Biosynthesis of riboflavin in plants. The ribA gene of Arabidopsis thaliana specifies a bifunctional GTP cyclohydrolase II/3,4-dihydroxy-2-butanone 4-phosphate synthase

A cDNA segment from Arabidopsis thaliana with similarity to the ribA gene o f Bacillus subtilis was sequenced. A similar gene was cloned from tomato. T he open reading frame of A. thaliana was fused to the malE gene of Escheric hia coil and was expressed in a recombinant E. coli strain. The recombinant fusion protein was purified and shown to have GTP cyclohydrolase II activi ty as well as 3,4-dihydroxy-2-butanone 4-phosphate synthase activity. The c ognate gene was amplified by polymerase chain reaction from chromosomal Ara bidopsis DNA and was shown to contain six introns. Intron 4 is located in t he region connecting the GTP cyclohydrolase II and 3,4-dihydroxy-2-butanone 4-phosphate synthase domain of the putative domains catalyzing the two rea ction steps. By comparison with the bacterial ribA gene, the Arabidopsis ge ne contains an additional 5' element specifying about 120 amino acid residu es. This segment contains numerous serine and threonine residues and does n ot show similarity with other known sequences. The N-terminal segment is no t required for catalytic activity and is likely to serve as signal sequence for import into chloroplasts. (C) 2000 Elsevier Science Ltd. All rights re served.