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.
Ståhlberg
J
J
Department of Molecular Biology, University of Uppsala, Uppsala, SE-751 24, Sweden.
Henriksson
H
H
Divne
C
C
Isaksson
R
R
Pettersson
G
G
Johansson
G
G
Jones
T A
TA
D000319:Adrenergic beta-Antagonists / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase; D002851:Chromatography, High Pressure Liquid; D018360:Crystallography, X-Ray; D015195:Drug Design; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011433:Propranolol / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013237:Stereoisomerism; D013379:Substrate Specificity; D014242:Trichoderma / Q000201:enzymology*
D000319:Adrenergic beta-Antagonists; D011433:Propranolol; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase