest molecular architecture adaptations required for a wide spectrum of activities on plant cell wall polysaccharides.

Cellulases from glycoside hydrolase family 7 (GH7) play crucial roles in plant lignocellulose deconstruction by fungi, but structural information available for GH7 fungal endoglucanases is limited when compared to the number of known sequences in the family. Here, we report the X-ray structure of the glycosylated catalytic domain (CD) of Trichoderma harzianum endoglucanase, ThCel7B, solved and refined at 2.9 Šresolution. Additionally, our extensive molecular dynamics simulations of this enzyme in complex with a variety of oligosaccharides provide a better understanding of its promiscuous hydrolytic activities on plant cell wall polysaccharides. The simulations demonstrate the importance of the hydrogen bond between substrate O2 hydroxyl in the subsite -1 and a side chain of catalytic Glu196 which renders ThCel7B capable to catalytically cleave cello and xylooligosaccharides, but not mannooligosaccharides. Moreover, detailed structural analyses and MD simulations revealed an additional binding pocket, suitable for accommodation of oligosaccharide decorations and/or substrates with mixed glycoside bonds that abuts onto the binding cleft close to subsite +2.

Functional characterization of a lytic polysaccharide monooxygenase from the thermophilic fungus Myceliophthora thermophila.

Thermophilic fungi are a promising source of thermostable enzymes able to hydrolytically or oxidatively degrade plant cell wall components. Among these enzymes are lytic polysaccharide monooxygenases (LPMOs), enzymes capable of enhancing biomass hydrolysis through an oxidative mechanism. Myceliophthora thermophila (synonym Sporotrichum thermophile), an Ascomycete fungus, expresses and secretes over a dozen different LPMOs. In this study, we report the overexpression and biochemical study of a previously uncharacterized LPMO (MtLPMO9J) from M. thermophila M77 in Aspergillus nidulans. MtLPMO9J is a single-domain LPMO and has 63% sequence similarity with the catalytic domain of NcLPMO9C from Neurospora crassa. Biochemical characterization of MtLPMO9J revealed that it performs C4-oxidation and is active against cellulose, soluble cello-oligosaccharides and xyloglucan. Moreover, biophysical studies showed that MtLPMO9J is structurally stable at pH above 5 and at temperatures up to 50°C. Importantly, LC-MS analysis of the peptides after tryptic digestion of the recombinantly produced protein revealed not only the correct processing of the signal peptide and methylation of the N-terminal histidine, but also partial autoxidation of the catalytic center. This shows that redox conditions need to be controlled, not only during LPMO reactions but also during protein production, to protect LPMOs from oxidative damage.

Biochemical and structural insights into a thermostable cellobiohydrolase from Myceliophthora thermophila.

Cellobiohydrolases hydrolyze cellulose, a linear polymer with glucose monomers linked exclusively by ß-1,4 glycosidic linkages. The widespread hydrogen bonding network tethers individual cellulose polymers forming crystalline cellulose, which prevent the access of hydrolytic enzymes and water molecules. The most abundant enzyme secreted by Myceliophthora thermophila M77 in response to the presence of biomass is the cellobiohydrolase MtCel7A, which is composed by a GH7-catalytic domain (CD), a linker, and a CBM1-type carbohydrate-binding module. GH7 cellobiohydrolases have been studied before, and structural models have been proposed. However, currently available GH7 crystal structures only define separate catalytic domains and/or cellulose-binding modules and do not include the full-length structures that are involved in shaping the catalytic mode of operation. In this study, we determined the 3D structure of catalytic domain using X-ray crystallography and retrieved the full-length enzyme envelope via small-angle X-ray scattering (SAXS) technique. The SAXS data reveal a tadpole-like molecular shape with a rigid linker connecting the CD and CBM. Our biochemical studies show that MtCel7A has higher catalytic efficiency and thermostability as well as lower processivity when compared to the well-studied TrCel7A from Trichoderma reesei. Based on a comparison of the crystallographic structures of CDs and their molecular dynamic simulations, we demonstrate that MtCel7A has considerably higher flexibility than TrCel7A. In particular, loops that cover the active site are more flexible and undergo higher conformational fluctuations, which might account for decreased processivity and enhanced enzymatic efficiency. Our statistical coupling analysis suggests co-evolution of amino acid clusters comprising the catalytic site of MtCel7A, which correlate with the steps in the catalytic cycle of the enzyme. DATABASE: The atomic coordinates and structural factors of MtCel7A have been deposited in the Protein Data Bank with accession number 5W11.

Identification of proteins associated with polyhydroxybutyrate granules from Herbaspirillum seropedicae SmR1--old partners, new players.

Herbaspirillum seropedicae is a diazotrophic ß-Proteobacterium found associated with important agricultural crops. This bacterium produces polyhydroxybutyrate (PHB), an aliphatic polyester, as a carbon storage and/or source of reducing equivalents. The PHB polymer is stored as intracellular insoluble granules coated mainly with proteins, some of which are directly involved in PHB synthesis, degradation and granule biogenesis. In this work, we have extracted the PHB granules from H. seropedicae and identified their associated-proteins by mass spectrometry. This analysis allowed us to identify the main phasin (PhaP1) coating the PHB granule as well as the PHB synthase (PhbC1) responsible for its synthesis. A phbC1 mutant is impaired in PHB synthesis, confirming its role in H. seropedicae. On the other hand, a phaP1 mutant produces PHB granules but coated mainly with the secondary phasin (PhaP2). Furthermore, some novel proteins not previously described to be involved with PHB metabolism were also identified, bringing new possibilities to PHB function in H. seropedicae.

Identification and characterization of PhbF: a DNA binding protein with regulatory role in the PHB metabolism of Herbaspirillum seropedicae SmR1.

BACKGROUND: Herbaspirillum seropedicae SmR1 is a nitrogen fixing endophyte associated with important agricultural crops. It produces polyhydroxybutyrate (PHB) which is stored intracellularly as granules. However, PHB metabolism and regulatory control is not yet well studied in this organism. RESULTS: In this work we describe the characterization of the PhbF protein from H. seropedicae SmR1 which was purified and characterized after expression in E. coli. The purified PhbF protein was able to bind to eleven putative promoters of genes involved in PHB metabolism in H. seropedicae SmR1. In silico analyses indicated a probable DNA-binding sequence which was shown to be protected in DNA footprinting assays using purified PhbF. Analyses using lacZ fusions showed that PhbF can act as a repressor protein controlling the expression of PHB metabolism-related genes. CONCLUSIONS: Our results indicate that H. seropedicae SmR1 PhbF regulates expression of phb-related genes by acting as a transcriptional repressor. The knowledge of the PHB metabolism of this plant-associated bacterium may contribute to the understanding of the plant-colonizing process and the organism's resistance and survival in planta.

Role of conserved cysteine residues in Herbaspirillum seropedicae NifA activity.

Herbaspirillum seropedicae is an endophytic diazotrophic bacterium that associates with economically important crops. NifA protein, the transcriptional activator of nif genes in H. seropedicae, binds to nif promoters and, together with RNA polymerase-sigma(54) holoenzyme, catalyzes the formation of open complexes to allow transcription initiation. The activity of H. seropedicae NifA is controlled by ammonium and oxygen levels, but the mechanisms of such control are unknown. Oxygen sensitivity is attributed to a conserved motif of cysteine residues in NifA that spans the central AAA+ domain and the interdomain linker that connects the AAA+ domain to the C-terminal DNA binding domain. Here we mutagenized this conserved motif of cysteines and assayed the activity of mutant proteins in vivo. We also purified the mutant variants of NifA and tested their capacity to bind to the nifB promoter region. Chimeric proteins between H. seropedicae NifA, an oxygen-sensitive protein, and Azotobacter vinelandii NifA, an oxygen-tolerant protein, were constructed and showed that the oxygen response is conferred by the central AAA+ and C-terminal DNA binding domains of H. seropedicae NifA. We conclude that the conserved cysteine motif is essential for NifA activity, although single cysteine-to-serine mutants are still competent at binding DNA.