| 1. |
- von Ossowski, I., et al.
(författare)
-
Engineering the exo-loop of Trichoderma reesei cellobiohydrolase, Ce17A. A comparison with Phanerochaete chrysosporium Cel7D
- 2003
-
Ingår i: Journal of Molecular Biology. - 0022-2836 .- 1089-8638. ; 333:4, s. 817-829
-
Tidskriftsartikel (refereegranskat)abstract
- The exo-loop of Trichoderma reesei cellobiohydrolase Cel7A forms the roof of the active site tunnel at the catalytic centre. Mutants were designed to study the role of this loop in crystalline cellulose degradation. A hydrogen bond to substrate made by a tyrosine at the tip of the loop was removed by the Y247F mutation. The mobility of the loop was reduced by introducing a new disulphide bridge in the mutant D241C/D249C. The tip of the loop was deleted in mutant Delta(G245-Y252). No major structural disturbances were observed in the mutant enzymes, nor was the thermostability of the enzyme affected by the mutations. The Y247F mutation caused a slight k(cat) reduction on 4-nitrophenyl lactoside, but only a small effect on cellulose hydrolysis. Deletion of the tip of the loop increased both k(cat) and K-M and gave reduced product inhibition. Increased activity was observed on amorphous cellulose, while only half the original activity remained on crystalline cellulose. Stabilisation of the exo-loop by the disulphide bridge enhanced the activity on both amorphous and crystalline cellulose. The ratio Glc(2)/(Glc(3) + Glc(1)) released from cellulose, which is indicative of processive action, was highest with Tr Cel7A wild-type enzyme and smallest with the deletion mutant on both substrates. Based on these data it seems that the exo-loop of Tr Cel7A has evolved to facilitate processive crystalline cellulose degradation, which does not require significant conformational changes of this loop.
|
|
| 2. |
|
|
| 3. |
|
|
| 4. |
- Furlanetto, Valentina, et al.
(författare)
-
Structural and Functional Characterization of a Gene Cluster Responsible for Deglycosylation of C-glucosyl Flavonoids and Xanthonoids by Deinococcus aerius
- 2024
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 436:9
-
Tidskriftsartikel (refereegranskat)abstract
- Plant C-glycosylated aromatic polyketides are important for plant and animal health. These are specialized metabolites that perform functions both within the plant, and in interaction with soil or intestinal microbes. Despite the importance of these plant compounds, there is still limited knowledge of how they are metabolized. The Gram-positive aerobic soil bacterium Deinococcus aerius strain TR0125 and other Deinococcus species thrive in a wide range of harsh environments. In this work, we identified a C-glycoside deglycosylation gene cluster in the genome of D. aerius. The cluster includes three genes coding for a GMC-type oxidoreductase (DaCGO1) that oxidizes the glucosyl C3 position in aromatic C-glucosyl compounds, which in turn provides the substrate for the C-glycoside deglycosidase (DaCGD; composed of α+β subunits) that cleaves the glucosyl-aglycone C–C bond. Our results from size-exclusion chromatography, single particle cryo-electron microscopy and X-ray crystallography show that DaCGD is an α2β2 heterotetramer, which represents a novel oligomeric state among bacterial CGDs. Importantly, the high-resolution X-ray structure of DaCGD provides valuable insights into the activation of the catalytic hydroxide ion by Lys261. DaCGO1 is specific for the 6-C-glucosyl flavones isovitexin, isoorientin and the 2-C-glucosyl xanthonoid mangiferin, and the subsequent C–C-bond cleavage by DaCGD generated apigenin, luteolin and norathyriol, respectively. Of the substrates tested, isovitexin was the preferred substrate (DaCGO1, Km 0.047 mM, kcat 51 min−1; DaCGO1/DaCGD, Km 0.083 mM, kcat 0.42 min−1).
|
|
| 5. |
- Gandini, Rosaria, et al.
(författare)
-
A Transmembrane Crenarchaeal Mannosyltransferase Is Involved in N-Glycan Biosynthesis and Displays an Unexpected Minimal Cellulose-Synthase-like Fold
- 2020
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 432:16, s. 4658-4672
-
Tidskriftsartikel (refereegranskat)abstract
- Protein glycosylation constitutes a critical post-translational modification that supports a vast number of biological functions in living organisms across all domains of life. A seemingly boundless number of enzymes, glycosyltransferases, are involved in the biosynthesis of these protein-linked glycans. Few glycanbiosynthetic glycosyltransferases have been characterized in vitro, mainly due to the majority being integral membrane proteins and the paucity of relevant acceptor substrates. The crenarchaeote Pyrobaculum calidifontis belongs to the TACK superphylum of archaea (Thaumarchaeota, Aigarchaeota, Crenarchaeota, Korarchaeota) that has been proposed as an eukaryotic ancestor. In archaea, N-glycans are mainly found on cell envelope surface-layer proteins, archaeal flagellins and pili. Archaeal N-glycans are distinct from those of eukaryotes, but one noteworthy exception is the high-mannose N-glycan produced by P. calidifontis, which is similar in sugar composition to the eukaryotic counterpart. Here, we present the characterization and crystal structure of the first member of a crenarchaeal membrane glycosyltransferase, PcManGT. We show that the enzyme is a GDP-, dolichylphosphate-, and manganese-dependent mannosyltransferase. The membrane domain of PcManGT includes three transmembrane helices that topologically coincide with "half' of the sixtransmembrane helix cellulose-binding tunnel in Rhodobacter spheroides cellulose synthase BcsA. Conceivably, this "half tunnel" would be suitable for binding the dolichylphosphate-linked acceptor substrate. The PcManGT gene (Pcal_0472) is located in a large gene cluster comprising 14 genes of which 6 genes code for glycosyltransferases, and we hypothesize that this cluster may constitute a crenarchaeal N-glycosylation (PNG) gene cluster.
|
|
| 6. |
- Hallberg, B. M., et al.
(författare)
-
Crystal structure of the flavoprotein domain of the extracellular flavocytochrome cellobiose dehydrogenase
- 2002
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 315:3, s. 421-434
-
Tidskriftsartikel (refereegranskat)abstract
- Cellobiose dehydrogenase (CDH) participates in the degradation of cellulose and lignin. The protein is an extracellular flavocytochrome with a b-type cytochrome domain (CYTcdh) connected to a flavodehydrogenase domain (DHcdh). DHcdh catalyses a two-electron oxidation at the anomeric C1 position of cellobiose to yield cellobiono-1,5-lactone, and the electrons are subsequently transferred from DHcdh to an acceptor, either directly or via CYTcdh. Here, we decribe the crystal structure of Phanerochaete chrysosporium DHcdh determined at 1.5 Angstrom resolution. DHcdh belongs to the GMC family of oxidoreductases, which includes glucose oxidase (GOX) and cholesterol oxidase (COX); however, the sequence identity with members of the family is low. The overall fold of DHcdh is p-hydroxybenzoate hydroxylase-like and is similar to, but also different from, that of GOX and COX. It is partitioned into an FAD-binding subdomain of alpha/beta type and a substrate-binding subdomain consisting of a seven-stranded beta sheet and six helices. Docking of CYTcdh, and DHcdh suggests that CYTcdh covers the active-site entrance in DHcdh, and that the resulting distance between the cofactors is within acceptable limits for inter-domain electron transfer. Based on docking of the substrate, cellobiose, in the active site of DHcdh, we propose that the enzyme discriminates against glucose by favouring interaction with the non-reducing end of cellobiose.
|
|
| 7. |
- Hallberg, Martin, et al.
(författare)
-
Crystal structure of the 270 kDa homotetrameric lignin-degrading enzyme pyranose 2-oxidase
- 2004
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 341:3, s. 781-796
-
Tidskriftsartikel (refereegranskat)abstract
- Pyranose 2-oxidase (P2Ox) is a 270 kDa homotetramer localized preferentially in the hyphal periplasmic space of lignocellulolytic fungi and has a proposed role in lignocellulose degradation to produce the essential co-substrate, hydrogen peroxide, for lignin peroxidases. P2Ox oxidizes D-glucose and other aldopyranoses regioselectively at C2 to the corresponding 2-keto sugars; however, for some substrates, the enzyme also displays specificity for oxidation at C3. The crystal structure of P2Ox from Trametes multicolor has been determined using single anomalous dispersion with mercury as anomalous scatterer. The model was refined at 1.8 Angstrom resolution to R and R-free values of 0.134 and 0.171, respectively. The overall fold of the P2Ox subunit resembles that of members of the glucose-methanol-choline family of long-chain oxidoreductases, featuring a flavin-binding Rossmann domain of class alpha/beta and a substrate-binding subdomain with a six-stranded central beta sheet and three U helices. The homotetramer buries a large internal cavity of roughly 15,000 Angstrom(3), from which the four active sites are accessible. Four solvent channels lead from the surface into the cavity through which substrate must enter before accessing the active site. The present structure shows an acetate molecule bound in the active site with the carboxylate group positioned immediately below the flavin N5 atom, and with one carboxylate oxygen atom interacting with the catalytic residues His548 and Asn593. The entrance to the active site is blocked by a loop (residues 452 to 461) with excellent electron density but elevated temperature factors. We predict that this loop is dynamic and opens to allow substrate entry and exit. In silico docking of D-glucose in the P2Ox active site shows that with the active-site loop in the closed conformation, monosaccharides cannot be accommodated; however, after removing the loop from the model, a tentative set of protein-substrate interactions for beta-D-glucose have been outlined.
|
|
| 8. |
|
|
| 9. |
|
|
| 10. |
- Stahlberg, J., et al.
(författare)
-
Structural basis for enantiomer binding and separation of a common beta-blocker : Crystal structure of cellobiohydrolase Cel7A with bound (S)-propranolol at 1.9 angstrom resolution
- 2001
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 305:1, s. 79-93
-
Tidskriftsartikel (refereegranskat)abstract
- Cellobiohydrolase Cel7A (previously called CBH 1), the major cellulase produced by the mould fungus Trichoderma reesei, has been successfully exploited as a chiral selector for separation of stereo-isomers of some important pharmaceutical compounds, e.g. adrenergic beta -blockers. Previous investigations, including experiments with catalytically deficient mutants of Cel7A, point unanimously to the active site as being responsible for discrimination of enantiomers. In this work the structural basis for enantioselectivity of basic drugs by Cel7A has been studied by X-ray crystallography. The catalytic domain of Cel7A was co-crystallised with the (S)-enantiomer of a common beta -blocker, propranolol, at pH 7, and the structure of the complex was determined and refined at 1.9 Angstrom resolution. Indeed, (S)-propranolol binds at the active site, in glucosyl-binding subsites -1/ + 1. The catalytic residues Glu212 and Glu217 make tight salt links with the secondary amino group of (S)-propranolol. The oxygen atom attached to the chiral centre of (S)-propranolol forms hydrogen bonds to the nucleophile Glu212 O-epsilon1 and to Gln175 N-epsilon2, whereas the aromatic naphthyl moiety stacks with the indole ring of Trp376 in site +1. The bidentate charge interaction with the catalytic glutamate residues is apparently crucial, since no enantioselectivity has been obtained with the catalytically deficient mutants E212Q and E217Q. Activity inhibition experiments with wild-type Cel7A were performed in conditions close to those used for crystallisation. Competitive inhibition constants for (R)- and (S)-propranolol were determined at 220 muM and 44 muM, respectively, corresponding to binding free energies of 20 kJ/ mol and 24 kJ/mol, respectively. The K-i value for (R)-propranolol was 57-fold lower than the highest concentration, 12.5 mM, used in co-crystallisation experiments. Still several attempts to obtain a complex with the (R)-enantiomer have failed. By using cellobiose as a selective competing ligand, the retention of the enantiomers of propranolol on the chiral stationary phase (CSP) based on Cel7A mutant D214N were resolved into enantioselective and non-selective binding. The enantioselective binding was weaker for both enantiomers on D214N-CSP than on wild-type-CSP.
|
|
