Evolution of vitamin B2 biosynthesis. A novel class of riboflavin synthase in Archaea

The open reading frame MJ1184 of Methanococcus jannaschii with similarity to riboflavin synthase of Methanothermobacter thermoautotrophicus was cloned into an expression vector but was poorly expressed in an Escherichia coli host strain. However, a synthetic open reading frame that was optimized for expression in E.coli directed the synthesis of abundant amounts of a protein with an apparent subunit mass of 17.5 kDa. The protein was purified to apparent homogeneity. Hydrodynamic studies indicated a relative mass of 88 kDa suggesting a homopentamer structure. The enzyme was shown to catalyze the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine at a rate of 24 nmol mg(-1) min(-1) at 40 degrees C. Divalent metal ions, preferably manganese or magnesium, are required for maximum activity. In contrast to pentameric archaeal type riboflavin synthases, orthologs from plants, fungi and eubacteria are trimeric proteins characterized by an internal sequence repeat with similar folding patterns. In these organisms the reaction is achieved by binding the two substrate molecules in an antiparallel orientation. With the enzyme of M.jannaschii, 13C NMR spectroscopy with 13C-labeled 6,7-dimethyl-8-ribityllumazine samples as substrates showed that the regiochemistry of the dismutation reaction is the same as observed in eubacteria and eukaryotes, however, in a non-pseudo-c2 symmetric environment. Whereas the riboflavin synthases of M.jannaschii and M.thermoautotrophicus are devoid of similarity with those of eubacteria and eukaryotes, they have significant sequence similarity with 6,7-dimethyl-8-ribityllumazine synthases catalyzing the penultimate step of riboflavin biosynthesis. 6,7-Dimethyl-8-ribityllumazine synthase and the archaeal riboflavin synthase appear to have diverged early in the evolution of Archaea from a common ancestor. Some Archaea have eubacterial type riboflavin synthases which may have been acquired by lateral gene transfer.     

Biosynthesis of riboflavin: cloning, sequencing, and expression of the gene coding for 3,4-dihydroxy-2-butanone 4-phosphate synthase of Escherichia coli.

3,4-Dihydroxy-2-butanone 4-phosphate is biosynthesized from ribulose 5-phosphate and serves as the biosynthetic precursor for the xylene ring of riboflavin. The gene coding for 3,4-dihydroxy-2-butanone 4-phosphate synthase of Escherichia coli has been cloned and sequenced. The gene codes for a protein of 217 amino acid residues with a calculated molecular mass of 23,349.6 Da. The enzyme was purified to near homogeneity from a recombinant E. coli strain and had a specific activity of 1,700 nmol mg-1 h-1. The N-terminal amino acid sequence and the amino acid composition of the protein were in agreement with the deduced sequence. The molecular mass as determined by ion spray mass spectrometry was 23,351 +/- 2 Da, which is in agreement with the predicted mass. The previously reported loci htrP, "luxH-like," and ribB at 66 min of the E. coli chromosome are all identical to the gene coding for 3,4-dihydroxy-2-butanone 4-phosphate synthase, but their role had not been hitherto determined. Sequence homology indicates that gene luxH of Vibrio harveyi and the central open reading frame of the Bacillus subtilis riboflavin operon code for 3,4-dihydroxy-2-butanone 4-phosphate synthase.     

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.     

Evolution of vitamin B2 biosynthesis: 6,7-dimethyl-8-ribityllumazine synthases of Brucella

The penultimate step in the biosynthesis of riboflavin (vitamin B2) 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 Km 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.     

蜡样芽孢杆菌ATCC14579核黄素操纵子的克隆及在枯草芽孢杆菌中的表达

利用BLAST从B．cereus ATCC14579的基因组中找到一段与枯草芽孢杆茵核黄素操纵子具有较高相似性的4．6kb大小的基因组DNA片段,该片段中含有完整的核黄素操纵子。该操纵子结构基因的编码产物的氨基酸序列与枯草芽孢杆菌核黄素操纵子相应结构基因的编码产物的氨基酸序列具有99％的同源性。该片段被克隆到大肠杆茵一枯草芽孢杆茵穿梭载体pHP13M中。表达分析的结果表明B．cereus ATCC14579核黄素操纵子可在大肠杆茵和枯草芽孢杆菌中表达。利用PCR方法用来自枯草杆菌的sac B基因的启动子替换B．cereus ATCC14579核黄素操纵子原有的启动子使其更好表达。替换启动子后的核黄素操纵子在本文使用的发酵条件下有较好的表达,核黄素产量从39．5mg／L增加到61．7mg/L.     

Crystal structure of an archaeal pentameric riboflavin synthase in complex with a substrate analog inhibitor: stereochemical implications

Whereas eubacterial and eukaryotic riboflavin synthases form homotrimers, archaeal riboflavin synthases from Methanocaldococcus jannaschii and Methanothermobacter thermoautrophicus are homopentamers with sequence similarity to the 6,7-dimethyl-8-ribityllumazine synthase catalyzing the penultimate step in riboflavin biosynthesis. Recently it could be shown that the complex dismutation reaction catalyzed by the pentameric M. jannaschii riboflavin synthase generates riboflavin with the same regiochemistry as observed for trimeric riboflavin synthases. Here we present crystal structures of the pentameric riboflavin synthase from M. jannaschii and its complex with the substrate analog inhibitor, 6,7-dioxo-8-ribityllumazine. The complex structure shows five active sites located between adjacent monomers of the pentamer. Each active site can accommodate two substrate analog molecules in anti-parallel orientation. The topology of the two bound ligands at the active site is well in line with the known stereochemistry of a pentacyclic adduct of 6,7-dimethyl-8-ribityllumazine that has been shown to serve as a kinetically competent intermediate. The pentacyclic intermediates of trimeric and pentameric riboflavin synthases are diastereomers.     

Isoleucyl-tRNA synthetase of Methanobacterium thermoautotrophicum Marburg. Cloning of the gene, nucleotide sequence, and localization of a base change conferring resistance to pseudomonic acid

The ileS gene encoding the isoleucyl-tRNA synthetase of the thermophilic archaebacterium Methanobacterium thermoautotrophicum Marburg was isolated and sequenced. ileS was closely flanked by an unknown open reading frame and by purL and thus is arranged differently from the organizations observed in several eubacteria or in Saccharomyces cerevisiae. The deduced amino acid sequence of isoleucyl-tRNA synthetase was compared with primary sequences of isoleucyl-, valyl-, leucyl-, and methionyl-tRNA synthetases from eubacteria and yeast. The archaebacterial enzyme fitted well into this group of enzymes. It contained the two short consensus sequences observed in class I aminoacyl-tRNA synthetases as well as regions of homology with enzymes of the isoleucine family. Comparison between the isoleucyl-tRNA synthetases of M. thermoautotrophicum yielded 36% amino acid identity with the yeast enzyme and 32% identity with the corresponding enzyme from Escherichia coli. The ileS gene of the pseudomonic acid-resistant M. thermoautotrophicum mutant MBT10 was also sequenced. The mutant enzyme had undergone a glycine to aspartic acid transition at position 590, in a conserved region comprising the KMSKS consensus sequence. The inhibition constants of pseudomonic acid, KiIle and KiATP, for the mutant enzyme were 10-fold higher than those determined for the wild-type enzyme. Both the mutant and the wild-type ileS gene were expressed in E. coli, and their products displayed the expected difference in sensitivity toward pseudomonic acid.     

枯草芽孢杆菌核黄素操纵子与ribC基因的修饰与遗传效应

[目的]研究核黄素操纵子(rib)组成型高表达,以及ribC基因低水平表达对枯草芽孢杆菌过量合成核黄素的影响.[方法]在染色体原位修饰启动子,用mRNA稳定子替换mRNA前导区,使rib操纵子组成型高表达;修饰ribC基因的启动子,降低ribC基因的表达水平.采用qRT-PCR方法,表征基因的相对表达水平;通过摇瓶发酵,测定重组菌的生物量和核黄素产量,表征相关基因修饰所表现的遗传效应.[结果]用gsiB mRNA稳定子替换核黄素操纵子的mRNA前导区,使其相对表达水平提高了约1 500倍.ribC基因启动子-35区的首个碱基由“T”突变为“C”,使ribC基因的表达水平下降了97％以上.得到的重组菌株LX34在补加蔗糖20 g/L的LB培养基上摇瓶发酵36 h,可积累核黄素2.1 g/L,同时生物量没有明显下降.[结论]使用gsiB mRNA稳定子,能够有效地提高目标基因或操纵子的表达水平;启动子-35区首个碱基的点突变,能够有效降低ribC基因的表达水平;rib操纵子过量表达和ribC基因低水平表达,使细胞能够过量合成并积累核黄素.     

Comparative studies of ribosomal proteins and their genes from Methanococcus vannielii and other organisms

Using data from a partial protein sequence analysis of ribosomal proteins derived from the archaebacterium Methanococcus vannielii, oligonucleotide probes were synthesized. The probes enabled us to localize several ribosomal protein genes and to determine their nucleotide sequences. The amino acid sequences that were deduced from the genes correspond to proteins L12 and L10 from the rif operon, according to the genome organization in Escherichia coli, and to proteins L23 and L2, which have comparable locations, as in the Escherichia coli S10 operon. Various degrees of similarity were found when the four proteins were compared with the corresponding ribosomal proteins of prokaryotic or eukaryotic organisms. The highest sequence homology was found in counterparts from other archaebacteria, such as Halobacterium marismortui, Halobacterium halobium, or Sulfolobus. In general, the M. vannielii protein sequences were more related to the eukaryotic kingdom than to the Gram-positive or Gram-negative eubacteria. On the other hand, the organization of the ribosomal protein genes clearly follows the operon structure of the Escherichia coli genome and is different from the monocistronic eukaryotic gene arrangements. The protein coding regions were not interrupted by introns. Furthermore, the Shine-Dalgarno type sequences of methanogenic bacteria are homologous with those of eubacteria, and also their terminator regions are similar.     

The structure of the N-terminal domain of riboflavin synthase in complex with riboflavin at 2.6A resolution

Riboflavin synthase of Escherichia coli is a homotrimer with a molecular mass of 70 kDa. The enzyme catalyzes the dismutation of 6,7-dimethyl-8-(1'-D-ribityl)-lumazine, affording riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. The N-terminal segment (residues 1-87) and the C-terminal segment (residues 98-187) form beta-barrels with similar fold and a high degree of sequence similarity. A recombinant peptide comprising amino acid residues 1-97 forms a dimer, which binds riboflavin with high affinity. Here, we report the structure of this construct in complex with riboflavin at 2.6A resolution. It is demonstrated that the complex can serve as a model for ligand-binding in the native enzyme. The structure and riboflavin-binding mode is in excellent agreement with structural information obtained from the native enzyme from Escherichia coli and riboflavin synthase from Schizosaccharomyces pombe. The implications for the binding specificity and the regiospecificity of the catalyzed reaction are discussed.     

Use of the riboflavin synthase gene (ribC) as a model for development of an essential gene disruption and complementation system for Haemophilus influenzae

We have developed a system for rapid and reliable assessment of gene essentiality in Haemophilus influenzae Rd strain KW20. We constructed two "suicide" complementation vectors (pASK5 and pASK6) containing 5' and 3' regions of the nonessential ompP1 gene flanking a multiple cloning site and a selectable marker (a chloramphenicol resistance gene or a tetracycline resistance cassette). Transformation of H. influenzae with the complementation constructs directs chromosomal integration of a gene of interest into the ompP1 locus, where the strong, constitutive ompP1 promoter drives its expression. This single-copy, chromosome-based complementation system is useful for confirming the essentiality of disrupted genes of interest. It allows genetic analysis in a background free of interference from any upstream or downstream genetic elements and enables conclusive assignment of essentiality. We validated this system by using the riboflavin synthase gene (ribC), a component of the riboflavin biosynthetic pathway. Our results confirmed the essentiality of ribC for survival of H. influenzae Rd strain KW20 and demonstrated that a complementing copy of ribC placed under control of the ompP1 promoter reverses the lethal phenotype of a strain with ribC deleted.     

A genome phylogeny for mitochondria among alpha-proteobacteria and a predominantly eubacterial ancestry of yeast nuclear genes

Analyses of 55 individual and 31 concatenated protein data sets encoded in Reclinomonas americana and Marchantia polymorpha mitochondrial genomes revealed that current methods for constructing phylogenetic trees are insufficiently sensitive (or artifact-insensitive) to ascertain the sister of mitochondria among the current sample of eight alpha-proteobacterial genomes using mitochondrially-encoded proteins. However, Rhodospirillum rubrum came as close to mitochondria as any alpha-proteobacterium investigated. This prompted a search for methods to directly compare eukaryotic genomes to their prokaryotic counterparts to investigate the origin of the mitochondrion and its host from the standpoint of nuclear genes. We examined pairwise amino acid sequence identity in comparisons of 6,214 nuclear protein-coding genes from Saccharomyces cerevisiae to 177,117 proteins encoded in sequenced genomes from 45 eubacteria and 15 archaebacteria. The results reveal that approximately 75% of yeast genes having homologues among the present prokaryotic sample share greater amino acid sequence identity to eubacterial than to archaebacterial homologues. At high stringency comparisons, only the eubacterial component of the yeast genome is detectable. Our findings indicate that at the levels of overall amino acid sequence identity and gene content, yeast shares a sister-group relationship with eubacteria, not with archaebacteria, in contrast to the current phylogenetic paradigm based on ribosomal RNA. Among eubacteria and archaebacteria, proteobacterial and methanogen genomes, respectively, shared more similarity with the yeast genome than other prokaryotic genomes surveyed.     

Characterization of Actinobacillus pleuropneumoniae riboflavin biosynthesis genes.

In this paper, we report the identification, cloning, and complete nucleotide sequence of four genes from Actinobacillus pleuropneumoniae that are involved in riboflavin biosynthesis. The cloned genes can specify production of large amounts of riboflavin in Escherichia coli, can complement several defined genetic mutations in riboflavin biosynthesis in E. coli, and are homologous to riboflavin biosynthetic genes from E. coli, Haemophilus influenzae, and Bacillus subtilis. The genes have been designated A. pleuropneumoniae ribGBAH because of their similarity in both sequence and arrangement to the B. subtilis ribGBAH operon.     

Sequence of the gene for ribosomal protein L23 from the archaebacterium Methanococcus vannielii

The N-terminal sequence of HPLC-purified protein L23 from the Methanococcus vannielii ribosome has been determined by automated liquid-phase Edman degradation. Using the N-terminal amino acid sequence, an oligonucleotide probe complementary to the 5'-end of the gene was synthesized. The 26-mer oligonucleotide, containing two inosines, was used for hybridization with digested M. vannielii chromosomal DNA. The hybridizing band from HpaII-digested genomic DNA was ligated into pUC18 to yield plasmid pMvaZ1 containing the entire gene of protein L23. The nucleotide sequence complemented the partial amino acid sequence, and the gene codes for a protein of 9824 Da. The amino acid sequence of protein L23 form M. vannielii was compared to that of ribosomal proteins from other archaebacteria as well as from eubacteria and eukaryotes. The number of identical amino acids is highest when the M. vannielii protein is compared to the homologous protein from yeast and lowest vs that from tobacco chloroplasts. Interestingly, the secondary structures of the proteins as predicted by computer programs are more conserved than the primary structures.     

The nucleotide sequence of the HEM1 gene and evidence for a precursor form of the mitochondrial 5-aminolevulinate synthase in Saccharomyces cerevisiae

The biosynthesis of yeast 5-aminolevulinate (ALA) synthase, a mitochondrial protein encoded by the nuclear HEM1 gene, has been studied in vitro in a cell-free translation system and in vivo in whole cells. In vitro translation of mRNA hybrid-selected by the cloned HEM1 gene, or of total RNA followed by immunoprecipitation with anti-(ALA synthase) antibody yielded a single polypeptide of higher molecular mass than the purified ALA synthase. This larger form, also seen in pulse-labeled cells, can be post-translationally processed by isolated mitochondria. These results show that the cytoplasmically made ALA synthase is synthesized with a cleavable extension which was estimated to be about 3.5 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The complete nucleotide sequence of the HEM1 gene and its flanking regions was determined. The 5' ends of the HEM1 mRNAs map from -76 to -63 nucleotides upstream of the translation initiation codon. The open reading frame of 1644 base pairs encodes a protein of 548 amino acids with a calculated Mr of 59,275. The predicted amino-terminal sequence of the protein is strongly basic (five basic and no acidic amino acids within the first 35 residues), rich in serine and threonine and must represent the transient presequence that targets this protein to the mitochondria. Comparison of deduced amino acid sequences indicates a clear homology between the mature yeast and chick embryo liver ALA synthases.     

Biosynthesis of vitamin B2: diastereomeric reaction intermediates of archaeal and non-archaeal riboflavin synthases

The dismutation of 6,7-dimethyl-8-ribityllumazine catalyzed by riboflavin synthase affords riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. A pentacyclic adduct of two 6,7-dimethyl-8-ribityllumazines has been identified earlier as a catalytically competent reaction intermediate of the Escherichia coli enzyme. Acid quenching of reaction mixtures of riboflavin synthase of Methanococcus jannaschii, a paralog of 6,7-dimethyl-8-ribityllumazine synthase devoid of similarity with riboflavin synthases of eubacteria and eukaryotes, afforded a compound whose optical absorption and NMR spectra resemble that of the pentacyclic E. coli riboflavin synthase intermediate, whereas the circular dichroism spectra of the two compounds have similar envelopes but opposite signs. Each of the compounds could serve as a catalytically competent intermediate for the enzyme by which it was produced, but not vice versa. All available data indicate that the respective pentacyclic intermediates of the M. jannaschii and E. coli enzymes are diastereomers.     

Isolation of the GSY1 gene encoding yeast glycogen synthase and evidence for the existence of a second gene

Glycogen synthase preparations from Saccharomyces cerevisiae contained two polypeptides of molecular weights 85,000 and 77,000. Oligonucleotides based on protein sequence were utilized to clone a S. cerevisiae glycogen synthase gene, GSY1. The gene would encode a protein of 707 residues, molecular mass 80,501 daltons, with 50% overall identity to mammalian muscle glycogen synthases. The amino-terminal sequence obtained from the 85,000-dalton species matched the NH2 terminus predicted by the GSY1 sequence. Disruption of the GSY1 gene resulted in a viable haploid with glycogen synthase activity, and purification of glycogen synthase from this mutant strain resulted in an enzyme that contained the 77,000-dalton polypeptide. Southern hybridization of genomic DNA using the GSY1 coding sequence as a probe revealed a second weakly hybridizing fragment, present also in the strain with the GSY1 gene disrupted. However, the sequences of several tryptic peptides derived from the 77,000-dalton polypeptide were identical or similar to the sequence predicted by the GSY1 gene. The data are explained if S. cerevisiae has two glycogen synthase genes encoding proteins with significant sequence similarity The protein sequence predicted by the GSY1 gene lacks the extreme NH2-terminal phosphorylation sites of the mammalian enzymes. The COOH-terminal phosphorylated region of the mammalian enzyme over-all displayed low identity to the yeast COOH terminus, but there was homology in the region of the mammalian phosphorylation sites 3 and 4. Three potential cyclic AMP-dependent protein kinase sites are located in this region of the yeast enzyme. The region of glycogen synthase likely to be involved in covalent regulation are thus more variable than the catalytic center of the molecule.