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  Biosynthesis of riboflavin - 6,7-dimethyl-8-ribityllumazine synthase of Schizosaccharomyces pombe

Fischer, M., Haase, I., Feicht, R., Richter, G., Gerhardt, S., Changeux, J. P., et al. (2002). Biosynthesis of riboflavin - 6,7-dimethyl-8-ribityllumazine synthase of Schizosaccharomyces pombe. European Journal of Biochemistry, 269(2), 519-526.

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Genre: Journal Article
Alternative Title : Eur. J. Biochem.

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 Creators:
Fischer, M., Author
Haase, I., Author
Feicht, R., Author
Richter, G., Author
Gerhardt, S.1, Author              
Changeux, J. P., Author
Huber, R.2, Author              
Bacher, A., Author
Affiliations:
1External Organizations, ou_persistent22              
2Huber, Robert / Structure Research, Max Planck Institute of Biochemistry, Max Planck Society, ou_1565155              

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Free keywords: biosynthesis of riboflavin; crystallization; 6;7-dimethyl-8- ribityllumazine synthase; mutagenesis; riboflavin binding
 Abstract: 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 degreesC). The protein has been crystallized in space group C222(1). The crystals diffract to a resolution of 2.4 Angstrom. 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 value of 13 000 nmol.mg(-1).h(-1) and K-m values of 5 and 67 mum for 5-amino-6- ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2- butanone 4-phosphate, respectively. The enzyme binds riboflavin wit a K-d of 1.2 muM. 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.

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Language(s): eng - English
 Dates: 2002-01
 Publication Status: Published in print
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: eDoc: 39030
ISI: 000173651700013
 Degree: -

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Title: European Journal of Biochemistry
  Alternative Title : Eur. J. Biochem.
Source Genre: Journal
 Creator(s):
Affiliations:
Publ. Info: -
Pages: - Volume / Issue: 269 (2) Sequence Number: - Start / End Page: 519 - 526 Identifier: ISSN: 0014-2956