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H Henriksson

Publications and source records attributed to H Henriksson.

13 recordsLinked to original sources
PubMed2001

Family 7 cellobiohydrolases from Phanerochaete chrysosporium: crystal structure of the catalytic module of Cel7D (CBH58) at 1.32 A resolution and homology models of the isozymes.

Cellobiohydrolase 58 (Cel7D) is the major cellulase produced by the white-rot fungus Phanerochaete chrysosporium, constituting approximately 10 % of the total secreted protein in liquid culture on cellulose. The enzyme is classified into family 7 of the glycosyl hydrolases, together with cellobiohydrolase I (Cel7A) and endoglucanase I (Cel7B) from Trichoderma reesei. Like those enzymes, it catalyses cellulose hydrolysis with net retention of the anomeric carbon configuration. The structure of the catalytic module (431 residues) of Cel7D was determined at 3.0 A resolution using the structure of Cel7A from T. reesei as a search model in molecular replacement, and ultimately refined at 1.32 A resolution. The core structure is a beta-sandwich composed of two large and mainly antiparallel beta-sheets packed onto each other. A long cellulose-binding groove is formed by loops on one face of the sandwich. The catalytic residues are conserved and the mechanism is expected to be the same as for other family members. The Phanerochaete Cel7D binding site is more open than that of the T. reesei cellobiohydrolase, as a result of deletions and other changes in the loop regions, which may explain observed differences in catalytic properties. The binding site is not, however, as open as the groove of the corresponding endoglucanase. A tyrosine residue at the entrance of the tunnel may be part of an additional subsite not present in the T. reesei cellobiohydrolase. The Cel7D structure was used to model the products of the five other family 7 genes found in P. chrysosporium. The results suggest that at least two of these will have differences in specificity and possibly catalytic mechanism, thus offering some explanation for the presence of Cel7 isozymes in this species, which are differentially expressed in response to various growth conditions.

Amino Acid Sequence↗
PubMed2001

Structural basis for enantiomer binding and separation of a common beta-blocker: crystal structure of cellobiohydrolase Cel7A with bound (S)-propranolol at 1.9 A resolution.

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 A 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 microM and 44 microM, 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.

Adrenergic beta-Antagonists↗
PubMed2000

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.

Cellulase↗
PubMed1999

Discrimination between enantioselective and non-selective binding sites on cellobiohydrolase-based stationary phases by site specific competing ligands.

A systematic study was performed to investigate the influence of cellobiose or lactose on the enantioselective retention behaviour of some beta-blockers in liquid chromatography using Cellobiohydrolase (CHB) I from Trichoderma reesei or Cellobiohydrolase 58 from Phanerochaete chrysosporium immobilized on silica as stationary phases. The results revealed that the retention could be described by the function [equation; see text] where the observed capacity factor corresponds to the sum of an enantioselective mode being influenced by a site specific competing ligand (competitor) and a non-selective mode unaffected by the competitor. A non-constrained non-linear least-square regression gave in all cases virtually identical nondisplacable capacity factors (k'ns) for both enantiomers of the same drug. The experimental capacity factors (k'(x,C)) of the enantiomers all show a close fit to the adapted function. The Kd values calculated for the competitor were also virtually identical for each pair of enantiomers and were in accordance with Ki data determined for the competitors in classical enzyme kinetics experiments, demonstrating that one unique site; namely, the catalytic site, was responsible for the enantioselective binding. Similar results were obtained with the resolution of rac-alprenolol and rac-metoprolol on CBH I phase.

Binding Sites↗
PubMed1999

Continuous beds for microchromatography: chromatofocusing and anion exchange chromatography.

A method was developed for the preparation of continuous beds derivatized with polyethyleneimine (PEI) for chromatofocusing and anion exchange chromatography in the capillary mode. First, a continuous bed activated by epoxy groups was synthesized inside a fused silica capillary and became at the same time covalently attached to the inner wall of the capillary. A PEI solution was then pumped through the continuous bed to allow the imine groups in PEI to react with the epoxy groups in the bed. Efficient immobilization of PEI was indicated by the high-resolution separation of standard proteins (hemoglobins C, S, F, and A) in both chromatofocusing and anion exchange chromatography on a capillary column prepared by this method.

Buffers↗
PubMed1999

Endoglucanase 28 (Cel12A), a new Phanerochaete chrysosporium cellulase.

A 28-kDa endoglucanase was isolated from the culture filtrate of Phanerochaete chrysosporium strain K3 and named EG 28. It degrades carboxymethylated cellulose and amorphous cellulose, and to a lesser degree xylan and mannan but not microcrystalline cellulose (Avicel). EG 28 is unusual among cellulases from aerobic fungi, in that it appears to lack a cellulose-binding domain and does not bind to crystalline cellulose. The enzyme is efficient at releasing short fibres from filter paper and mechanical pulp, and acts synergistically with cellobiohydrolases. Its mode of degrading filter paper appears to be different to that of endoglucanase I from Trichoderma reesei. Furthermore, EG 28 releases colour from stained cellulose beads faster than any other enzyme tested. Peptide mapping suggests that it is not a fragment of another known endoglucanases from P. chrysosporium and peptide sequences indicate that it belongs to family 12 of the glycosyl hydrolases. EG 28 is glycosylated. The biological function of the enzyme is discussed, and it is hypothesized that it is homologous to EG III in Trichoderma reesei and the role of the enzyme is to make the cellulose in wood more accessible to other cellulases.

Amino Acid Sequence↗
PubMed1998

Assessment of functional ability in patients with scleroderma: a proposed new disability assessment instrument.

OBJECTIVE: To develop a simple assessment of functional ability in patients with scleroderma and to examine its reliability both as a patient self-administered instrument and when administered by a trained observer. METHODS: An 11 item, 4 grade, functional assessment questionnaire was developed after extensive consultation with patients, physiotherapists (PT), and occupational therapists (OT) with the aim of including all functional areas of relevance. The instrument was self-administered by patients after an interval of 7 days. In the interval, the patients were assessed using the same instrument by direct observation from both a PT and an OT. Forty-seven patients with scleroderma, of varying severity, were recruited from 2 centers. Results were similar for both centers and data were pooled for analysis. RESULTS: Agreement between the patients' first and 2nd assessment was good for all questions (estimated kappas 0.69 to 0.94) with no evidence of an order effect. Agreement was also good between therapists (estimated kappas 0.47 to 0.81). There was poor agreement between patients and therapists, with patients rating their disability substantially higher compared to the standardized therapist assessment. CONCLUSION: This assessment schedule has high face and content validity and has excellent reliability both between trained therapists and within patients over a short time period. Its administration either as a self-report or by a therapist depends, in part, on the type of investigation undertaken.

Disability Evaluation↗
PubMed1996

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 beta-strands forming a 40 A 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.

Alprenolol↗
PubMed1975

[Focus on the aged].

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Activities of Daily Living↗
PubMedDate not supplied

Fine needle biopsy as a method for following ploidy changes in the Dunning R-3327 rat prostate tumour.

The use of fine needle biopsies for the collection of cells for flow cytometry was investigated using the Altman Dunning R-3327 rat prostate tumour. Five rats were castrated 150 days after tumour implantation. Multiple fine needle biopsies were performed 138, 194 and 220 days post implantation and the results compared to those from intact animals. A small aneuploid peak was found at the 2.8c position in intact animals. Although tumour growth was slowing down in castrates, indicating hormone independency but sensitivity, no change in flow cytometric profile could be demonstrated. This indicates that, in this model, no correlation between aneuploidy and hormone independence exists. The fine needle biopsy method proved to be easy and reliable for tumours of above 1 cm3 in volume.

Animals↗