Glycosylation Is Vital for Industrial Performance of Hyperactive Cellulases

In the terrestrial biosphere, biomass deconstruction is conducted by microbes employing a variety of complementary strategies, many of which remain to be discovered. Moreover, the biofuels industry seeks more efficient (and less costly) cellulase formulations upon which to launch the nascent sustainable bioenergy economy. The glycan decoration of fungal cellulases has been shown to protect these enzymes from protease action and to enhance binding to cellulose. We show here that thermal tolerant bacterial cellulases are glycosylated as well, although the types and extents of decoration differ from their Eukaryotic counterparts. Our major findings are that glycosylation of CelA is uniform across its three linker peptides and composed of mainly galactose disaccharides (which is unique) and that this glycosylation dramatically impacts the hydrolysis of insoluble substrates, proteolytic and thermal stability, and substrate binding and changes the dynamics of the enzyme. This study suggests that the glycosylation of CelA is crucial for its exceptionally high cellulolytic activity on biomass and provides the robustness needed for this enzyme to function in harsh environments including industrial settings

Performance of ab initio and density functional methods for conformational equilibria of CnH2n+2 alkane isomers (n=2-8)

Conformational energies of n-butane, n-pentane, and n-hexane have been calculated at the CCSD(T) level and at or near the basis set limit. Post-CCSD(T) contribution were considered and found to be unimportant. The data thus obtained were used to assess the performance of a variety of density functional methods. Double-hybrid functionals like B2GP-PLYP and B2K-PLYP, especially with a small Grimme-type empirical dispersion correction, are capable of rendering conformational energies of CCSD(T) quality. These were then used as a `secondary standard' for a larger sample of alkanes, including isopentane and the branched hexanes as well as key isomers of heptane and octane. Popular DFT functionals like B3LYP, B3PW91, BLYP, PBE, and PBE0 tend to overestimate conformer energies without dispersion correction, while the M06 family severely underestimates GG interaction energies. Grimme-type dispersion corrections for these overcorrect and lead to qualitatively wrong conformer orderings. All of these functionals also exhibit deficiencies in the conformer geometries, particularly the backbone torsion angles. The PW6B95 and, to a lesser extent, BMK functionals are relatively free of these deficiencies. Performance of these methods is further investigated to derive conformer ensemble corrections to the enthalpy function, $H_{298}-H_0$, and the Gibbs energy function, ${\rm gef}(T)\equiv - [G(T)-H_0]/T$, of these alkanes. While $H_{298}-H_0$ is only moderately sensitive to the level of theory, ${\rm gef}(T)$ exhibits more pronounced sensitivity. Once again, double hybrids acquit themselves very well.Comment: J. Phys. Chem. A, revised [Walter Thiel festschrift

Glycosylation Is Vital for Industrial Performance of Hyperactive Cellulases

In the terrestrial biosphere, biomass deconstruction is conducted by microbes employing a variety of complementary strategies, many of which remain to be discovered. Moreover, the biofuels industry seeks more efficient (and less costly) cellulase formulations upon which to launch the nascent sustainable bioenergy economy. The glycan decoration of fungal cellulases has been shown to protect these enzymes from protease action and to enhance binding to cellulose. We show here that thermal tolerant bacterial cellulases are glycosylated as well, although the types and extents of decoration differ from their Eukaryotic counterparts. Our major findings are that glycosylation of CelA is uniform across its three linker peptides and composed of mainly galactose disaccharides (which is unique) and that this glycosylation dramatically impacts the hydrolysis of insoluble substrates, proteolytic and thermal stability, and substrate binding and changes the dynamics of the enzyme. This study suggests that the glycosylation of CelA is crucial for its exceptionally high cellulolytic activity on biomass and provides the robustness needed for this enzyme to function in harsh environments including industrial settings

Benchmark thermochemistry of the C_nH_{2n+2} alkane isomers (n=2--8) and performance of DFT and composite ab initio methods for dispersion-driven isomeric equilibria

The thermochemistry of linear and branched alkanes with up to eight carbons has been reexamined by means of W4, W3.2lite and W1h theories. `Quasi-W4' atomization energies have been obtained via isodesmic and hypohomodesmotic reactions. Our best atomization energies at 0 K (in kcal/mol) are: 1220.04 n-butane, 1497.01 n-pentane, 1774.15 n-hexane, 2051.17 n-heptane, 2328.30 n-octane, 1221.73 isobutane, 1498.27 isopentane, 1501.01 neopentane, 1775.22 isohexane, 1774.61 3-methylpentane, 1775.67 diisopropyl, 1777.27 neohexane, 2052.43 isoheptane, 2054.41 neoheptane, 2330.67 isooctane, and 2330.81 hexamethylethane. Our best estimates for $\Delta H^\circ_{f,298K}$ are: -30.00 n-butane, -34.84 n-pentane, -39.84 n-hexane, -44.74 n-heptane, -49.71 n-octane, -32.01 isobutane, -36.49 isopentane, -39.69 neopentane, -41.42 isohexane, -40.72 3-methylpentane, -42.08 diisopropyl, -43.77 neohexane, -46.43 isoheptane, -48.84 neoheptane, -53.29 isooctane, and -53.68 hexamethylethane. These are in excellent agreement (typically better than 1 kJ/mol) with the experimental heats of formation at 298 K obtained from the CCCBDB and/or NIST Chemistry WebBook databases. However, at 0 K a large discrepancy between theory and experiment (1.1 kcal/mol) is observed for only neopentane. This deviation is mainly due to the erroneous heat content function for neopentane used in calculating the 0 K CCCBDB value. The thermochemistry of these systems, especially of the larger alkanes, is an extremely difficult test for density functional methods. A posteriori corrections for dispersion are essential. Particularly for the atomization energies, the B2GP-PLYP and B2K-PLYP double-hybrids, and the PW6B95 hybrid-meta GGA clearly outperform other DFT functionals.Comment: (J. Phys. Chem. A, in press

Comparing Residue Clusters from Thermophilic and Mesophilic Enzymes Reveals Adaptive Mechanisms.

Understanding how proteins adapt to function at high temperatures is important for deciphering the energetics that dictate protein stability and folding. While multiple principles important for thermostability have been identified, we lack a unified understanding of how internal protein structural and chemical environment determine qualitative or quantitative impact of evolutionary mutations. In this work we compare equivalent clusters of spatially neighboring residues between paired thermophilic and mesophilic homologues to evaluate adaptations under the selective pressure of high temperature. We find the residue clusters in thermophilic enzymes generally display improved atomic packing compared to mesophilic enzymes, in agreement with previous research. Unlike residue clusters from mesophilic enzymes, however, thermophilic residue clusters do not have significant cavities. In addition, anchor residues found in many clusters are highly conserved with respect to atomic packing between both thermophilic and mesophilic enzymes. Thus the improvements in atomic packing observed in thermophilic homologues are not derived from these anchor residues but from neighboring positions, which may serve to expand optimized protein core regions

Anchor residues are conserved in atomic packing.

<p>(A) The thermostable GH9 (PDB 4dod) is shown in surface representation, with anchor residues that are seen in a larger number of clusters shown in stick representation. Residues exhibiting the largest ΔSASA_1.4, which are never anchor residues, are colored red. (B) Sequence positions from 4dod are binned by the number of clusters in which they are found. The heat scale indicates ΔSASA_1.4. Importantly, blue is not observed as there are no mesophilic clusters with significantly better atomic packing relative to the matched thermophilic cluster. (C) Sequence positions from the representative set of structures are binned by the number of motifs in which they are found (x-axis), with ΔSASA_1.4 shown for each paired position (y-axis). A white symbol indicates sequence conservation, and gray indicates the sequence differs at that position.</p

Thermophilic enzyme clusters display closer atomic packing compared to mesophilic enzyme clusters for most enzyme pairs evaluated.

<p>(A) SASA_1.4 values for clusters from the representative thermophilic-mesophilic structure pairs are shown, with thermophilic clusters shown in red, mesophilic clusters in green and the difference, ΔSASA_1.4, in blue. Values are sorted by ΔSASA_1.4. (B) SASA_1.4 values are shown comparing clusters from the thermophilic (PDB 1a5z) and mesophilic (PDB 6ldh) lactate dehydrogenase enzymes, which have a difference in optimum activity temperature of 30°C. (C) the thermophilic (PDB 1a5z) and mesophilic (PDB 5ldh) lactate dehydrogenase enzymes, with a difference in optimum activity temperature of 48°C, (D) and the thermophilic (PDB 1a5z) and psychrophilic (PDB 1ldh) lactate dehydrogenase enzymes, with a difference in optimum activity temperature of 70°C.</p

Identifying equivalent clusters in homologous proteins allows for direct comparison of local environments.

<p>(A) A cartoon depiction of cluster of adjacent residues is shown (red circle). (B) Structural alignment of paired enzymes is shown, with PDB 1vbr in orange and 2uwf in gray. The structurally aligned residues for the paired enzymes are shown beneath. (C) Differences in atomic packing is depicted with alternate sequences shown in stick and sphere representation on PDB 2wva.</p

The backbone can move significantly in the structurally equivalent clusters.

<p>(A) Three Cα atoms from a paired cluster are shown in red spheres (thermophilic enzyme) and purple spheres (mesophilic enzyme). The atoms are labeled a, b and c for the thermophilic enzyme and a’, b’ and c’ for the mesophilic enzyme. The Euclidian distances between Cα atoms are shown for each enzyme, with the distance differences at right. (B) The sum of the absolute values for the distance differences (red), and the average distance differences (blue) for each representative cluster are shown, sorted by summed or averaged distances.</p

Evaluating the potential for epistasis.

<p>(A) The number of residues in each motif are determined for all representative thermophilic-mesophilic structure pairs and binned according to the motif size. (B) The number of residue substitutions, given as Hamming distance, in each equivalent thermophilic-mesophilic motif is determined and binned.</p