Phylogenetic analyses and comparative genomics of vitamin B6 (pyridoxine) and pyridoxal phosphate biosynthesis pathways

Vitamin B6 in its active form pyridoxal phosphate is an essential coenzyme of many diverse enzymes. Biochemistry, enzymology and genetics of de novo vitamin B6 biosynthesis have been primarily investigated in Escherichia coli. Database searches revealed that the key enzymes involved in ring closure of the aromatic pyridoxin ring (PdxA; PdxJ) are present mainly in genomes of bacteria constituting the gamma subdivision of proteobacteria. The distribution of DXS, a transketolase-like enzyme involved in vitamin B6 biosynthesis as well as in thiamine and isoprenoid biosynthesis and the distribution of vitamin B6 modifying enzymes (PdxH: oxidase; PdxK: kinase) was also analyzed. These enzymes are also present in the genomes of animals. Two recent papers (Ehrenshaft et al., 1999, Proc. Natl. Acad. Sci. USA. 96: 9374-9378; Osmani et al., 1999, J. Biol. Chem. 274: 23565-23569) show the involvement of an extremely conserved protein (a member of the UPF0019 or SNZ family) found in all three domains of life (bacteria, archaea, eukarya) in an alternative vitamin B6 biosynthesis pathway. Members of this family were previously identified as a stationary phase inducible protein in yeast, as an ethylene responsible protein in plants and in a marine sponge, as a singlet oxygen resistance protein in Cercospora nicotianae and as a cumene hydroperoxide and H2O2 inducible protein in Bacillus subtilis. In yeast, the SNZ protein interacts with another protein called SNO which also represents a member of a highly conserved protein family (called UPF0030 or SNO family). Phylogenetic trees for the DXS, PdxA, PdxJ, PdxH, PdxK, SNZ and SNO protein families are presented and possible implications of the two different vitamin B6 biosynthesis pathways in cellular metabolism are discussed. A radically different view of bacterial evolution (Gupta, 2000, Crit. Rev. Microbiol. 26: 111-131) which proposes a linear rather than a treelike evolutionary relationship between procaryotic species indicates that the gamma subdivision of proteobacteria represents the most recently evolved bacterial lineage. This proposal might help to explain why the PdxA/PdxJ pathway is largely restricted to this subdivision.     

Biosynthesis of vitamin B6 in Rhizobium: in vitro synthesis of pyridoxine from 1-deoxy-D-xylulose and 4-hydroxy-L-threonine

Pyridoxine (vitamin B6) in Rhizobium is synthesized from 1-deoxy-D-xylulose and 4-hydroxy-L-threonine. To define the pathway enzymatically, we established an enzyme reaction system with a crude enzyme solution of R. meliloti IFO14782. The enzyme reaction system required NAD+, NADP+, and ATP as coenzymes, and differed from the E. coli enzyme reaction system comprising PdxA and PdxJ proteins, which requires only NAD+ for formation of pyridoxine 5'-phosphate from 1-deoxy-D-xylulose 5-phosphate and 4-(phosphohydroxy)-L-threonine.     

Multistate binding in pyridoxine 5'-phosphate synthase: 1.96 A crystal structure in complex with 1-deoxy-D-xylulose phosphate

We report the 1.96 A crystal structure of pyridoxine 5'-phosphate synthase (PdxJ) in complex with 1-deoxy-D-xylulose phosphate (dXP). The octameric enzyme possesses eight distinct binding sites, and three different binding states are observed. The observation of these three states supports a mechanism in which precise conformational changes of a peptide loop and groups of active site residues modulate binding and specificity. The differences in protein conformation when one or two substrates are bound can be correlated with a condensation mechanism that leads productively to the formation of pyridoxine 5'-phosphate (PNP). "Snapshots" of the progression from the apo form to a singly occupied "transitional binding" state and, subsequently, to a fully occupied, reactive state are revealed and indicate how the enzyme structure can be related to a plausible catalytic mechanism and, moreover, to favorable energetics of reaction.     

T-T interactions in the induction of antigen-specific human suppressor T lymphocytes in vitro.

We intended to investigate whether the suppression of antigen-induced antibody responses in vitro in man by T suppressor cells required contact of T suppressor cells with target cells or whether this effect was mediated by factors released by T suppressor cells. To this end supernatants of antigen-induced T suppressor cells were tested (by a plaque forming cell assay) for their capacity to suppress antibody responses of autologous and allogeneic human peripheral blood lymphocytes. We have shown that supernatants of antigen-specific T suppressor cells, designated as TsF24: a) can suppress an antibody response of autologous but not allogeneic lymphocytes to the inducing antigen; b) are antigen-specific in their effect; and 3) are produced by radiosensitive T cells. Furthermore, the target of the factor is a radiosensitive T cell. These findings taken together indicate that, in the generation of T-effector suppressor cells in man, T-T interactions occur, and in addition, that cellfree factors may be involved in these interactions.     

A mutation in the gene for trigger factor suppresses the defect in cell division in the divE42 mutant of Escherichia coli K12

A total of sixteen spontaneously generated, independent suppressor mutants was isolated from a mutant (divE42) of Escherichia coli K12 that is defective in cell division. One of the suppressor mutants, designated TR4, had a novel phenotype: it was able to grow at 42° C but not at 32° C. The Kohara genomic library was screened for complementing clones. Clone 148 was able to complement the mutation responsible for the cold-sensitive phenotype, and the gene for trigger factor (tig), which encodes a ribosome-associated peptidyl-prolyl cis/trans isomerase, was identified as the mutated gene by deletion analysis with the insert DNA from clone 148. DNA sequencing revealed that the mutation in the tig gene of the TR4 suppressor mutant was a single nucleotide insertion (+A) at a distance of 834 nucleotides from the initiation codon for this enzyme. When the wild-type tig gene was introduced into the TR4 suppressor mutant, the bacteria were able to grow at 32° C but not at 42° C, an indication that the intergenic suppressor mutation was recessive to the wild-type allele. A model is proposed that accounts for the phenotypes of the divE42 mutant and the TR4 suppressor mutant. Received: 3 March 1998 / Accepted: 14 July 1998     

ARF tumor suppression in the nucleolus

Since its discovery close to twenty years ago, the ARF tumor suppressor has played a pivotal role in the field of cancer biology. Elucidating ARF's basal physiological function in the cell has been the focal interest of numerous laboratories throughout the world for many years. Our current understanding of ARF is constantly evolving to include novel frameworks for conceptualizing the regulation of this critical tumor suppressor. As a result of this complexity, there is great need to broaden our understanding of the intricacies governing the biology of the ARF tumor suppressor. The ARF tumor suppressor is a key sensor of signals that instruct a cell to grow and proliferate and is appropriately localized in nucleoli to limit these processes. This article is part of a Special Issue entitled: Role of the Nucleolus in Human Disease.     

Mutation probe of gene structure in E. coli: suppressor mutations in the seven-tRNA operon

Summary Cells defective in uracil-DNA glycosylase (ung:: Tn10) were used in two ways to reveal differences in select point mutations (GC to AT transitions) within the seven-tRNA operon of E. coli. The mutations were indicated as de novo or converted glutamine tRNA suppressor mutations in the genes glnU and/or glnV: (1) the kinetics of photoenzymatic monomerization of pyrimidine dimers quantitated by ung-dependent UV mutagenesis indicated more rapid repair of dimers at sites for converted suppressor mutation than of dimers at sites for de novo suppressor mutation, and (2) spontaneous deamination of cytosine was considerably more frequent at sites for converted suppressor mutation than at sites for de novo suppressor mutation. To explain these results we suggest the physical structure of the DNA in vivo is different at different sites in the seven-tRNA operon. The non-transcribed strand including specifically the anticodon region of the site for converted suppressor mutation may frequently be looped out in a single strand so that a T=C dimer is more accessible to DNA photolyase or a free cytosine residue of non-irradiated DNA is in an aqueous environment conducive to deamination. In addition, we analysed the spontaneous de novo suppressor mutation data to determine an estimate for the in vivo rate of cytosine deamination in double strand DNA of 3.2×10¹³/sec.     

A mutation in the gene for trigger factor suppresses the defect in cell division in the divE42 mutant of Escherichia coli K12

A total of sixteen spontaneously generated, independent suppressor mutants was isolated from a mutant (divE42) of Escherichia coli K12 that is defective in cell division. One of the suppressor mutants, designated TR4, had a novel phenotype: it was able to grow at 42°?C but not at 32°?C. The Kohara genomic library was screened for complementing clones. Clone 148 was able to complement the mutation responsible for the cold-sensitive phenotype, and the gene for trigger factor (tig), which encodes a ribosome-associated peptidyl-prolyl cis/trans isomerase, was identified as the mutated gene by deletion analysis with the insert DNA from clone 148. DNA sequencing revealed that the mutation in the tig gene of the TR4 suppressor mutant was a single nucleotide insertion (+A) at a distance of 834 nucleotides from the initiation codon for this enzyme. When the wild-type tig gene was introduced into the TR4 suppressor mutant, the bacteria were able to grow at 32°?C but not at 42°?C, an indication that the intergenic suppressor mutation was recessive to the wild-type allele. A model is proposed that accounts for the phenotypes of the divE42 mutant and the TR4 suppressor mutant.     

Modulation of the immunosuppressive effects of splenic macrophages in Fischer rats bearing adenocarcinoma 13762

Summary The nature of spleen cells in Fischer rats bearing a large size (>1 cm diameter) mammary adenocarcinoma 13762A (MAC) which block the immunostimulating capacities of MTP² (a synthetic immunomodulator) and suppress proliferation in vitro of splenic T and B lymphocytes by their respective mitogens was investigated. Splenic macrophages were recognized as the suppressor cells by (a) restoration of mitogenic responses by depletion of macrophages from spleen cell suspensions and (b) continued suppressor activity in spleen cell suspensions of tumor bearers devoid of viable T lymphocytes. Macrophage contact with T lymphocytes was required for the inhibition of T lymphocyte proliferation by concanavalin A as shown by (a) the absence of suppressor activity in supernatants derived from cultured suppressor macrophages, (b) lowering of the suppressor activity of intact macrophages after treatment with neuraminidase, (c) lowering of the suppressor activity of macrophages by addition of red cells to spleen cultures of tumor bearers indicating red cell interference with macrophage-T cell interaction and (d) lack of inhibiting action of suppressor macrophages on allogenic T lymphocyte proliferation showing macrophage T cell recognition for suppression.Animals bearing a large size tumor exhibited spleen hypertrophy and an increase in macrophage:lymphocyte ratio and a decrease in red cell:lymphocyte ratio. Splenic macrophages did not appear to be implicated in blocking antitumor immunity induction since (a) suppressor macrophages were absent in spleens during the inductive phase of the immune response and (b) MAC implanted in allogenic Wistar rats grew to about 2 cm diameter, induced splenic suppressor macrophages but the tumor was later rejected by the animals. Collectively the results suggest that suppressor macrophages are the result of increasing tumor volume rather than its cause.This study was supported by a grant from the National Cancer Institute of Canada Abbreviations used: Con A, Concanavalin A; LPS, lipopolysaccharide; PHA, phytohemagglutinin; MTP, maltose tetrapalmitate; MAC, mammary adenocarcinoma 13762; RPMI, Roswell Park Memorial Institute; TBR, tumor bearing rat; RBC, red blood cell     

A partial characterization of suppressor cells in rat fetal liver cells

Rat fetal liver cells (FLC) obtained at 18–20 days gestation suppressed mixed lymphocyte reactions(MLR) of adult lymph node cells. The suppression was not strain specific: both syngeneic and allogeneic FLC were capable of suppressing the MLR. The same suppressor activity was observed with fetal spleen cells but not with fetal thymus cells. Removal of phagocytic cells from FLC failed to inhibit the suppressor activity. The suppressor cells were separated into two different types by BSA density gradient: one is radiosensitive, the other radioresistant. A stronger suppressor activity was observed in radiosensitive cells. The suppressor cells belonged to the fraction agglutinated with peanut agglutinin. The data suggest that the suppressor cells in rat FLC may be a proliferating blastoid-type cell rather than mature lymphocytes or mature macrophages.     

Differential activation of cytotoxic and suppressor T cells against syngeneic tumors in the mouse.

The cytotoxic T cell against a methylcholanthrene-induced sarcoma, S1509a, was induced in syngeneic mice by deliberate immunization with mitomycin C (MMC)-treated live tumor cells. The soluble tumor antigen (STA) extracted from the same tumor by 3 M KCl was, however, unable to induce the cytotoxic T cell upon immunization, although it was able to activate predominantly the suppressor T cell that then specifically suppressed the effect of the cytotoxic T cell against the homologous tumor. The suppressor T cell generated by STA had the same characteristics as those found in tumor-bearing animals: 1) The suppressor T cell has a very strict specificity against individual tumors; 2) The cell expresses cell surface determinants controlled by genes in the I-J subregion of the mouse H-2 complex. The activity of the cytotoxic T cell was completely inhibited by live tumor cells but not by STA, whereas that of the suppressor T cell was neutralized by STA. The results that cytotoxic and suppressor T cells are activated under different conditions, and that the antigenic determinants recognizable by these two cell types are not the same. The soluble extract contains only the determinants recognizable by the suppressor T cell, and the cytotoxic T cell can be activated only by the determinants associated with self antigen present on the surface of live tumor cells.     

Structural basis for the function of pyridoxine 5'-phosphate synthase

BACKGROUND: Pyridoxal 5'-phosphate is the active form of vitamin B(6) that acts as an essential, ubiquitous coenzyme in amino acid metabolism. In Escherichia coli, the pathway of the de novo biosynthesis of vitamin B(6) results in the formation of pyridoxine 5'-phosphate (PNP), which can be regarded as the first synthesized B(6) vitamer. PNP synthase (commonly referred to as PdxJ) is a homooctameric enzyme that catalyzes the final step in this pathway, a complex intramolecular condensation reaction between 1-deoxy-D-xylulose-5'-phosphate and 1-amino-acetone-3-phosphate. RESULTS: The crystal structure of E. coli PNP synthase was solved by single isomorphous replacement with anomalous scattering and refined at a resolution of 2.0 A. The monomer of PNP synthase consists of one compact domain that adopts the abundant TIM barrel fold. Intersubunit contacts are mediated by three additional helices, respective to the classical TIM barrel helices, generating a tetramer of symmetric dimers with 422 symmetry. In the shared active sites of the active dimers, Arg20 is directly involved in substrate binding of the partner monomer. Furthermore, the structure of PNP synthase with its physiological products, PNP and P(i), was determined at 2.3 A resolution, which provides insight into the dynamic action of the enzyme and allows us to identify amino acids critical for enzymatic function. CONCLUSION: The high-resolution structures of the free enzyme and the enzyme-product complex of E. coli PNP synthase suggest essentials of the enzymatic mechanism. The main catalytic features are active site closure upon substrate binding by rearrangement of one C-terminal loop of the TIM barrel, charge-charge stabilization of the protonated Schiff-base intermediate, the presence of two phosphate binding sites, and a water channel that penetrates the beta barrel and allows the release of water molecules in the closed state. All related PNP synthases are predicted to fold into a similar TIM barrel pattern and have comparable active site architecture. Thus, a common mechanism can be anticipated.     

Mechanism for the suppression of gamma-interferon responsiveness in mice after thermal injury

As reported previously, gamma-interferon production was decreased after the administration of inducers to thermally injured mice as compared with noninjured controls. Similarly, spleen cells from injured mice had decreased ability to produce interferon in vitro after stimulation with inducers. The present study demonstrated that the decrease in interferon production was associated with the presence of suppressor cells in the spleen of burned mice that were capable of inhibiting interferon production by normal splenic lymphocytes in vitro. Passive transfer of spleen cells containing suppressor cell activity derived from injured mice induced suppression in normal mice, and the time of the appearance of suppressor cell activity in injured mouse spleens closely approximated the time of the appearance of the suppression of interferon production observed in mice after thermal injury. The suppressor cells were characterized as a population of macrophages by the following: they adhered to plastic surface and could be removed from spleen cells by carbonyl-iron treatment; treatment of plastic-adherent cells with anti-Thy-1.2 and anti-mouse immunoglobulin antisera followed by complement failed to abrogate the suppression produced by these cells.     

A defect in the suppressor circuits among OKT4+ cell populations in patients with systemic lupus erythematosus occurs independently of a defect in the OKT8+ suppressor T cell function

The autologous mixed lymphocyte reaction (MLR) is thought to be part of a regulatory role of T cells on B cell function. OKT4+, but not OKT8+, cells can proliferate in response to autologous non-T cells. Moreover, the OKT4+ cell population activated early in the course of autologous MLR functioned as inducer cells for the differentiation of B cells, whereas later in the response, the activated OKT4+ cells were particularly enriched in suppressor cells. A part of the autologous MLR appears to be an important pathway for the activation of feedback suppression mechanisms among cells contained within the OKT4+ populations. Patients with systemic lupus erythematosus (SLE) were studied with regard to the following OKT4+ cell functions in vitro after activation in the autologous MLR: a) proliferative response, and b) helper and suppressor activities for differentiation of B cells. A marked reduction in the proliferative response of OKT4+ cells was observed in SLE patients. SLE OKT4+ cells activated in the autologous MLR could function as helper cells but could not exert any suppressor activity. This OKT4+ cell abnormality was present regardless of the disease activity, and occurred in the absence of autoantibodies including anti-T cell antibodies. Instead, SLE anti-T cell antibodies could preferentially eliminate cells bearing the OKT8+ phenotype characteristic of suppressor cells in populations of normal T cells. These results suggest that the defect in the suppressor circuits among OKT4+ cell populations is intrinsic to SLE lymphocytes and that the OKT8+ suppressor T cell defect is caused by antibodies produced by the B cells of SLE patients.     

Biosynthesis of Vitamin B6 in Rhizobium: In Vitro Synthesis of Pyridoxine from 1-Deoxy-D-xylulose and 4-Hydroxy-L-threonine

Pyridoxine (vitamin B₆) in Rhizobium is synthesized from 1-deoxy-D-xylulose and 4-hydroxy-L-threonine. To define the pathway enzymatically, we established an enzyme reaction system with a crude enzyme solution of R. meliloti IFO14782. The enzyme reaction system required NAD⁺, NADP⁺, and ATP as coenzymes, and differed from the E. coli enzyme reaction system comprising PdxA and PdxJ proteins, which requires only NAD⁺ for formation of pyridoxine 5′-phosphate from 1-deoxy-D-xylulose 5-phosphate and 4-(phosphohydroxy)-L-threonine.     

Deficiencies in suppressor T cell activity seen in patients with active systemic lupus erythematosus are due to the dilution of normally functioning suppressor T cells by nonsuppressor T cells

Concanavalin A (Con A)-activated T lymphocytes from patients with active, but not inactive, systemic lupus erythematosus (SLE) failed to express normal suppressor activity, regardless of the phenotype of CD4+ or CD8+. Con A-activated CD4+ or CD8+ T lymphocytes from the SLE patients and from normal controls were further separated into two populations, using the autologous erythrocyte rosette technique. One population very rich in cells capable of forming rosettes with autologous erythrocytes from the active patients showed the same degree of suppressor activity, as did that from normal controls; the CD4+ or CD8+ population poor in autorosetting cells derived from Con A-activated T lymphocytes from both the controls and patients did not express suppressor activity. Moreover, when autorosetting T cells from the active patients and nonrosetting cells from the same patients were mixed at a normal ratio (4:6), normal suppressor activity could be restored. It was notable that the frequency of autorosette-forming cells was markedly reduced in the Con A-activated T lymphocytes from the active, but not inactive, SLE patients, regardless of the phenotype of CD4+ or CD8+. These findings indicate the presence of a normally functioning suppressor T cell population in patients with active SLE. It seems that the lack of suppressor T cell function in patients with active SLE is due to the dilution of a few normal suppressor T cells by large numbers of nonsuppressor T lymphocytes.     

Effect of three suppressors on the expression of powdery mildew resistance genes in barley

Three recessive mutagen-induced alleles that partially suppress the phenotypic expression of the semidominant powdery mildew resistance gene Mla12 have been studied. When each suppressor is present in homozygous condition, the infection type 0, conferred by gene Mla12 when homozygous, is changed to intermediate infection types. The three suppressor lines were crossed with seven near-isogenic lines with different powdery mildew resistance genes and one, M100, was crossed with nine additional lines. Seedlings of parents and from the F1and F2 generations were tested with powdery mildew isolates that possessed the appropriate avirulence and virulence genes. The segregation of phenotypes in the F2 generation disclosed that the three suppressors affected the phenotypic expression of three resistance genes, whereas that of four resistance genes remained unaffected. The suppressor in mutant M100 affected the phenotypic expression of 9 of the 10 additional resistance genes present. It is suggested that the three suppressors are mutationally modified genes involved in host defence processes. This implies that different resistance genes employ different, but overlapping, spectra of defence processes, or signal transduction pathways. Key words : barley, Hordeum vulgare, powdery mildew, Erysiphe graminis hordei, mutation, resistance, suppressor.     

Autoreactive T cells in mercury-induced autoimmunity. Ability to induce the autoimmune disease

It has been previously shown that autoreactive T cells appear during mercury-induced autoimmunity in Brown-Norway (BN) rats. In the present work, it is shown that: 1) T cells and T helper cells from HgCl2-injected BN rats are able to actively transfer autoimmunity in normal BN rats; the disease transferred is exacerbated when recipients are treated with the antisuppressor/cytotoxic T cell monoclonal antibody (OX8); 2) normal T cells preincubated with HgCl2 are also able to transfer the disease in OX8-treated but not in T cell-depleted rats; and 3) T cells from HgCl2-injected BN rats also transferred the disease in both normal and T cell depleted rats. It is concluded that: 1) autoreactive T cells, and presumably anti-Ia T cells are involved in the pathogenesis of mercury-induced autoimmunity; 2) these autoreactive T cells induce suppressor/cytotoxic T cells to proliferate in normal syngeneic recipients; the fact that this T cell subset did not proliferate in HgCl2-injected BN rats suggests that HgCl2 also affects T suppressor cells; and 3) mercury-induced autoimmunity could result from the additive effect of the emergence of autoreactive T cells and of a defect at the T suppressor level.     

Splicing of a yeast proline tRNA containing a novel suppressor mutation in the anticodon stem

The intron-containing proline tRNAUGG genes in Saccharomyces cerevisiae can mutate to suppress +1 frameshift mutations in proline codons via a G to U base substitution mutation at position 39. The mutation alters the 3' splice junction and disrupts the bottom base-pair of the anticodon stem which presumably allows the tRNA to read a four-base codon. In order to understand the mechanism of suppression and to study the splicing of suppressor pre-tRNA, we determined the sequences of the mature wild-type and mutant suppressor gene products in vivo and analyzed splicing of the corresponding pre-tRNAs in vitro. We show that a novel tRNA isolated from suppressor strains is the product of frameshift suppressor genes. Sequence analysis indicated that suppressor pre-tRNA is spliced at the same sites as wild-type pre-tRNA. The tRNA therefore contains a four-base anticodon stem and nine-base anticodon loop. Analysis of suppressor pre-tRNA in vitro revealed that endonuclease cleavage at the 3' splice junction occurred with reduced efficiency compared to wild-type. In addition, reduced accumulation of mature suppressor tRNA was observed in a combined cleavage and ligation reaction. These results suggest that cleavage at the 3' splice junction is inefficient but not abolished. The novel tRNA from suppressor strains was shown to be the functional agent of suppression by deleting the intron from a suppressor gene. The tRNA produced in vivo from this gene is identical to that of the product of an intron+ gene, indicating that the intron is not required for proper base modification. The product of the intron- gene is a more efficient suppressor than the product of an intron+ gene. One interpretation of this result is that inefficient splicing in vivo may be limiting the steady-state level of mature suppressor tRNA.     

A suppressor of mutations in the region adjacent to iterons of pSC101 ori.

Some single-base changes in a 14-bp region (the downstream region) adjacent to three repeated sequences (iterons) in pSC101 ori are very deleterious for replication. We isolated a host suppressor mutation for one of these mutations and found that the suppressor suppressed all the mutations tested in the downstream region. The nucleotide sequence of the suppressor revealed that the suppressor gene was identical to dksA, which encodes a multicopy suppressor of the heat shock gene dnaK.