tailieunhanh - Báo cáo khoa học: Biosynthesis of riboflavin 6,7-Dimethyl-8-ribityllumazine synthase of Schizosaccharomyces pombe

A cDNA sequence fromSchizosaccharomyces pombewith similarity to 6,7-dimethyl-8-ribityllumazine synthase was expressed in a recombinant Escherichia colistrain. The recombinant protein is ahomopentamer of 17-kDa subunits with an apparent molecular mass of 87 kDa as determined by sedimentation equilibriumcentrifugation (it sediments at an apparent velocity of S at 20°C). The protein has been crystallized in space group C2221 . | Eur. J. Biochem. 269 519-526 2002 FEBS 2002 Biosynthesis of riboflavin 6 7-Dimethyl-8-ribityllumazine synthase of Schizosaccharomyces pombe Markus Fischer1 Ilka Haase1 Richard Feicht1 Gerald Richter1 Stefan Gerhardt2 Jean-Pierre Chanaeux3 Robert Huber2 and Adelbert Bacher1 1Institut fur Organische Chemie und Biochemie Technische Universitat Munchen Germany 2 Department of Protein Crystallography Max-Planck-Institute of Biochemistry Martinsried Germany 3Department of Molecular Neurobiology Institut Pasteur Paris France A cDNA sequence from Schizosaccharomyces pombe with similarity to 6 7-dimethyl-8-ribityllumazine synthase was expressed in a recombinant Escherichia coli strain. The recombinant protein is a homopentamer of 17-kDa subunits with an apparent molecular mass of 87 kDa as determined by sedimentation equilibrium centrifugation it sediments at an apparent velocity of S at 20 C . The protein has been crystallized in space group C2221. The crystals diffract to a resolution of A. The enzyme catalyses the formation of 6 7-dimethyl-8-ribityllumazine from 5-amino-6-ribityl-amino-2 4 1H 3H -pyrimidinedione and 3 4-dihydroxy-2-butanone 4-phosphate. Steady-state kinetic analysis afforded a vmax value of 13 000 nmol-mg_1 h-1 and Km values of 5 and 67 M for 5-amino-6-ribitylamino-2 4 1H 3H -pyrimidinedione and 3 4-dihydroxy-2-butanone 4-phosphate respectively. The enzyme binds riboflavin with a Ki of M. The fluorescence quantum yield of enzyme bound riboflavin is 2 as compared with that of free riboflavin. The protein riboflavin complex displays an optical transition centered around 530 nm as shown by absorbance and CD spectrometry which may indicate a charge transfer complex. Replacement of tryptophan 27 by tyrosine or phenylalanine had only minor effects on the kinetic properties but complexes of the mutant proteins did not show the anomalous long wavelength absorbance of the wild-type protein. The replacement of tryptophan 27 by aliphatic amino acids .