Evolution of Vitamin B₂Biosynthesis: 6,7-Dimethyl-8-Ribityllumazine Synthases ofBrucella

Abstract

The penultimate step in the biosynthesis of riboflavin (vitamin B 2 ) involves the condensation of 3,4-dihydroxy-2-butanone 4-phosphate with 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, which is catalyzed by 6,7-dimethyl-8-ribityllumazine synthase (lumazine synthase). Pathogenic Brucella species adapted to an intracellular lifestyle have two genes involved in riboflavin synthesis, ribH1 and ribH2, which are located on different chromosomes. The ribH2 gene was shown previously to specify a lumazine synthase (type II lumazine synthase) with an unusual decameric structure and a very high K m for 3,4-dihydroxy-2-butanone 4-phosphate. Moreover, the protein was found to be an immunodominant Brucella antigen and was able to generate strong humoral as well as cellular immunity against Brucella abortus in mice. We have now cloned and expressed the ribH1 gene, which is located inside a small riboflavin operon, together with two other putative riboflavin biosynthesis genes and the nusB gene, specifying an antitermination factor. The RibH1 protein (type I lumazine synthase) is a homopentamer catalyzing the formation of 6,7-dimethyl-8-ribityllumazine at a rate of 18 nmol mg ؊1 min ؊1 . Sequence comparison of lumazine synthases from archaea, bacteria, plants, and fungi suggests a family of proteins comprising archaeal lumazine and riboflavin synthases, type I lumazine synthases, and the eubacterial type II lumazine synthases.Vitamin B 2 (riboflavin) (compound 6 [ Fig. 1]) is the precursor of flavin mononucleotide and flavin adenine dinucleotide, essential cofactors for a wide variety of redox enzymes. Moreover, they are involved in numerous other physiological processes involving light sensing, bioluminescence, circadian time keeping, and DNA repair (for a review, see reference 39). The vitamin is biosynthesized by plants, fungi, and certain microorganisms but must be obtained from dietary sources and/or the intestinal flora by animals.The pathways of riboflavin biosynthesis in microorganisms and plants have been reviewed recently (9, 10). The final steps are catalyzed by 6,7-dimethyl-8-ribityllumazine synthase (lumazine synthase [compound VI]) and riboflavin synthase (compound VII). More specifically, lumazine synthase catalyzes the condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (substrate 2) with 3,4-dihydroxy-2-butanone 4-phosphate (substrate 4), resulting in the pteridine derivative, substrate 5 (Fig. 1).Lumazine synthases from a variety of eubacteria (including Escherichia coli, Bacillus subtilis, Mycobacterium tuberculosis, and the hyperthermophile Aquifex aeolicus), archaea (Methanococcus jannaschii), fungi (Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Magnaporthe grisea), and a plant (spinach) have been studied in some detail (11, 16, 22, 32, 34, 35, 40-42, 44, 56, 57). The enzymes from fungi and from M. tuberculosis are C5-symmetric homopentamers, and the lumazine synthases of plants, most eubacteria, and archaea are 532-symmetric, hollow capsids, which are best described as dode...

Identification and Characterization of RibN, a Novel Family of Riboflavin Transporters from Rhizobium leguminosarum and Other Proteobacteria

“…In previous work, we demonstrated that the B. abortus 2308 riboflavin biosynthesis pathway harbors two paralogs for the lumazine synthase RibH. In particular, the ribH2 gene contains an FMN riboswitch and encodes a functional lumazine synthase with a novel quaternary arrangement that is essential for B. abortus intracellular survival and virulence (7,(26)(27)(28). We sought to determine the arrangement of riboflavin biosynthesis genes and FMN riboswitches in the genome of R. leguminosarum strain 3841 (29).…”

Genetic Control of Biosynthesis and Transport of Riboflavin and Flavin Nucleotides and Construction of Robust Biotechnological Producers

“…The pentameric enzymes of S. cerevisiae and Brucella abortus contain inserts of 4 amino acids between helices a4 and a5, which is hypothesized as being responsible for their inability to form an icosahedral capsid as a consequence of steric hindrance (116,231). Pathogenic bacteria belonging to the genus Brucella contain 2 types of lumazine synthases; one type has very low enzymatic activity and an unusual decametric structure that appeared to be an immunodominant antigen (556).…”

Structural and Kinetic Properties of Lumazine Synthase Isoenzymes in the Order Rhizobiales

“…17 Here, we further study this enzyme by describing its 3D crystallographic structure at 2.22 Å. Static lightscattering (SLS) experiments were also performed to complement the original data. In this sense, an average molecular weight (M r ) of 80,100 was determined (corresponding to 4.8 monomers), which is in good agreement with the 84,000 value calculated from its sequence.…”

“…Additionally, Mesorhizobium loti also encodes its own RibH1 and RibH2 enzymes, which were assigned as part of the Type-I and Type-II LSs, respectively (Table 1). 17 In contrast, Agrobacterium tumefaciens only encodes a Type-I LS that is called RibH.…”

“…16 Next, we have demonstrated that B. abortus RibH1 is pentameric in solution and also shows very low LS catalytic activity. 17 The existence of two enzymes for the same putative function in a single microorganism had prompted us to investigate this occurrence in other species and to perform an exhaustive phylogenetic analysis of the LS family. 17 This analysis allowed us to postulate the existence of two types of eubacterial, fungal and plant LS: Type I LSs, the great majority of LSs studied to date, and Type II LSs, which are present only in some eubacteria that also have a Type-I LS.…”