Evolution of vitamin B2 biosynthesis: riboflavin synthase of Arabidopsis thaliana and its inhibition by riboflavin.
A synthetic gene specifying the catalytic domain of the Arabidopsis thaliana riboflavin synthase was expressed with high efficiency in a recombinant Escherichia coli strain. The recombinant pseudomature protein was shown to convert 6,7-dimethyl-8-ribityllumazine into riboflavin at a rate of 0.027 s-1 at 25 degrees C. The protein sediments at a rate of 3.9 S. Sedimentation equilibrium analysis afforded a molecular mass of 67.5 kDa, indicating a homotrimeric structure, analogous to the riboflavin synthases of Eubacteria and fungi. The protein binds its product riboflavin with relatively high affinity (Kd =1.1 microM). Product inhibition results in a characteristic sigmoidal velocity versus substrate concentration relationship. Characterization of the enzyme/product complex by circular dichroism and UV absorbance spectroscopy revealed a shift of the absorption maxima of riboflavin from 370 and 445 to 399 and 465 nm, respectively. Complete or partial sequences for riboflavin synthase orthologs were analyzed from 11 plant species. In each case for which the complete plant gene sequence was available, the catalytic domain was preceded by a sequence of 1-72 amino acid residues believed to function as plastid targeting signals. Comparison of all available riboflavin synthase sequences indicates that hypothetical gene duplication conducive to the two-domain architecture occurred very early in evolution.
Fischer
Markus
M
Lehrstuhl für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstr. 4, D-85747 Garching, Germany. markus.fischer@ch.tum.de
Haase
Ilka
I
Feicht
Richard
R
Schramek
Nicholas
N
Köhler
Peter
P
Schieberle
Peter
P
Bacher
Adelbert
A
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D001483:Base Sequence; D020134:Catalytic Domain; D002942:Circular Dichroism; D019295:Computational Biology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics; D011621:Pteridines / Q000737:chemistry; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D012256:Riboflavin / Q000737:chemistry*; D012258:Riboflavin Synthase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet
D010940:Plant Proteins; D011621:Pteridines; D011994:Recombinant Proteins; C001061:6,7-dimethyl-8-ribityllumazine; D012258:Riboflavin Synthase; D012256:Riboflavin