Evolution of Vitamin B2 Biosynthesis: 6,7-Dimethyl-8-Ribityllumazine Synthases of Brucella

The penultimate step in the biosynthesis of riboflavin (vitaminB₂) involves the condensation of 3,4-dihydroxy-2-butanone 4-phosphatewith 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, whichis catalyzed by 6,7-dimethyl-8-ribityllumazine synthase (lumazinesynthase). Pathogenic Brucella species adapted to an intracellularlifestyle have two genes involved in riboflavin synthesis, ribH1and ribH2, which are located on different chromosomes. The ribH2gene was shown previously to specify a lumazine synthase (typeII lumazine synthase) with an unusual decameric structure anda very high K_m for 3,4-dihydroxy-2-butanone 4-phosphate. Moreover,the protein was found to be an immunodominant Brucella antigenand was able to generate strong humoral as well as cellularimmunity against Brucella abortus in mice. We have now clonedand expressed the ribH1 gene, which is located inside a smallriboflavin operon, together with two other putative riboflavinbiosynthesis genes and the nusB gene, specifying an antiterminationfactor. The RibH1 protein (type I lumazine synthase) is a homopentamercatalyzing the formation of 6,7-dimethyl-8-ribityllumazine ata rate of 18 nmol mg^–1 min^–1. Sequence comparisonof lumazine synthases from archaea, bacteria, plants, and fungisuggests a family of proteins comprising archaeal lumazine andriboflavin synthases, type I lumazine synthases, and the eubacterialtype II lumazine synthases.

INTRODUCTION

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Introduction

Vitamin B₂ (riboflavin) (compound 6 [Fig. 1]) is the precursorof flavin mononucleotide and flavin adenine dinucleotide, essentialcofactors for a wide variety of redox enzymes. Moreover, theyare involved in numerous other physiological processes involvinglight sensing, bioluminescence, circadian time keeping, andDNA repair (for a review, see reference 39). The vitamin isbiosynthesized by plants, fungi, and certain microorganismsbut must be obtained from dietary sources and/or the intestinalflora by animals.

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FIG. 1. Biosynthesis of riboflavin in eubacteria. I, GTP cyclohydrolase II; II, 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate deaminase; III, 5-amino-6-ribosylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate reductase; IV, hypothetical phosphatase; V, 3,4-dihydroxy-2-butanone 4-phosphate synthase; VI, 6,7-dimethyl-8-ribityllumazine synthase; VII, riboflavin synthase; 1, GTP; 2, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione; 3, ribulose 5'-phosphate; 4, 3,4-dihydroxy-2-butanone 4-phosphate; 5, 6,7-dimethyl-8-ribityllumazine; 6, riboflavin.

The pathways of riboflavin biosynthesis in microorganisms andplants have been reviewed recently (9, 10). The final stepsare catalyzed by 6,7-dimethyl-8-ribityllumazine synthase (lumazinesynthase [compound VI]) and riboflavin synthase (compound VII).More specifically, lumazine synthase catalyzes the condensationof 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (substrate2) 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 Escherichiacoli, Bacillus subtilis, Mycobacterium tuberculosis, and thehyperthermophile Aquifex aeolicus), archaea (Methanococcus jannaschii),fungi (Saccharomyces cerevisiae, Schizosaccharomyces pombe,and Magnaporthe grisea), and a plant (spinach) have been studiedin some detail (11, 16, 22, 32, 34, 35, 40-42, 44, 56, 57).The enzymes from fungi and from M. tuberculosis are C5-symmetrichomopentamers, and the lumazine synthases of plants, most eubacteria,and archaea are 532-symmetric, hollow capsids, which are bestdescribed as dodecamers of pentamers. The subunit folds of theseenzymes and the topology of the pentamer moieties are closelysimilar. The topologically equivalent active sites (5 in thecase of the pentameric enzymes and 60 in the case of the icosahedralenzymes) are invariably located at interfaces between adjacentsubunits in the pentamer moieties. Recently, it was found thatpentameric riboflavin synthases of archaea are closely relatedto 6,7-dimethyl-8-ribityllumazine synthases (15, 25, 45).

Brucellosis is a disease of humans and livestock that is causedby closely related Brucella species adapted to intracellularlife within the cells of a variety of mammals; the main pathogenicspecies for domestic animals are Brucella abortus, Brucellamelitensis, and Brucella suis. Goldbaum and coworkers have shownthat an 18-kDa B. abortus antigen with sequence similarity tolumazine synthases is a serological marker of active diseasein human brucellosis patients (17, 19). Immunization with theprotein has been shown to induce both cellular and humoral immuneresponses in mice. Moreover, the generation of protective immunityhas also been observed in this model (2). Hence, the 18-kDaprotein is of considerable immunologic interest and has beensuggested to be a general carrier for the engineering of subunitvaccines (36). In preliminary enzymatic studies, this proteinwas shown to catalyze the formation of 6,7-dimethyl-8-ribityllumazine(compound 5), albeit at a low rate. Recently, the catalyticproperties of this protein have been analyzed in closer detail(31). The three-dimensional structure of the 18-kDa antigenhas been studied in considerable detail by Goldbaum and coworkers,and the protein has been reported to be a homodecamer whichis best described as a D5-symmetric dimer of pentamers (31,58). The 18-kDa antigen of B. abortus is the first lumazinesynthase that has been reported to exhibit this quaternary arrangement.

Brucella spp. are known to share a genome topology characterizedby two circular chromosomes with approximate sizes of 2.1 MDa(chromosome I) and 1.2 MDa (chromosome II). The recently publishedB. abortus genome sequence indicates that the 18-kDa antigenis specified by the ribH2 gene, located on chromosome II ofB. abortus. Moreover, a second gene (designated ribH1) withsequence similarity to lumazine synthase was found on chromosomeI.

This paper describes the identification, cloning, and expressionof the ribH1 gene of B. abortus and provides a biochemical characterizationof the encoded type I lumazine synthase. Based on this study,we suggest that the decameric protein encoded by the ribH2 genebe designated type II lumazine synthase. We also describe thegenomic organization of the ribH1 and ribH2 genes and providea phylogenetic analysis of lumazine synthases and related pentamericriboflavin synthases derived from different organisms.

MATERIALS AND METHODS

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Materials and Methods

Materials. 5-Amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (substrate2) and 6,7-dimethyl-8-ribityllumazine (substrate 5) were synthesizedaccording to published procedures (1, 49). Recombinant 3,4-dihydroxy-2-butanone4-phosphate synthase of E. coli was used for preparation of3,4-dihydroxy-2-butanone 4-phosphate (substrate 4) (46).

Cloning and expression of the ribH1 gene of B. abortus. The ribH1 gene was amplified by PCR using B. abortus chromosomalDNA as the template and the oligonucleotides RibH1-Ndel-Vo 5'-ATAATAATACATATGGAGTTTCTCATGTCCAAGCAC-3'and RibH1-HindIII-Hi 5'-TATTATTATAAGCTTAGGCTCCGAATTTTTTGCGCAGGC-3'as primers. The amplicon was digested with NdeI and HindIII.The resulting fragment was purified with an agarose gel extractionkit (QIAGEN, Hilden, Germany) and was then ligated to the plasmidpT7-7 (51), which had been treated with the same restrictionenzymes. The ligation mixture was transformed into E. coli strainXL1-Blue (3), resulting in the recombinant strain XL-1-pT7-7-BARibH1.The reisolated plasmid was sequenced by the method of Sanger(47) (GATC Biotech, Konstanz, Germany) and was transformed intoE. coli BL21(DE3) competent cells (50) (Stratagene, La Jolla,CA), resulting in the recombinant E. coli strain BL21(DE3)-pT7-7-BARibH1.

Bacterial culture. E. coli BL21(DE3)-pT7-7-BARibH1 was grown to an optical density(at 600 nm) of 1.0 in LB medium containing 100 µg of ampicillinper ml at 37°C with shaking (150 rpm). An aliquot (5 ml)of this culture was diluted into 500 ml of medium, and incubationwith shaking was continued to an optical density of 1.0. Isopropyl-ß-thiogalactosidewas added to a final concentration of 1 mM, and the suspensionwas incubated for 4 h at 37°C with shaking (150 rpm).

Purification of type I lumazine synthase of B. abortus. Frozen bacterial cell mass was thawed in 50 mM potassium phosphate,pH 7.0. The suspension was ultrasonically treated and centrifuged.The supernatant was loaded on top of a Q-Sepharose column (1.8by 25 cm) that had been equilibrated with 50 mM potassium phosphate,pH 7.0. The column was developed with a linear gradient of 0.05to 1.0 M potassium phosphate, pH 7.0. Fractions were combinedand concentrated by ultrafiltration. The solution was loadedon top of a Superdex-200 column (2.6 by 60 cm), which was developedwith 100 mM potassium phosphate, pH 7.0. Fractions were combinedand concentrated by ultrafiltration.

Assay of lumazine synthase activity. Steady-state kinetic experiments were performed as describedpreviously (12). Corrected initial rates were fitted to theHill equation (equation 1) or equation 2 (see Results), whichrepresents a classical substrate inhibition model, with theprogram package Origin or the program DynaFit, version 3.28.024(33).

Analytical ultracentrifugation. Experiments were performed with an analytical ultracentrifuge,Optima XL-A, from Beckman Instruments (Palo Alto, CA), equippedwith absorbance and interference optics. Aluminum double-sectorcells equipped with quartz windows were used throughout. Theprotein concentration was monitored photometrically at 280 nm.For boundary sedimentation experiments, a solution containing100 mM potassium phosphate, pH 5.0, 300 mM sodium chloride,and 1.2 mg of protein per ml was centrifuged at 59,000 rpm and20°C. Sedimentation equilibrium experiments were performedwith a solution containing 100 mM potassium phosphate, pH 5.0,300 mM sodium chloride, and 0.4 mg of protein per ml. Sampleswere centrifuged at 10,000 rpm and 4°C for 72 h. The partialspecific volume was estimated from the amino acid composition,resulting in a value of 0.733 ml g^–1 (37).

Phylogenetic analysis. Sequences used for the phylogenetic analysis of lumazine synthaseproteins were obtained from the HMM library, the genome assignmentserver Superfamily 1.69, and the NCBI database. The amino acidsequences were aligned with the ClustalX (version 1.81) programfor multiple sequence alignment (53). Phylogenetic analysisof this alignment was inferred with the maximum-likelihood heuristicalgorithm implemented by PHYML, version 2.4.4 (21), under theJTT substitution model (26). The reliability of tree nodes wasanalyzed by generating 1,000 bootstrap trees (8).

RESULTS

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Results

Genomic organization of B. abortus lumazine synthase genes. The sequences of the complete genomes of B. abortus, B. suis,and B. melitensis were recently published (4, 23, 43) and showthat the 18-kDa lumazine synthase antigen (type II lumazinesynthase) is specified by the ribH2 gene, which is located onchromosome II. In all Brucella species analyzed, open readingframes (ORFs) with similarity to formate dehydrogenase (BMEII0588,BAb2-0534) and a sugar binding periplasmic precursor (BMEII0590)were found, and ORFs with unknown function surround the ribH2gene (Fig. 2). This group of genes does not appear to have acommon transcriptional control, as judged by analysis of theORF orientation and promoter localization. Moreover, analysisof the 5' untranslated region suggests that the ribH2 gene isunder the regulatory control of a putative RFN element thatis believed to sense the flavin mononucleotide concentration(54, 55).

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FIG. 2. Topology of riboflavin biosynthesis operons. Sequences were derived from fully sequenced genomes available from GenBank. Orientations of the genes involved in riboflavin biosynthesis or adjacent to those genes are given as arrows (not drawn to scale). Organisms (accession numbers) are as follows: Brucella meliloti 16 M chromosome I (NC 003317), Brucella suis 1330 chromosome I (NC 004310), Brucella abortus biovar 1 strain 9-941 chromosome I (NC 006932), Sinorhizobium meliloti 1021 (NC_003047), Mesorhizobium loti MAFF303099 (NC 002678), Bartonella henselae strain Houston-1 (NC 005956), Rhodopseudomonas palustris CGA009 (NC 005296), Bradyrhizobium japonicum USDA 110 (NC 004463), Caulobacter crescentus CB15 (NC_002696), Xanthomonas axonopodis pv. citri strain 306 (NC 003919), Xanthomonas oryzae KACC10331 (NC 006834), Xylella fastidiosa Temecula1 (NC_004556), Pseudomonas syringae pv. tomato strain DC3000 (NC_004578), Pseudomonas putida KT2440 (NC 002947), Methylococcus capsulatus strain Bath (NC 002977), Acinetobacter sp. ADP1 (NC 005966), Escherichia coli K-12 (NC_000913), Shigella sonnei Ss046 (NC 007384), Shigella flexneri 2a strain 301 (NC_004337), Salmonella enterica subsp. enterica serovar Choleraesuis strain SC-B67 (NC_006905), Yersinia pestis KIM (NC_004088), Photorhabdus luminescens subsp. laumondii TTO1 (NC 005126), Haemophilus influenzae 86-028NP (NC_007146), Geobacter sulfurreducens PCA (NC 002939), Aquifex aeolicus VF5 (NC 000918), Helicobacter pylori J99 (NC_000921), Candidatus (Blochmannia) floridanus (NC_005061), Buchnera aphidicola strain Sg (Schizaphis graminum) (NC_004061), Caulobacter crescentus CB15 (NC_002696), Ralstonia solanacearum GMI1000 (NC 003295), Chromobacterium violaceum ATCC 12472 (NC 005085), Azoarcus sp. EbN1 (NC 006513), Nitrosomonas europaea ATCC 19718 (NC 004757), Bordetella bronchiseptica RB50 (NC 002927), B. meliloti 16 M chromosome II (NC 003318), Brucella suis 1330 chromosome II (NC 004311), B. abortus biovar 1 strain 9-941 chromosome II (NC 006933), M. loti MAFF303099 (NC 002678), S. meliloti 1021 (NC_003047), R. palustris CGA009 (NC 005296), B. japonicum USDA 110 (NC 004463), P. syringae pv. tomato strain DC3000 (NC_004578).

Surprisingly, closer study of the three sequenced Brucella genomesrevealed the presence of a second gene locus with sequence similarityto lumazine synthase genes. This gene is located on chromosomeI and appears to be part of an operon-like gene arrangement(Fig. 2). This operon comprises four ORFs with sequence similarityto ribD (specifying a putative bifunctional deaminase/reductase),ribH (specifying a putative lumazine synthase), ribB (specifyinga putative riboflavin synthase), and nusB (specifying a putativeantitermination factor). The operon is preceded by a gene ofunknown function, possibly a transcriptional regulator, withsequence similarity to the ytcG gene of B. subtilis. The predictedroles of these gene products in the riboflavin biosynthesispathway are illustrated in Fig. 1. The B. abortus operon asa whole has similarity to the rib operons of various other eubacteria,most notably to an E. coli operon that has been studied in somedetail (42, 46, 52).

Figure 2 shows the genomic localizations of ribH genes froma variety of organisms. Besides Brucella, other organisms (Mesorhizobiumloti, Sinorhizobium meliloti, Rhodopseudomonas palustris, Bradyrhizobiumjaponicum, and Pseudomonas syringae) also have two differentORFs with sequence similarity to lumazine synthase (designatedribH1 and ribH2). This analysis could not detect any organismpresenting only the ribH2 gene; moreover, the ribH2 gene wasnever found within an operon, with the exception of in Caulobactercrescentus. In the C. crescentus genome, we found a rib operonwith a lumazine synthase resembling the ribH2 gene, whereasa gene with similarity to the ribH1 genes is located at a differentsite on the same chromosome; this putative ribH1 gene is probablynot part of a larger transcription unit. Nine organisms, includingBrucella, have shortened rib operons without ribA genes (bifunctionalGTP cyclohydrolase II/3,4-dihydroxy-2-butanone 4-phosphate synthase);isolated ribA genes are located elsewhere on chromosome I. Interestingly,Pseudomonas syringae harbors a ribA gene within a rib operonand another gene coding for an additional monofunctional GTPcyclohydrolase II. In Sinorhizobium meliloti, the genes ribD/ribBand ribH/nusB are separated by three ORFs with unknown functionsand opposite directions spanning 2,555 bp. Buchnera aphidicolais the only organism in this study that has separate genes fordeaminase and reductase (ribD1 and ribD2), though arranged intandem.

In many cases, the genes in the respective operons overlap bya few base pairs. Notably, in almost all operons (32 of 33 analyzedgenomes), the nusB gene appears downstream of the rib genes,in most cases followed by thiL, which specifies thiamine monophosphatekinase (Fig. 2). The rib genes are invariably oriented in thesame direction as nusB and thiL, although the functional relevanceof this association is still unknown.

The amino acid sequence identity between the type I and typeII lumazine synthases of B. abortus is 21% (Fig. 3). Interestingly,both lumazine synthases have higher sequence similarities withother lumazine synthases, e.g., B. subtilis (type I, 33%; typeII, 24%) and E. coli (type I, 37%; type II, 29%) than with eachother.

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FIG. 3. Sequence alignment of lumazine synthases from eubacteria and yeast and an archaeal riboflavin synthase. BabRibH2, type II lumazine synthase of B. abortus (P61711); BabRibH1, type I lumazine synthase of B. abortus (Q57DY1); BsuLS, lumazine synthase of Bacillus subtilis (P11998); AaeLS, lumazine synthase of Aquifex aeolicus (O66529); SpoLS, lumazine synthase of Schizosaccharomyces pombe (Q9UUB1); MjaRS, riboflavin synthase of Methanocaldococcus jannaschii (Q58584). Identical residues are printed in white on black, and similar residues are shaded in gray.

Quaternary structure of the type I lumazine synthase of B. abortus. For a detailed characterization, the ribH1 gene of B. abortuswas cloned into an expression plasmid under the control of aT7 RNA polymerase promoter and lacZ operator. In a recombinantE. coli strain, the expression construct directed the formationof a 17-kDa protein, as judged by sodium dodecyl sulfate-polyacrylamidegel electrophoresis (data not shown). The recombinant proteinwas purified to apparent homogeneity by anion-exchange chromatography,followed by gel permeation chromatography. Edman degradationof the N terminus of the recombinant protein resulted in thesequence MEFLMSKHEADA, in agreement with the translated ORF(Fig. 3), thus confirming the integrity of the protein sequenceat the N terminus.

The type I lumazine synthase of B. abortus sediments at an apparentvelocity of 5.9S at 20°C (Fig. 4A). In comparison, it shouldbe noted that pentameric lumazine synthases from the yeastsS. cerevisiae and S. pombe have similar apparent sedimentationcoefficients, of 5.5S and 5.0S, respectively (11, 42). Sedimentationequilibrium experiments indicated a molecular mass of 88 kDa,with an ideal monodisperse model used for calculation (Fig.4B). The calculated subunit molecular mass of 17,599 Da indicatesa pentameric mass of 88 kDa, in excellent agreement with theexperimental data.

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FIG. 4. Analytical ultracentrifugation analysis of the type I lumazine synthase of B. abortus. (A) For boundary sedimentation centrifugation analysis, a solution containing 100 mM potassium phosphate, pH 5.0, 300 mM sodium chloride, and 1.2 mg of protein per ml was centrifuged at 59,000 rpm and 20°C for 3 h. The protein concentration was monitored photometrically (280 nm) at intervals of 5 min. (B) For sedimentation equilibrium centrifugation analysis, a solution containing 100 mM potassium phosphate, pH 5.0, 300 mM sodium chloride, and 0.4 mg of protein per ml was centrifuged at 10,000 rpm and 4°C for 72 h.

Functional characterization of the type I lumazine synthase of B. abortus. Enzymatic studies show that the type I lumazine synthase catalyzesthe formation of 6,7-dimethyl-8-ribityllumazine (substrate 5)at a rate of about 18 ± 2 nmol mg^–1 min^–1at 37°C (Table 1). Notably, this value is about 1 orderof magnitude below the catalytic rates of lumazine synthasesfrom other mesophilic microorganisms and from spinach (Table1). The kinetic data for the substrate, 3,4-dihydroxy-2-butanone4-phosphate (substrate 4), were fitted to the Hill equation(equation 1),

Formula 1 (1)
whereV is the reaction velocity, V_max is the maximum reaction velocity,S is the substrate concentration, K is the reaction constant,and n is the Hill coefficient, resulting in a K value of 125± 10 µM for substrate 4.

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TABLE 1. Properties of lumazine synthases

Steady-state kinetic experiments showed a decreasing reactionvelocity at high concentrations of the substrate 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione.The data could be best approximated with the model representedby equation 2, indicating a K_m of 90 ± 16 µM andan inhibition constant of 370 ± 70 µM (Table 1):

Formula 2 (2)
where V is the reaction velocity,V_max is the maximum reaction velocity, S is the concentrationof substrate 2, K_m is the Michaelis-Menten constant, and K_Sis the substrate inhibition constant.

As shown in Table 1, the affinity for 3,4-dihydroxy-2-butanone4-phosphate (substrate 4) is in the same range as the valuesreported for other lumazine synthases. In contrast, the K_m valuefor substrate 2 is about 10 times higher than that observedfor other orthologs.

Table 2 summarizes the genomic localization and the structuraland biochemical characterization of B. abortus type I and typeII lumazine synthases.

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TABLE 2. Summary of genomic, structural, and biochemical knowledge about B. abortus lumazine synthases

Structure-based phylogenetic analysis of lumazine synthases. We conducted a phylogenetic analysis comparing atypical, duplicatedgenes of archaea and bacteria with structurally characterizedpentameric and icosahedral lumazine synthases. From this analysis,depicted in Fig. 5, a completely new picture emerges, showingthat in addition to eubacterial type I lumazine synthases andarchaeal lumazine synthases, this folding gave origin to atleast two new evolutionarily related functions: the archaealriboflavin synthases, which are lumazine synthase-like riboflavinsynthases with no detectable lumazine synthase activity (13,15, 45), and the eubacterial type II lumazine synthases.

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FIG. 5. Phylogenetic analysis of the quaternary arrangements of lumazine synthases or related pentameric riboflavin synthases derived from different organisms, showing a distribution into four clearly defined branches: (i) type II lumazine synthases (decameric), (ii) archaeal pentameric riboflavin synthases, (iii) archaeal lumazine synthases, and (iv) type I eubacterial, fungal, and plant lumazine synthases. The corresponding organisms are indicated as follows (references describing the structures of the enzymes involved in this study are given in brackets): Aae, A. aeolicus (lumazine synthase [56, 57]); Afu, Archaeoglobus fulgidus; Ape, Aeropyrum pernix; Bab, B. abortus; Basu, B. subtilis (lumazine synthase [34, 35]); Bja, Bradyrhizobium japonicum; Bme, B. melitensis; Bsu, B. suis (RibH2 protein [58]); Ccr, Caulobacter crescentus; Eco, E. coli (lumazine synthase [42]); Fac, Ferroplasma acidarmanus; Mac, Methanosarcina acetivorans; Mgr, M. grisea (lumazine synthase [44]); Mja, Methanococcus jannaschii (riboflavin synthase [15, 45], lumazine synthase [22]); Mka, Methanopyrus kandleri; Mlo, M. loti; Mma, Methanosarcina mazei; Mth, Methanobacterium thermoautotrophicum; Mtu, M. tuberculosis (lumazine synthase [41]); Nfa, Nocardia farcinica; Pae, Pyrobaculum aerophilum; Pfl, Pseudomonas fluorescens; Pfu, Pyrococcus furiosus; Psy, P. syringae; Rpa, R. palustris; Sce, S. cerevisiae (lumazine synthase [40]); Sipo, Silicibacter pomeroyi; Sme, S. meliloti; Sol, S. oleracea (lumazine synthase [44]); Spo, S. pombe (lumazine synthase [16, 32]); Sso, Sulfolobus solfataricus; Sto, Sulfolobus tokodaii.

Eubacteria that harbor a ribH2 gene have a type I lumazine synthaseof apparent pentameric arrangement, with the exception of thatof Pseudomonas syringae, which is clustered with the icosahedrallumazine synthases. In addition, pentameric type I lumazinesynthases from some of the ${alpha}$ -Proteobacteria (Brucella, Rhizobium,and Rhodobacter) diverge from pentameric fungal and yeast lumazinesynthases (S. cerevisiae, S. pombe, and M. grisea).

DISCUSSION

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Discussion

Genomic analysis of Brucella identified two related ORFs (ribH1and ribH2) coding for two proteins designated type I and typeII lumazine synthases, respectively. The similarity betweenthe type I and type II lumazine synthases, which are locatedon two different chromosomes, is quite low (Fig. 3). Hence,we must assume that either the separation of both lumazine synthasegenes occurred very early in evolution or one of the ribH geneswas acquired prior to ${alpha}$ -proteobacterial speciation by lateralgene transfer. At this time, there is no experimental evidencethat would permit a decision.

Recent studies have shown the existence of a family of pentamericlumazine synthase-like riboflavin synthases (Fig. 5 and 6).These enzymes have been found exclusively in archaea, whichhave also been shown to be devoid of riboflavin synthases ofthe trimeric eubacterium/yeast/plant type. The cavity harboringthe active site of the pentameric riboflavin synthase of M.jannaschii is similar to that of the lumazine synthases. Thebinding mode of the acceptor lumazine molecule in pentamericriboflavin synthase closely resembles the pyrimidinedione-bindingsite of the lumazine synthases (Fig. 6). Notably, these archaealriboflavin synthases are evolutionarily old and have no detectablelumazine synthase activity (13, 15, 45). Completely sequencedarchaeal genomes typically comprise sets of two similar genes,coding for a lumazine synthase-like riboflavin synthase anda regular lumazine synthase. Interestingly, present-day lumazinesynthases have retained the capacity to bind riboflavin. Forexample, the lumazine synthase of the yeast S. pombe is yellowcolored due to the presence of a tightly bound riboflavin (11).

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FIG. 6. Structural comparison of type I and type II lumazine synthases and archaeal riboflavin synthase. (Top) Structural superposition of riboflavin synthase from M. jannaschii (MjaRS; PDB entry code 2B99 [45]), type I lumazine synthase from S. pombe (SpoLS; PDB entry code 1KYZ [16]), and type II lumazine synthase from B. abortus (BabRibH2; PDB entry code 1T13 [31]). The active sites are formed by two adjacent monomers of SpoLS with bound riboflavin (green; residues E17 to D112 and S113 to L158), BabRibH2 with bound 5-nitro-6-(D-ribitylamino)-2,4(1H,3H)-pyrimidinedione (gray; residues S12 to E106 and T107 to L156), and MjaRS with bound 6,7-dioxo-8-ribityllumazine, resembling the acceptor lumazine molecule (red; residues T2 to M90 and T91 to Y135). Secondary structure element labeling refers to SpoLS. The five topologically equivalent active sites of pentameric lumazine synthases are located at the interfaces between adjacent monomers of the pentamer; two of them (A and B) are shown. Ligands are drawn in the respective colors. (Bottom) Enlarged ligand-binding sites of all three enzymes, with Trp27 from SpoLS, Trp22 from BabRibH2, and Phe12 from MjaRS.

The situation in the archaea is comparable to that in the eubacteria,with type I and type II lumazine synthases as we describe inthe present work (Fig. 5). However, there is no evidence thateither type I or type II lumazine synthase has any riboflavinsynthase activity (results not shown).

It should be noted that the eubacterial type I lumazine synthasesthat have been reported in the literature have low catalyticactivities, in the range of about 200 to 300 nmol mg^–1min^–1 when assayed near the optimum growth temperatureof the cognate species (Table 1). By comparison with these data,the activity of the B. abortus type I lumazine synthase is morethan 10-fold lower.

The generally low catalytic activity of lumazine synthases doesnot result from a particularly large free energy barrier ofthe reaction catalyzed. Quite to the contrary, the condensationof 3,4-dihydroxy-2-butanone 4-phosphate (substrate 4) and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione(substrate 2) is characterized by such a low energy barrierthat it can proceed at an appreciable rate at room temperaturein aqueous solution and at neutral pH (30). Thus, it is mandatorythat blank assays without enzyme be run in order to correctfor the contribution of the uncatalyzed reaction in lumazinesynthase activity measurements (30). In the case of the lumazinesynthase of B. abortus, with its particularly low catalyticrate, correction for the contribution of the uncatalyzed reactionbecomes most important.

The low catalytic activities of the lumazine synthases are inno way unique among the enzymes of the riboflavin pathway. Allactivity values for the entire riboflavin pathways in E. coli,B. subtilis, and yeast are in the range of nanomoles per milligramper minute (5-7, 11, 12, 14, 20, 24, 28, 38, 46, 48). Since,at least in the case of lumazine synthase and riboflavin synthase,the inherent free energy barriers of the catalyzed reactionscannot be the reason for these low rates, we must assume, forlack of other arguments, that the selective pressure controllingthis pathway favors the evolution of catalysts with low reactionrates. In fact, riboflavin is required in only small amounts,and excess production would unnecessarily deplete the precursorpools. Still, it remains an open question why the catalyticactivity of the B. abortus type I enzyme is at the lower endof all documented lumazine synthases.

An earlier study showed that the decameric arrangement of typeII B. abortus lumazine synthase is related to a very high K_mfor 3,4-dihydroxy-2-butanone 4-phosphate (substrate 4) (31).The in vivo concentration of 3,4-dihydroxy-2-butanone 4-phosphateis unknown. However, unless we assume that it is in the samenumerical range as the K_m of the type II enzyme, we must assumethat the bulk of the 6,7-dimethyl-8-ribityllumazine (substrate6) would be generated by the enzyme with the lower K_m value,i.e., the type I lumazine synthase, whereas the type II enzymecould at best supply a minor amount of the overall riboflavinproduction.

Thus, the question of which selective pressures could have preventedthe loss of the ribH2 gene (in case both lumazine synthase genesare evolutionarily old in Brucella-related organisms) or couldhave favored its more recent acquisition by horizontal genetransfer is still open. Interestingly, the type II lumazinesynthase is an immunodominant antigen of B. abortus, and thereis unpublished evidence that links this protein to Brucellavirulence, suggesting that the type II lumazine synthase hasevolved for a new, as-yet-unknown function.

ACKNOWLEDGMENTS

This work was supported by a Howard Hughes Medical Instituteinternational grant (to F.A.G.) and by a grant from the AgenciaNacional de Promoción Científica y Tecnológica(ANPCyT) República Argentina. F.A.G. is a member of theresearch career of CONICET, and V.Z. and S.K. are recipientsof a fellowship from CONICET. This work was supported by theFonds der Chemischen Industrie and the Hans-Fischer-GesellschafteV. M.F. and F.A.G. acknowledge support for exchange visitsbetween laboratories by the BMBF and SECyT (project ARG 04/Z06).

We acknowledge R. Ugalde, D. Comerci, and Ines Marchesini forgenomic B. abortus DNA and for early genomic analysis and DianaPosadas for helping us in the phylogenetic analysis.

FOOTNOTES

* Corresponding author. Mailing address for Fernando Alberto Goldbaum: Patricias Argentinas 435 (C1405BWE), Buenos Aires, Argentina. Phone: (5411) 5238-7500. Fax: (5411) 5238-7501. E-mail: fgoldbaum{at}leloir.org.ar. Mailing address for Markus Fischer: Lehrstuhl für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstr. 4, D-85747 Garching, Germany. Phone: 49-89-289-13336. Fax: 49-89-289-13363. E-mail: markus.fischer{at}ch.tum.de.

REFERENCES

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