Evidence for involvement of the phenylpropanoid pathway in the biosynthesis of the norlignan agatharesinol

In order to study the biosynthesis of agatharesinol, a norlignan, l-phenylalanine-[ring-2,3,4,5,6-2H] and trans-cinnamic acid-[ring-13C6] were administered to fresh sapwood sticks of Cryptomeria japonica (sugi, Japanese cedar), that is, the labeled precursors were allowed to be absorbed through the tangential section of the wood sticks. The wood sticks were then maintained in high humidity desiccators for approximately 20 d after which ethyl acetate (EtOAc) extracts of the wood sticks were analyzed by gas chromatography-mass spectrometry (GC-MS). Native agatharesinol (trimethylsilylated) produces an m/z 369 ion and an m/z 484 ion that are characteristic of its structure. Agatharesinol formed in the sapwood sticks treated with the deuterium-labeled l-phenylalanine generated both of these ions together with m/z 373 and 377 ions (m/z 369+4 and +8, respectively), and also m/z 488 and 492 ions (m/z 484+4 and +8, respectively). Generation of m/z 373 and 488 ions is attributed to the substitution by deuterium of the four hydrogen atoms of either of the p-hydroxyphenyl rings of agatharesinol, and that of m/z 377 and 492 ions is attributed to the substitution by deuterium of the eight hydrogen atoms of both p-hydroxyphenyl rings. In the administration of the 13C-labeled trans-cinnamic acid, m/z 375 and 381 ions (m/z 369+6 and +12, respectively), and also m/z 490 and 496 ions (m/z 484+6 and +12, respectively) were found, indicating that either aromatic ring or both aromatic rings of agatharesinol were 13C-labeled. Consequently, assimilation of the labeled precursors into agatharesinol was clearly detected, and an experimental procedure for studies on the biosynthesis was developed.     

Evolution of albinism in cave planthoppers by a convergent defect in the first step of melanin biosynthesis

Albinism, the reduction or loss of melanin pigment, is found in many diverse cave‐dwelling animals. The mechanisms responsible for loss of melanin pigment are poorly understood. In this study we use a melanogenic substrate assay to determine the position where melanin synthesis is blocked in independently evolved cave planthoppers from Hawaii and Croatia. In this assay, substrates of enzymes responsible for melanin biosynthesis are added to fixed specimens in vitro and their ability to rescue black melanin pigmentation is determined. L‐tyrosine, the first substrate in the pathway, did not produce melanin pigment, whereas L‐DOPA, the second substrate, restored black pigment. Substrates in combination with enzyme inhibitors were used to test the possibility of additional downstream defects in the pathway. The results showed that downstream reactions leading from L‐DOPA and dopamine to DOPA‐melanin and dopamine‐melanin, the two types of insect melanin, are functional. It is concluded that albinism is caused by a defect in the first step of the melanin synthesis pathway in cave‐adapted planthoppers from widely separated parts of the world. However, Western blots indicated that tyrosine hydroxylase (TH), the only enzyme shown to operate at the first step in insects, is present in Hawaiian cave planthoppers. Thus, an unknown factor(s) operating at this step may be important in the evolution of planthopper albinism. In the cavefish Astyanax mexicanus, a genetic defect has also been described at the first step of melanin synthesis suggesting convergent evolution of albinism in both cave‐adapted insects and teleosts.     

The role of pH in the melanin biosynthesis pathway

Having oxidized 3,4-dihydroxyphenylalanine (dopa) with sodium periodate or mushroom tyrosinase in a pH range from 3.5 to 6.0, it has been possible to detect spectrophotometrically 4-(2-carboxy-2-aminoethyl)-1,2-benzoquinone with the amino group protonated (o-dopaquinone-H+), a postulated intermediate in the melanogenesis pathway. When the pH value was greater than 4, the final product obtained was 2-carboxy-2,3-dihydroindole-5,6-quinone (dopachrome); however, for pH values lower than 4, two different products were identified by means of cyclic voltammetry: 5-(2-carboxy-2-aminoethyl)-2-hydroxy-1,4-benzoquinone and dopachrome. These products appeared when oxidation was achieved with the enzyme as well as with periodate. This suggests that two chemical pathways can arise from alpha-dopaquinone-H+, whose relative importance is determined by the pH. The steps of these pathways would be dopa leads to o-dopaquinone-H+ leads to o-dopaquinone leads to leukodopachrome leads to dopachrome, for the first one, and dopa leads to o-dopaquinone-H+ leads to 2,4,5-trihydroxyphenylalanine leads to 5-(2-carboxy-2-aminoethyl)-2-hydroxy-1,4-benzoquinone very slowly leads to intermediate compound leads to dopachrome, for the second one. The stoichiometry for the conversion of dopaquinone-H+ into dopachrome for pH values greater than 4 followed equation of 2 o-dopaquinone-H+ leads to dopa + dopachrome. No participation of oxygen was detected in the conversion of leukodopachrome (2,3-dihydro-5,6-dihydroxyindole-2-carboxylate) into dopachrome.     

The second naphthol reductase of fungal melanin biosynthesis in Magnaporthe grisea: tetrahydroxynaphthalene reductase

Mutants of Magnaporthe grisea harboring a defective gene for 1,3, 8-trihydroxynaphthalene reductase retain the capability to produce scytalone, thus suggesting the existence of a second naphthol reductase that can catalyze the reduction of 1,3,6, 8-tetrahydroxynaphthalene to scytalone within the fungal melanin biosynthetic pathway. The second naphthol reductase gene was cloned from M. grisea by identification of cDNA fragments with weak homology to the cDNA of trihydroxynaphthalene reductase. The amino acid sequence for the second naphthol reductase is 46% identical to that of trihydroxynaphthalene reductase. The second naphthol reductase was produced in Esherichia coli and purified to homogeneity. Substrate competition experiments indicate that the second reductase prefers tetrahydroxynaphthalene over trihydroxynaphthalene by a factor of 310; trihydroxynaphthalene reductase prefers trihydroxynaphthalene over tetrahydroxynaphthalene by a factor of 4.2. On the basis of the 1300-fold difference in substrate specificities between the two reductases, the second reductase is designated tetrahydroxynaphthalene reductase. Tetrahydroxynaphthalene reductase has a 200-fold larger K(i) for the fungicide tricyclazole than that of trihydroxynaphthalene reductase, and this accounts for the latter enzyme being the primary physiological target of the fungicide. M. grisea mutants lacking activities for both trihydroxynaphthalene and tetrahydroxynaphthalene reductases do not produce scytalone, indicating that there are no other metabolic routes to scytalone.     

Analysis of a kinetic model for melanin biosynthesis pathway.

The kinetic behavior of the melanin biosynthesis pathway from L-tyrosine up to dopachrome has been studied from experimental and simulation assays. The reaction mechanism proposed is based on a single active site of tyrosinase. The diphenolase and monophenolase activities of tyrosinase involve one single (oxidase) and two overlapped (hydroxylase and oxidase) catalytic cycles, respectively. The stoichiometry of the pathway implies that one molecule of tyrosinase must accomplish two turnovers in the hydroxylase cycle for each one in the oxidase cycle. Furthermore, the steady-state rates of dopachrome production and O2 consumption from tyrosine and L-dopa, also fulfill the stoichiometry of the pathway: VO2T/VDCT = 1.5 and VO2T/VDCD = 1.0, where T represents L-tyrosine, DC represents dopachrome, and D represents L-dopa. It has been ascertained by high performance liquid chromatography that in the steady-state, a quantity of dopa is accumulated ([D]ss) which fulfills the constant ratio [D]ss = R[T]0. Taking this ratio into account, an analytical expression has been deduced for the monophenolase activity of tyrosinase. In this expression kcatT congruent to (2/3)k3(K1/K2)R, revealing that kcatT is not a true catalytic constant, since it also depends on equilibrium constants and on the experimental R = 0.057. This low value explains the lower catalytic efficiency of tyrosinase on tyrosine than on dopa, (VmaxT/KmT)/(VmaxD/KmD) congruent to (2/3)R, since a significant portion of tyrosinase is scavenged from the catalytic turnover as dead-end complex EmetT in the steady-state of the monophenolase activity of tyrosinase.     

Characterisation of the enzymatic complex for the first step in glycosylphosphatidylinositol biosynthesis

The mammalian N-acetylglucosaminyl transferase for the first step in glycosylphosphatidylinositol biosynthesis has been shown to consist of at least four components: PIG-A, PIG-C, PIG-H and GPI1. Here, the enzymatic complex is further characterised. PIG-A protein, which is thought to represent the catalytic subunit of the complex, was expressed in an epitope-tagged form in the PIG-A deficient JY5 lymphoblastoid cell line. Subcellular localisation of this protein was studied using immunofluorescence and immunoelectron microscopy. The protein was localised to both perinuclear and mitochondria-associated lamellae of the endoplasmic reticulum. Using affinity chromatography, epitope-tagged PIG-A protein was partially purified. To identify regions that might be involved in the catalytic process, computer-aided comparison was performed between PIG-A and 26 distantly related glycosyl transferases. A number of residues in the membrane-proximal region of the cytoplasmic domain (230-340) were found highly conserved. Finally, a topological model of the four partners participating in the enzymatic complex is introduced to provide a working model for further structural and functional analysis.     

蛋白-O-岩藻糖基转移酶1基因CfPOFUT1在果生炭疽菌中的功能分析

【目的】蛋白-O-岩藻糖基转移酶1 (protein O-fucosyltransferase 1,POFUT1)是催化蛋白质O-岩藻糖基化的关键酶,在动物和人体内被证明调控一系列的生理病理过程,然而POFUT1基因在果生炭疽菌乃至真菌中还未见报道。本研究旨在克隆果生炭疽菌中CfPOFUT1基因,并分析其生物学功能。【方法】利用RT-PCR技术扩增CfPOFUT1的基因并进行生物信息学分析,构建了CfPOFUT1基因的沉默和过表达载体,通过PEG介导法将载体导入原生质体中获得CfPOFUT1基因的沉默和过表达突变体。测定了野生型菌株、CfPOFUT1沉默菌株和过表达菌株在PDA上的菌丝生长、分生孢子产生、萌发与附着胞形成、胁迫应答和致病力、杀菌剂敏感性等生物学表型。【结果】与野生型菌株相比,基因过表达突变体产孢量显著增加,致病力增强,对嘧菌酯敏感性降低,但对多菌灵和咪鲜胺敏感性增强。基因沉默突变体产孢量减少,细胞壁完整性、内质网应激敏感性提高,致病力减弱,对嘧菌酯敏感性提高,但对多菌灵和咪鲜胺敏感性降低。【结论】CfPOFUT1基因参与调控果生炭疽菌分生孢子产量,细胞壁完整性、内质网对应激和药剂敏感性,并对其致病性也具有一定的影响。     

Ribose biosynthesis and evidence for an alternative first step in the common aromatic amino acid pathway in Methanococcus maripaludis.

An acetate-requiring mutant of Methanococcus maripaludis allowed efficient labeling of riboses following growth in minimal medium supplemented with [2-(13)C]acetate. Nuclear magnetic resonance and mass spectroscopic analysis of purified cytidine and uridine demonstrated that the C-1' of the ribose was about 67% enriched for 13C. This value was inconsistent with the formation of erythrose 4-phosphate (E4P) exclusively by the carboxylation of a triose. Instead, these results suggest that either (i) E4P is formed by both the nonoxidative pentose phosphate and triose carboxylation pathways or (ii) E4P is formed exclusively by the nonoxidative pentose phosphate pathway and is not a precursor of aromatic amino acids.     

The biosynthesis of melanin in Alternaria

A melanin which is insoluble in strong alkali has been isolated from Alternaria mycelium. Alkali fusion of the pigment produced p-hydroxybenzoa     

The first cytochrome P450 in ferns. Evidence for its involvement in phytoecdysteroid biosynthesis in Polypodium vulgare

The fern Polypodium vulgare is a phytoecdysteroid (PE)-producing plant. Cultures of P. vulgare prothalus produce PE, whereas prothalus-derived callus cultures do not. However, this callus line can transform topically applied ecdysone (E) to 20-hydroxyecdysone (20E), which is the last step in the biosynthetic pathway of the main plant PE. This hydroxylation is catalysed by a cytochrome P450 enzyme. E treatment of the callus line results in an increased amount of P450, showing a linear correspondence between the amount of P450 and in vivo E 20-hydroxylation activity, estimated by measuring the bioconversion of E to 20E. This activity can be inhibited by molecules that bind to the P450-heme group. E shows a P450-substrate-binding spectrum with microsomes that overexpress the P450 protein. Finally, a P450 protein was purified from E-treated calli, this being the first P450 to be described in the pterydophyte group.     

Interactions of two selected field isolates of Colletotrichum gloeosporioides f. sp. aeschynomene on Aeschynomene virginica

The fungal plant pathogen, Colletotrichum gloeosporioides f. sp. aeschynomene (CGA), has been used successfully since 1982 to control northern jointvetch (NJV), Aeschynomene virginica, in rice and soybeans in the USA as COLLEGO^®, a bioherbicidal formulation of a single isolate of this pathogen. The interactions and fitness of different field isolates of this fungus and effective bioherbicide have not been fully examined. We examined CGA population structure and inter-isolate competition using molecular markers in field and greenhouse experiments. NJV plants were first inoculated with one isolate or a mixture of two isolates, Cla-5a and 3-1-3, followed by a challenge inoculation containing either one or both isolates, 4 or 5 days later. Plants first inoculated with Cla-5a alone, or in mixture with 3-1-3, had Cla-5a comprising approximately 80% of the population after 21 days regardless of challenge inoculation. In contrast, when the primary treatment was 3-1-3 alone, wounding, or benzodiathiazole, 3-1-3 detection only approached 50% of the population following challenge inoculation. Primary inoculation with Cla-5a significantly reduced the number of lesions caused by 3-1-3 challenge, whereas primary inoculation with 3-1-3 did not significantly affect the number of lesions caused by Cla-5a challenge. Size of lesions caused by the two isolates following challenge inoculations and dispersal of the isolates from lesions to healthy plants were not affected by the primary inoculation. These results suggest a specific interaction by Cla-5a with the host defense response creating a more favorable environment for its own colonization of NJV to the exclusion of 3-1-3.     

Properties and inhibition of the first two enzymes of the non-mevalonate pathway of isoprenoid biosynthesis

Enzymes of the 1-deoxy-D-xylulose 5-phosphate/2-C-methylerythritol 4-phosphate (DOXP/MEP) pathway are targets for new herbicides and antibacterial drugs. Until now, no inhibitors for the DOXP synthase have been known of. We show that one of the breakdown products of the herbicide clomazone affects the DOXP synthase. One inhibitor of the non-mevalonate pathway, fosmidomycin, blocks the DOXP reductoisomerase (DXR) of plants and bacteria. The I(50) values of plants are, however, higher than those found for the DXR of Escherichia coli. The DXR of plants, isolated from barley seedlings, shows a pH optimum of 8.1, which is typical for enzymes active in the chloroplast stroma.     

Agrobacterium tumefaciens TR-DNA encodes a pathway for agropine biosynthesis

Summary A pathway for biosynthesis of the crown-gall opine, agropine is proposed and three potential new precursors characterised. The location of genes involved in the three steps of this pathway was determined by site directed insertions and deletions in the T_R region of the octopine Ti plasmid, pTiB6Tra^c. The proposed biosynthetic pathway for agropine which involves three T-DNA genes is in contrast to the biosynthesis of octopine and nopaline where single T-DNA genes are involved.     

5-Aminolevulinate synthase and the first step of heme biosynthesis

5-Aminolevulinate synthase catalyzes the condensation of glycine and succinyl-CoA to yield 5-aminolevulinate. In animals, fungi, and some bacteria, 5-aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway. Mutations on the human erythroid 5-aminolevulinate synthase, which is localized on the X-chromosome, have been associated with X-linked sideroblastic anemia. Recent biochemical and molecular biological developments provide important insights into the structure and function of this enzyme. In animals, two aminolevulinate synthase genes, one housekeeping and one erythroid-specific, have been identified. In addition, the isolation of 5-aminolevulinate synthase genomic and cDNA clones have permitted the development of expression systems, which have tremendously increased the yields of purified enzyme, facilitating structural and functional studies. A lysine residue has been identified as the residue involved in the Schiff base linkage of the pyridoxal 5-phosphate cofactor, and the catalytic domain has been assigned to the C-terminus of the enzyme. A conserved glycine-rich motif, common to all aminolevulinate synthases, has been proposed to be at the pyridoxal 5phosphate-binding site. A heme-regulatory motif, present in the presequences of 5-aminolevulinate synthase precursors, has been shown to mediate the inhibition of the mitochondrial import of the precursor proteins in the presence of heme. Finally, the regulatory mechanisms, exerted by an iron-responsive element binding protein, during the translation of erythroid 5-aminolevulinate synthase mRNA, are discussed in relation to heme biosynthesis.     

Evidence for lipoxygenase pathway involvement in allergic tracheal contraction

Challenge of actively sensitized guinea-pig trachea in vitro led to a contraction which was enhanced by the cyclo-oxygenase inhibitors, indomethacin and sodium meclofenamate. Cyclo-oxygenase inhibitors eliminated the release of PGE-like material induced by arachidonic acid (AA), histamine, and antigen challenge. AA (10 μg./ml.) and PGE₂ (100 ng./ml.) usually relaxed the trachea, whereas in the presence of cyclo-oxygenase inhibitors a contraction occurred. Phenidone and ETYA, which also blocked the lipoxygenase pathway of AA metabolism inhibited the enhancement of allergic tracheal contraction induced by cyclo-oxygenase inhibitors, decreased the time that the trachea remained contracted, and also eliminated the contraction induced by AA and PGE₂. Thus, cyclo-oxygenase inhibitors may enhance allergic tracheal contraction by diverting AA metabolism into the lipoxygenase pathway and a product of the latter pathway, possibly SRS-A, may be responsible for the enhancement and for the prolonged phase of allergic tracheal contraction. An analogous mechanism may account for aspirin-induced asthma in man.     

Evidence for lipoxygenase pathway involvement in allergic tracheal contraction

Challenge of actively sensitized guinea-pig trachea in vitro led to a contraction which was enhanced by the cyclo-oxygenase inhibitors, indomethacin and sodium meclofenamate. Cyclo-oxygenase inhibitors eliminated the release of PGE-like material induced by arachidonic acid (AA), histamine, and antigen challenge. AA (10 microgram./ml.) and PGE2 (100 ng./ml.) usually relaxed the trachea, whereas in the presence of cyclo-oxygenase inhibitors a contraction occurred. Phenidone and ETYA, which also blocked the lipoxygenase pathway of AA metabolism inhibited the enhancement of allergic tracheal contraction induced by cyclo-oxygenase inhibitors, decreased the time that the trachea remained contracted, and also eliminated the contraction induced by AA and PGE2. Thus, cyclo-oxygenase inhibitors may enhance allergic tracheal contraction by diverting AA metabolism into the lipoxygenase pathway and product of the latter pathway, possibly SRS-A, may be responsible for the enhancement and for the prolonged phase of allergic tracheal contraction. An analogous mechanism may account for aspirin-induced asthma in man.     

Biochemical characterization of the first step in sulfonolipid biosynthesis in Alistipes finegoldii

Sulfonolipids are unusual lipids found in the outer membranes of Gram-negative bacteria in the phylum Bacteroidetes. Sulfonolipid and its deacylated derivative, capnine, are sulfur analogs of ceramide-1-phosphate and sphingosine-1-phosphate, respectively; thus, sulfonolipid biosynthesis is postulated to be similar to the sphingolipid biosynthetic pathway. Here, we identify the first enzyme in sulfonolipid synthesis in Alistipes finegoldii as the product of the alfi_1224 gene, cysteate acyl-acyl carrier protein (ACP) transferase (SulA). We show SulA catalyzes the condensation of acyl-ACP and cysteate (3-sulfo-alanine) to form 3-ketocapnine. Acyl-CoA is a poor substrate. We show SulA has a bound pyridoxal phosphate (PLP) cofactor that undergoes a spectral redshift in the presence of cysteate, consistent with the transition of the lysine–aldimine complex to a substrate–aldimine complex. Furthermore, the SulA crystal structure shows the same prototypical fold found in bacterial serine palmitoyltransferases (Spts), enveloping the PLP cofactor bound to Lys251. We observed the SulA and Spt active sites are identical except for Lys281 in SulA, which is an alanine in Spt. Additionally, SulA(K281A) is catalytically inactive but binds cysteate and forms the external aldimine normally, highlighting the structural role of the Lys281 side chain in walling off the active site from bulk solvent. Finally, the electropositive groove on the protein surface adjacent to the active site entrance provides a landing pad for the electronegative acyl-ACP surface. Taken together, these data identify the substrates, products, and mechanism of SulA, the PLP-dependent condensing enzyme that catalyzes the first step in sulfonolipid synthesis in a gut commensal bacterium.