Biosynthesis of riboflavin: 6,7-dimethyl-8-ribityllumazine synthase of Schizosaccharomyces pombe.
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 5.0 S at 20 degrees C). The protein has been crystallized in space group C2221. The crystals diffract to a resolution of 2.4 A. The enzyme catalyses the formation of 6,7-dimethyl-8-ribityllumazine from 5-amino-6-ribitylamino-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 microm 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 Kd of 1.2 microm. 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 substantially reduced the affinity of the enzyme for riboflavin and for the substrate, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione.
Fischer
Markus
M
Institut für Organische Chemie und Biochemie, Technische Universität München, Germany. markus.fischer@ch.tum.de
Haase
Ilka
I
Feicht
Richard
R
Richter
Gerald
G
Gerhardt
Stefan
S
Changeux
Jean-Pierre
JP
Huber
Robert
R
Bacher
Adelbert
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D012256:Riboflavin / Q000096:biosynthesis*; D012568:Schizosaccharomyces / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization
D017931:DNA Primers; D009097:Multienzyme Complexes; C044822:6,7-dimethyl-8-ribityllumazine synthase; D012256:Riboflavin