Exo-exo synergy between Cel6A and Cel7A from Hypocrea jecorina: Role of carbohydrate binding module and the endo-lytic character of the enzymes.

Synergy between cellulolytic enzymes is essential in both natural and industrial breakdown of biomass. In addition to synergy between endo- and exo-lytic enzymes, a lesser known but equally conspicuous synergy occurs among exo-acting, processive cellobiohydrolases (CBHs) such as Cel7A and Cel6A from Hypocrea jecorina. We studied this system using microcrystalline cellulose as substrate and found a degree of synergy between 1.3 and 2.2 depending on the experimental conditions. Synergy between enzyme variants without the carbohydrate binding module (CBM) and its linker was strongly reduced compared to the wild types. One plausible interpretation of this is that exo-exo synergy depends on the targeting role of the CBM. Many earlier works have proposed that exo-exo synergy was caused by an auxiliary endo-lytic activity of Cel6A. However, biochemical data from different assays suggested that the endo-lytic activity of both Cel6A and Cel7A were 103 -104 times lower than the common endoglucanase, Cel7B, from the same organism. Moreover, the endo-lytic activity of Cel7A was 2-3-fold higher than for Cel6A, and we suggest that endo-like activity of Cel6A cannot be the main cause for the observed synergy. Rather, we suggest the exo-exo synergy found here depends on different specificities of the enzymes possibly governed by their CBMs. Biotechnol. Bioeng. 2017;114: 1639-1647. © 2017 Wiley Periodicals, Inc.

Oligosaccharide and substrate binding in the starch debranching enzyme barley limit dextrinase.

Complete hydrolytic degradation of starch requires hydrolysis of both the α-1,4- and α-1,6-glucosidic bonds in amylopectin. Limit dextrinase (LD) is the only endogenous barley enzyme capable of hydrolyzing the α-1,6-glucosidic bond during seed germination, and impaired LD activity inevitably reduces the maltose and glucose yields from starch degradation. Crystal structures of barley LD and active-site mutants with natural substrates, products and substrate analogues were sought to better understand the facets of LD-substrate interactions that confine high activity of LD to branched maltooligosaccharides. For the first time, an intact α-1,6-glucosidically linked substrate spanning the active site of a LD or pullulanase has been trapped and characterized by crystallography. The crystal structure reveals both the branch and main-chain binding sites and is used to suggest a mechanism for nucleophilicity enhancement in the active site. The substrate, product and analogue complexes were further used to outline substrate binding subsites and substrate binding restraints and to suggest a mechanism for avoidance of dual α-1,6- and α-1,4-hydrolytic activity likely to be a biological necessity during starch synthesis.

Probing substrate interactions in the active tunnel of a catalytically deficient cellobiohydrolase (Cel7).

Cellobiohydrolases break down cellulose sequentially by sliding along the crystal surface with a single cellulose strand threaded through the catalytic tunnel of the enzyme. This so-called processive mechanism relies on a complex pattern of enzyme-substrate interactions, which need to be addressed in molecular descriptions of processivity and its driving forces. Here, we have used titration calorimetry to study interactions of cellooligosaccharides (COS) and a catalytically deficient variant (E212Q) of the enzyme Cel7A from Trichoderma reesei. This enzyme has ∼10 glucopyranose subsites in the catalytic tunnel, and using COS ligands with a degree of polymerization (DP) from 2 to 8, different regions of the tunnel could be probed. For COS ligands with a DP of 2-3 the binding constants were around 10(5) m(-1), and for longer ligands (DP 5-8) this value was ∼10(7) m(-1). Within each of these groups we did not find increased affinity as the ligands got longer and potentially filled more subsites. On the contrary, we found a small but consistent affinity loss as DP rose from 6 to 8, particularly at the higher investigated temperatures. Other thermodynamic functions (ΔH, ΔS, and ΔCp) decreased monotonously with both temperature and DP. Combined interpretation of these thermodynamic results and previously published structural data allowed assessment of an affinity profile along the length axis of the active tunnel.

Reversibility of substrate adsorption for the cellulases Cel7A, Cel6A, and Cel7B from Hypocrea jecorina.

Adsorption of cellulases on the cellulose surface is an integral part of the catalytic mechanism, and a detailed description of the adsorption process is therefore required for a fundamental understanding of this industrially important class of enzymes. However, the mode of adsorption has proven intricate, and several key questions remain open. Perhaps most notably it is not clear whether the adsorbed enzyme is in dynamic equilibrium with the free population or irreversibly associated with no or slow dissociation. To address this, we have systematically investigated adsorption reversibility for two cellobiohydrolases (Cel7A and Cel6A) and one endoglucanase (Cel7B) on four types of pure cellulose substrates. Specifically, we monitored dilution-induced release of adsorbed enzyme in samples that had previously been brought to a steady state (constant concentration of free enzyme). In simple dilution experiments (without centrifugation), the results consistently showed full reversibility. In contrast to this, resuspension of enzyme-substrate pellets separated by centrifugation showed extensive irreversibility. We conclude that these enzymes are in a dynamic equilibrium between free and adsorbed states but suggest that changes in the physical properties of cellulose caused by compaction of the pellet hampers subsequent release of adsorbed enzyme. This latter effect may be pertinent to both previous controversies in the literature on adsorption reversibility and the development of enzyme recycling protocols in the biomass industry.

In situ stability of substrate-associated cellulases studied by DSC.

This work shows that differential scanning calorimetry (DSC) can be used to monitor the stability of substrate-adsorbed cellulases during long-term hydrolysis of insoluble cellulose. Thermal transitions of adsorbed enzyme were measured regularly in subsets of a progressing hydrolysis, and the size of the transition peak was used as a gauge of the population of native enzyme. Analogous measurements were made for enzymes in pure buffer. Investigations of two cellobiohydrolases, Cel6A and Cel7A, from Trichoderma reesei, which is an anamorph of the fungus Hypocrea jerorina, showed that these enzymes were essentially stable at 25 °C. Thus, over a 53 h experiment, Cel6A lost less than 15% of the native population and Cel7A showed no detectable loss for either the free or substrate-adsorbed state. At higher temperatures we found significant losses in the native populations, and at the highest tested temperature (49 °C) about 80% Cel6A and 35% of Cel7A was lost after 53 h of hydrolysis. The data consistently showed that Cel7A was more long-term stable than Cel6A and that substrate-associated enzyme was less long-term stable than enzyme in pure buffer stored under otherwise equal conditions. There was no correlation between the intrinsic stability, specified by the transition temperature in the DSC, and the long-term stability derived from the peak area. The results are discussed with respect to the role of enzyme denaturation for the ubiquitous slowdown observed in the enzymatic hydrolysis of cellulose.

Crystal structures of the all-cysteinyl-coordinated D14C variant of Pyrococcus furiosus ferredoxin: [4Fe-4S] ↔ [3Fe-4S] cluster conversion.

The structure of the all-cysteinyl-coordinated D14C variant of [4Fe-4S] ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus has been determined to 1.7 Å resolution from a crystal belonging to space group C222(1) with two types of molecules, A and B, in the asymmetric unit. A and B molecules have different crystal packing and intramolecular disulfide bond conformation. The crystal packing reveals a ß-sheet interaction between A molecules in adjacent asymmetric units, whereas B molecules are packed as monomers in a less rigid position next to the A-A extended ß-sheet dimers. The A molecules contain an intramolecular disulfide bond in a double conformation with 60% occupancy left-handed and 40% occupancy right-handed spiral conformation, whereas B molecules have an intramolecular disulfide bond in a right-handed spiral conformation. The cluster in D14C [4Fe-4S] P. furiosus ferredoxin was chemically oxidized at pH 5.8 to [3Fe-4S]. For purification at pH 8.0, two forms of the protein are obtained. Mass spectrometric analysis shows that the two forms are the D14C [3Fe-4S] P. furiosus ferredoxin monomer and a disulfide-bonded dimer of D14C [3Fe-4S] P. furiosus ferredoxin. When oxidization and purification are carried out at pH 5.8, only the monomer is obtained. The crystal structure of D14C [3Fe-4S] P. furiosus ferredoxin monomer was determined to 2.8 Å resolution from a crystal belonging to space group P2(1)2(1)2(1) with two molecules in the asymmetric unit. The molecules resemble molecule A of D14C [4Fe-4S] P. furiosus ferredoxin and electron density clearly shows the presence of a [3Fe-4S] cluster.

Expression, purification and enzymatic characterization of the catalytic domains of human tryptophan hydroxylase isoforms.

Tryptophan hydroxylase exists in two isoforms: Isoform 1 catalyses the first and rate-limiting step in the synthesis of serotonin in the peripheral parts of the body while isoform 2 catalyses this step in the brain. The catalytic domains of human tryptophan hydroxylase 1 and 2 have been expressed, purified and the kinetic properties have been studied and are compared. Substrate inhibition by tryptophan is observed for isoform 1 but not for isoform 2. Large differences are observed in the K (m,tetrahydrobiopterin) values for the two isoforms, being >10 times larger for isoform 1 compared to isoform 2.

Crystal structure of tryptophan hydroxylase with bound amino acid substrate.

Tryptophan hydroxylase (TPH) is a mononuclear non-heme iron enzyme, which catalyzes the reaction between tryptophan, O 2, and tetrahydrobiopterin (BH 4) to produce 5-hydroxytryptophan and 4a-hydroxytetrahydrobiopterin. This is the first and rate-limiting step in the biosynthesis of the neurotransmitter and hormone serotonin (5-hydroxytryptamine). We have determined the 1.9 A resolution crystal structure of the catalytic domain (Delta1-100/Delta415-445) of chicken TPH isoform 1 (TPH1) in complex with the tryptophan substrate and an iron-bound imidazole. This is the first structure of any aromatic amino acid hydroxylase with bound natural amino acid substrate. The iron coordination can be described as distorted trigonal bipyramidal coordination with His273, His278, and Glu318 (partially bidentate) and one imidazole as ligands. The tryptophan stacks against Pro269 with a distance of 3.9 A between the iron and the tryptophan Czeta3 atom that is hydroxylated. The binding of tryptophan and maybe the imidazole has caused the structural changes in the catalytic domain compared to the structure of the human TPH1 without tryptophan. The structure of chicken TPH1 is more compact, and the loops of residues Leu124-Asp139 and Ile367-Thr369 close around the active site. Similar structural changes are seen in the catalytic domain of phenylalanine hydroxylase (PAH) upon binding of substrate analogues norleucine and thienylalanine to the PAH.BH 4 complex. In fact, the chicken TPH1.Trp.imidazole structure resembles the PAH.BH 4.thienylalanine structure more (root-mean-square deviation for Calpha atoms of 0.90 A) than the human TPH1 structure (root-mean-square deviation of 1.47 A).