Hongbin Henriksson

Crystal Structures of a Poplar Xyloglucan Endotransglycosylase Reveal Details of Transglycosylation Acceptor Binding

Xyloglucan endotransglycosylases (XETs) cleave and religate xyloglucan polymers in plant cell walls via a transglycosylation mechanism. Thus, XET is a key enzyme in all plant processes that require cell wall remodeling. To provide a basis for detailed structure–function studies, the crystal structure of Populus tremula x tremuloides XET16A (PttXET16A), heterologously expressed in Pichia pastoris, has been determined at 1.8-Å resolution. Even though the overall structure of PttXET16A is a curved β-sandwich similar to other enzymes in the glycoside hydrolase family GH16, parts of its substrate binding cleft are more reminiscent of the distantly related family GH7. In addition, XET has a C-terminal extension that packs against the conserved core, providing an additional β-strand and a short α-helix. The structure of XET in complex with a xyloglucan nonasaccharide, XLLG, reveals a very favorable acceptor binding site, which is a necessary but not sufficient prerequisite for transglycosylation. Biochemical data imply that the enzyme requires sugar residues in both acceptor and donor sites to properly orient the glycosidic bond relative to the catalytic residues.

Enzymatic properties of native and deglycosylated hybrid aspen (Populus tremula×tremuloides) xyloglucan endotransglycosylase 16A expressed in Pichia pastoris

The cDNA encoding a xyloglucan endotransglycosylase, PttXET16A, from hybrid aspen (Populus tremulaxtremuloides) has been isolated from an expressed sequence tag library and expressed in the methylotrophic yeast Pichia pastoris. Sequence analysis indicated a high degree of similarity with other proteins in the XTH (xyloglucan transglycosylase/hydrolase) gene subfamily of GH16 (glycoside hydrolase family 16). In addition to the conserved GH16 catalytic sequence motif, PttXET16A contains a conserved N-glycosylation site situated proximal to the predicted catalytic residues. MS analysis indicated that the recombinant PttXET16A expressed in P. pastoris is heterogeneous due to the presence of variable N-glycosylation and incomplete cleavage of the alpha-factor secretion signal peptide. Removal of the N-glycan by endoglycosidase H treatment did not influence the catalytic activity significantly. Similarly, site-directed mutagenesis of Asn93 to serine to remove the N-glycosylation site resulted in an enzyme which was comparable with the wild-type enzyme in specific activity and thermal stability but had clearly reduced solubility. Hydrolytic activity was detected neither in wild-type PttXET16A before or after enzymatic deglycosylation nor in PttXET16A N93S (Asn93-->Ser) mutant.

The active sites of cellulases are involved in chiral recognition: a comparison of cellobiohydrolase 1 and endoglucanase 1

The cellulases cellobiohydrolase 1 (CBH 1) and endoglucanase 1 (EG 1) from the fungus Trichoderma reesei are closely related with 40% sequence identity and very similar in structure. In CBH 1 the active site is enclosed by long loops and some antiparallel β‐strands forming a 40 Å long tunnel, whereas in EG 1 part of those loops are missing so that the enzyme has a more common active site groove. Both enzymes were immobilized on silica and these materials were used as chiral stationary phases for chromatographic separation of the enantiomers of two chiral drugs, propranolol and alprenolol. The CBH 1 phase showed much better resolution than did the EG 1 phase, suggesting that the tunnel structure of the protein may play an important role in the chiral separation. The chiral compounds were found to be competitive inhibitors of both enzymes when p‐nitrophenyl lactoside (pNPL) was used as substrate. (S)‐enantiomers showed stronger inhibitory effects and also longer retention time on the stationary phases than the (R)‐enantiomers. The consistency between kinetic data and retention on the stationary phases clearly shows that the enzymatically active sites of CBH 1 and EG 1 are involved in chiral recognition.

N-linked glycosylation of native and recombinant cauliflower xyloglucan endotransglycosylase 16A.

The gene encoding a XET (xyloglucan endotransglycosylase) from cauliflower ( Brassica oleracea var. botrytis ) florets has been cloned and sequenced. Sequence analysis indicated a high degree of similarity to other XET enzymes belonging to glycosyl hydrolase family 16 (GH16). In addition to the conserved GH16 catalytic sequence motif EIDFE, there exists one potential N-linked glycosylation site, which is also highly conserved in XET enzymes from this family. Purification of the corresponding protein from extracts of cauliflower florets allowed the fractionation of a single, pure glycoform, which was analysed by MS techniques. Accurate protein mass determination following the enzymic deglycosylation of this glycoform indicated the presence of a high-mannose-type glycan of the general structure GlcNAc2Man6. LC/MS and MS/MS (tandem MS) analysis provided supporting evidence for this structure and confirmed that the glycosylation site (underlined) was situated close to the predicted catalytic residues in the conserved sequence YLSSTNNEHDEIDFEFLGNRTGQPVILQTNVFTGGK. Heterologous expression in Pichia pastoris produced a range of protein glycoforms, which were, on average, more highly mannosylated than the purified native enzyme. This difference in glycosylation did not influence the apparent enzymic activity of the enzyme significantly. However, the removal of high-mannose glycosylation in recombinant cauliflower XET by endoglycosidase H, quantified by electrospray-ionization MS, caused a 40% decrease in the transglycosylation activity of the enzyme. No hydrolytic activity was detected in native or heterologously expressed BobXET16A, even when almost completely deglycosylated.

Cellobiohydrolase 58 (P.c. Cel 7D) is complementary to the homologous CBH I (T.r. Cel 7A) in enantioseparations.

Cellobiohydrolase 58 (EC 3.2.1.91, P.c. Cel 7D) from Phanerochaete chrysosporium was immobilized on silica and the resulting material, CBH 58-silica, was then used as a chiral stationary phase (CSP) in liquid chromatographic separations of enantiomers. The enantioselectivities obtained on CBH 58-silica were compared with those on CBH I-silica (a phase based on a corresponding cellulase from Trichoderma reesei). CBH 58-silica displayed higher selectivity than CBH I-silica for the more hydrophilic compounds, such as atenolol and metoprolol, although great similarities in chiral separation of beta-adrenergic antagonists were found between the two phases. None of the acidic compounds tested could be resolved on the CBH 58 phase. Moreover, the solutes were retained more on the CBH 58 phase in general, indicating an improved application potential in bioanalysis. Addition of cellobiose or lactose, both of which are inhibitors of cellulases, to the mobile phase impaired the enantioselectivity, indicating an overlap of the enantioselective and catalytic sites. The chiral analytes also functioned as competitive inhibitors and their inhibition constants were determined.

The catalytic amino-acid residues in the active site of cellobiohydrolase 1 are involved in chiral recognition

The catalytic amino-acid residues in the active site of cellobiohydrolase 1 are involved in chiral recognition

Hemicellulase activity of aerobic fungal cellulases

Summary Cellulases isolated from Trichoderma reesei and Phanerochaete chrysosporium were screened for hemicellulolytic, pectinolytic and cellulolytic activity using locust bean mannan, birchwood xylan, citrus fruit pectin and carboxymethylated cellulose (CMC) as substrates. The purpose of this work was to choose appropriate enzymes to include in a “miniature cellulase system” with minimal hemicellulase activity for the preparation of lignin-carbohydrate complexes (LCCs). The endoglucanases showed CMC activity whereas activity towards the substrate was not detected for the CBHs. Xylanase activity was observed for EG I and EG 38 whereas mannanase activity was observed for EG 44. None of the enzymes degraded pectin. The results suggest that CBH I, CBH II, CBH 58, EG II and EG III are good candidates for the effective preparation of LCCs. The possible biological function for the hemicellulolytic activity of cellulases is discussed.

Studies on the enantioselective retention mechanisms of cellobiohydrolase I (CBH I) by covalent modification of the intact and fragmented protein

Studies on the enantioselective retention mechanisms of cellobiohydrolase I (CBH I) by covalent modification of the intact and fragmented protein

Microcalorimetric studies on the complex formation between cellobiohydrolase I (CBH I) from Trichoderma reesei and the (R)- and (S)-enantiomers of the β-receptor blocking agent alprenolol

Abstract The thermodynamic quantities for the complex formation between the enantiomers of the β-blocking drug alprenolol and cellobiohydrolase I (CBH I), that earlier has been used as a chiral selector for aminoalcohols, revealed positive ΔH0 — values in all cases implying an entropy driven process. Association constants (Ka) for cellulase and the (R)- and (S)-enantiomers of alprenolol were determined by isothermal titration microcalorimetry and the inhibition constants (Ki) by enzyme inhibition experiments. Both inhibition experiments and microcalorimetry revealed that the affinity between the enantiomers of alprenolol and CBH I was higher in sodium phosphate buffer than in potassium phosphate buffer. This result was in agreement with previously reported liquid chromatographic separations of enantiomers using a chiral stationary phase based on CBH I immobilized to silica particles. The best fit of the isothermal titration data corresponded to a 1:1 binding isotherm.

Crystallization and preliminary X-ray analysis of a xyloglucan endotransglycosylase from Populus tremula x tremuloides.

Xyloglucan endotransglycosylases (XETs) cleave and religate xyloglucan polymers in plant cell walls. Recombinant XET from poplar has been purified from a Pichia pastoris expression system and crystallized. Two different crystal forms were obtained by vapour diffusion from potassium sodium tartrate and from an imidazole buffer using sodium acetate as a precipitant. Data were collected from these crystal forms to 3.5 and 2.1 A resolution, respectively. The first crystal form was found to belong to space group P3(1)21 or P3(2)21 (unit-cell parameters a = 98.6, b = 98.6, c = 98.5 A) and the second crystal form to space group P6(3) (unit-cell parameters a = 188.7, b = 188.7, c = 46.1 A).