Sesamin is known as a specific inhibitor of Δ-desaturation, the conversion from dihoo-γ-linolenic acid (20: 3(n-6)) to arachidonic acid (20:4(n-6)). In the previous paper, we reported that sesamin ...inhibited Δ-desaturation of n-6 fatty acids in rat hepatocytes but not that of n-3 fatty acids, from 20: 4(n-3) to 20: 5(n-3). Then, we studied the effects of sesamin on Δ-desaturation of n-6 and n-3 fatty acids in vivo. Rats were fed two types of diets containing sesamin (0.5% w/w) for 4 weeks as follows: in experiment 1 (Exp. 1) γ-linolenic acid-rich diet and in experiment 2 (Exp. 2) a-linolenic acid-rich diet. The fatty acid composi-tion of liver lipids was compared to those of control groups without sesamin. In both Exps. 1 and 2, sesamin increased the liver weight and phospholipid contents in liver. In Exp. 2, sesamin increased n-6 fatty acids and decreased n-3 fatty acids even though the diet was rich in n-3 fatty acids. Sesamin enhanced the composition ratio of 20: 3(n-6) in both Exps. 1 and 2. Decrease of Δ-desaturation index of n-6 fatty acid, the ratio of 20:4(n-6)/20: 3(n-6), by the administration of sesamin suggested that sesamin inhibited the Δ-desaturation of n-6 fatty acids in the liver. On the contrary, the Δ-desaturation index of n- 3 fatty acids, the ratio of 20: 5(n-3) to 18: 3(n-3), was increased by sesamin administration in the liver of rats fed a-linolenic acid-rich diet (Exp. 2). These results sug-gested that sesamin inhibited the Δ-desaturation of n-6 fatty acids but not that of n-3 fatty acids in vivo similarly to results from rat hepatocytes.
An analysis of the ABBA family of aromatic prenyltransferases was conducted. further structural and biochemical characterization of additional members of the ABBA family of aromatic PTases, such as ...the novel fungal members, will afford a more complete understanding of the key features involved in substrate selection and specificity/promiscuity.
2-Phospho-4-(cytidine 5′-diphospho)-2-
C-methyl-
d-erythritol was transformed to 2-
C-methyl-
d-erythritol 2,4-cyclodiphosphate by a novel
Escherichia coli enzyme involved in the nonmevalonate ...pathway.
Gibberellins (GAs) are diterpene plant hormones essential for many developmental processes. Although the GA biosynthesis pathway has been well studied, our knowledge on its early stage is still ...limited. There are two possible routes for the biosynthesis of isoprenoids leading to GAs, the mevalonate (MVA) pathway in the cytosol and the methylerythritol phosphate (MEP) pathway in plastids. To distinguish these possibilities, metabolites from each isoprenoid pathway were selectively labeled with (13)C in Arabidopsis seedlings. Efficient (13)C-labeling was achieved by blocking the endogenous pathway chemically or genetically during the feed of a (13)C-labeled precursor specific to the MVA or MEP pathways. Gas chromatography-mass spectrometry analyses demonstrated that both MVA and MEP pathways can contribute to the biosyntheses of GAs and campesterol, a cytosolic sterol, in Arabidopsis seedlings. While GAs are predominantly synthesized through the MEP pathway, the MVA pathway plays a major role in the biosynthesis of campesterol. Consistent with some crossover between the two pathways, phenotypic defects caused by the block of the MVA and MEP pathways were partially rescued by exogenous application of the MEP and MVA precursors, respectively. We also provide evidence to suggest that the MVA pathway still contributes to GA biosynthesis when this pathway is limiting.
Natural rubber is synthesized as rubber particles in the latex, the fluid cytoplasm of laticifers, of Hevea brasiliensis. Although it has been found that natural rubber is biosynthesized through the ...mevalonate pathway, the involvement of an alternative 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is uncertain. We obtained all series of the MEP pathway candidate genes by analyzing expressed sequence tag (EST) information and degenerate PCR in H. brasiliensis. Complementation experiments with Escherichia coli mutants were performed to confirm the functions of the MEP pathway gene products of H. brasiliensis together with those of Arabidopsis thaliana, and it was found that 1-deoxy-D-xylulose-5-phosphate reductoisomerase, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase, and 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase of H. brasiliensis were functionally active in the E. coli mutants. Gene expression analysis revealed that the expression level of the HbDXS2 gene in latex was relatively high as compared to those of other MEP pathway genes. However, a feeding experiment with 1-sup(13)C 1-deoxy-D-xylulose triacetate, an intermediate derivative of the MEP pathway, indicated that the MEP pathway is not involved in rubber biosynthesis, but is involved in carotenoids biosynthesis in H. brasiliensis.
A gene cluster encoding five enzymes of the mevalonate pathway had been cloned from Streptomyces sp. strain CL190. This gene cluster contained an additional ORF, orfD, encoding an unknown protein ...that was detected in some archaebacteria and some Gram-positive bacteria including Staphylococcus aureus. The recombinant product of orfD was purified as a soluble protein and characterized. The molecular mass of the enzyme was estimated to be 37 kDa by SDS-polyacrylamide gel electrophoresis and 155 kDa by gel filtration chromatography, suggesting that the enzyme is most likely to be a tetramer. The purified enzyme contained flavin mononucleotide (FMN) with the amount per tetramer being 1.4 to 1.6 mol/mol. The enzyme catalyzed the isomerization of isopentenyl diphosphate (IPP) to produce dimethylallyl diphosphate (DMAPP) in the presence of both FMN and NADPH. The Escherichia coli plasmid expressing orfD could complement the disrupted IPP isomerase gene in E. coli. These results indicate that orfD encodes an unusual IPP isomerase showing no sequence similarity to those of IPP isomerases identified to date. Based on the difference in enzymatic properties, we classify the IPP isomerases into two types: Type 2 for FMN- and NAD(P)H-dependent enzymes, and type 1 for the others. In view of the critical role of this isomerase in S. aureus and of the different enzymatic properties of mammalian (type 1) and S. aureus (type 2) isomerases, this unusual enzyme is considered to be a suitable molecular target for the screening of antibacterial drugs specific to S. aureus.
The discovery of the 2-C-methyl-D-erythritol-4-phosphate pathway for the biosynthesis of isoprenoids raises the important question of the nature and regulation of the enzymes involved in this ...pathway. CLA1, a gene previously isolated from Arabidopsis, encodes the first enzyme of the 2-C-methyl-D-erythritol-4-phosphate pathway, 1-deoxy-D-xylulose-5-phosphate synthase. We demonstrate this enzyme activity by complementation of the cla1-1 mutant phenotype and by direct enzymatic assays. Based on mRNA and protein expression patterns this enzyme is expressed mainly in developing photosynthetic and non-photosynthetic tissues. The β-glucuronidase expression pattern driven from the CLA1 gene regulatory region supports the northern and protein data while also showing that this gene has some level of expression in most tissues of the plant. A mutation in the CLA1 gene interferes with the normal development of chloroplasts and etioplasts, but does not seem to affect amyloplast structure. Microscopic analysis also shows a pleiotropic effect of the CLA1 gene mutation in mesophyll tissue formation.
A gene cluster encoding five enzymes of the mevalonate pathway had
been cloned from
Streptomyces
sp. strain CL190. This
gene cluster contained an additional ORF,
orfD
, encoding
an unknown protein ...that was detected in some archaebacteria and some
Gram-positive bacteria including
Staphylococcus aureus
.
The recombinant product of
orfD
was purified as a
soluble protein and characterized. The molecular mass of the enzyme was
estimated to be 37 kDa by SDS-polyacrylamide gel electrophoresis and
155 kDa by gel filtration chromatography, suggesting that the enzyme is
most likely to be a tetramer. The purified enzyme contained flavin
mononucleotide (FMN) with the amount per tetramer being 1.4 to 1.6
mol/mol. The enzyme catalyzed the isomerization of isopentenyl
diphosphate (IPP) to produce dimethylallyl diphosphate (DMAPP) in the
presence of both FMN and NADPH. The
Escherichia coli
plasmid expressing
orfD
could complement the disrupted
IPP isomerase gene in
E. coli
. These results indicate
that
orfD
encodes an unusual IPP isomerase showing no
sequence similarity to those of IPP isomerases identified to date.
Based on the difference in enzymatic properties, we classify the IPP
isomerases into two types: Type 2 for FMN- and NAD(P)H-dependent
enzymes, and type 1 for the others. In view of the critical role of
this isomerase in
S. aureus
and of the different
enzymatic properties of mammalian (type 1) and
S. aureus
(type 2) isomerases, this unusual enzyme is considered to be a suitable
molecular target for the screening of antibacterial drugs specific to
S. aureus
.
Human Vγ2Vδ2 T cells are stimulated by prenyl pyrophosphates, such as isopentenyl pyrophosphate (IPP), and play important roles in mediating immunity against microbial pathogens and have potent ...anti-tumor activity. (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) has been identified as a metabolite in the 2-C-methyl-D-erythritol-4 phosphate (MEP) pathway for isoprenoid biosynthesis that is used by many bacteria and protozoan parasites. We find that HMBPP is the major Vγ2Vδ2 T-cell antigen for many bacteria, including Mycobacterium tuberculosis, Yersinia enterocolitica and Escherichia coli. HMBPP was a 30 000-fold more potent antigen than IPP. Using mutant bacteria, we show that bacterial antigen levels for Vγ2Vδ2 T cells are controlled by MEP pathway enzymes and find no evidence for the production of 3-formyl-1-butyl pyrophosphate. Moreover, HMBPP reactivity required only germ line-encoded Vγ2Vδ2 TCR elements and is present at birth. Importantly, we show that bacterial HMBPP levels correlated with their ability to expand Vγ2Vδ2 T cells in vivo upon engraftment into severe combined immunodeficiency–beige mice. Thus, the production of HMBPP by a microbial-specific isoprenoid pathway plays a major role in determining whether bacteria will stimulate Vγ2Vδ2 T cells in vivo. This preferential stimulation by a common microbial isoprenoid metabolite allows Vγ2Vδ2 T cells to respond to a broad array of pathogens using this pathway.
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