Biosynthesis of bisorbicillinoid in Trichoderma sp. USF-2690; evidence for the biosynthetic pathway,via sorbicillinol,of sorbicillin,bisorbicillinol, bisorbibutenolide,and bisorbicillinolide

An incorporation study of [1-(13)C] and [1,2-(13)C2] labeled sodium acetates into sorbicillinol 1 established a ring closure system between C-1 and C-6 and the positions that were oxidized and/or methylated on a hexaketide chain. Subsequent investigations, using 13C-labeled 1 prepared from [1-(13)C] labeled sodium acetate, clearly demonstrated that both bisorbicillinol 2 and sorbicillin 6 incorporated 13C-labeled 1 into their carbon skeletons. 13C-labeled bisorbicillinols 2 derived from [1-(13)C]- and [2-(13)C]-labeled sodium acetates clearly indicate that these were on the biosynthetic route from 1 to bisorbibutenolide (bislongiquinolide) 3 and bisorbicillinolide 4 via 2 as a branching point in the fungus.     

Role of polyisoprenoids in tobacco resistance against biotic stresses

Infection with avirulent pathogens, tobacco mosaic virus (TMV) or Pseudomonas syringae pv. tabaci induced accumulation of polyisoprenoid alcohols, solanesol and a family of polyprenols [from polyprenol composed of 14 isoprene units (Pren-14) to -18, with Pren-16 dominating] in the leaves of resistant tobacco plants Nicotiana tabacum cv. Samsun NN. Upon TMV infection, solanesol content was increased seven- and eight-fold in the inoculated and upper leaves, respectively, while polyprenol content was increased 2.5- and 2-fold in the inoculated and upper leaves, respectively, on the seventh day post-infection. Accumulation of polyisoprenoid alcohols was also stimulated by exogenously applied hydrogen peroxide but not by exogenous salicylic acid (SA). On the contrary, neither inoculation of the leaves of susceptible tobacco plants nor wounding of tobacco leaves caused an increase in polyisoprenoid content. Taken together, these results indicate that polyisoprenoid alcohols might be involved in plant resistance against pathogens. A putative role of accumulated polyisoprenoids in plant response to pathogen attack is discussed. Similarly, the content of plastoquinone (PQ) was increased two-fold in TMV-inoculated and upper leaves of resistant plants. Accumulation of PQ was also stimulated by hydrogen peroxide, bacteria ( P.  syringae ) and SA. The role of PQ in antioxidant defense in cellular membranous compartments is discussed in the context of the enzymatic antioxidant machinery activated in tobacco leaves subjected to viral infection. Elevated activity of several antioxidant enzymes (ascorbate peroxidase, guaiacol peroxidase, glutathione reductase and superoxide dismutase, especially the CuZn superoxide dismutase isoform) and high, but transient elevation of catalase was found in inoculated leaves of resistant tobacco plants but not in susceptible plants.     

Recovery of solanesol from tobacco as a value-added byproduct for alternative applications

Solanesol in the waste streams of a bioprocess designed for alternative applications of low-alkaloid tobacco was recovered using three different extraction methods. Compared to the conventional heat-reflux extraction (HRE) and ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE) using 1:3 hexane:ethanol (v/v) as the solvent after saponification treatment of tobacco biomass was found the most effective in terms of solanesol yield, processing time, and volume of solvent consumed. Quantification of solanesol was achieved by optimizing the mobile phase at 60/40 acetonitrile–isopropanol and lowering the oven temperature to 22 °C using a standard reverse-phase high performance liquid chromatography (RP-HPLC). The total solanesol recovered from tobacco biomass and chloroplast accounted for 30% (w/w) of the total solanesol in the fresh leaves. Since solanesol is the precursor of metabolically active quinones such as coenzyme Q10 and vitamin K analogues, extraction of solanesol from tobacco bioprocess waste is a feasible operation and could leverage the overall profitability of biorefining tobacco for alternative, value-added uses.     

Biosynthesis of theobroxide and its related compounds, metabolites of Lasiodiplodia theobromae

Administration of (13)C labeled acetates ([1-(13)C], [2-(13)C] and [1,2-(13)C(2)] to Lasiodiplodia theobromae showed the tetraketide origins of both theobroxide, a potato-tuber inducing substance [1, (1S, 2R, 5S, 6R)-3-methyl-7-oxa-bicyclo[4.1.0]hept-3-en-2,5-diol]) and its carbonyldioxy derivative [2, (1S, 4R, 5S, 6R)-7,9-dioxa-3-methyl-8-oxobicyclo [4.3.0]-2-nonene-4,5-diol]. The incorporation of acetate-derived hydrogen into 1 and 2 was studied using [2-(2)H(3), 2-(13)C]acetate. Three and one deuterium atoms were incorporated at one methyl and epoxy carbons, respectively. The observed loss of deuterium atoms from the methyl group suggests a considerable amount of exchange from the methyl group of [2-(2)H(3), 2-(13)C]acetate during biosynthesis of 1 and 2. Incorporation of [1-(13)C]- and [1,2-(13)C(2)]acetates indicates the carbonyl carbon of the carbonyldioxy derivative is derived from the carboxy carbon of the precursor.     

Metabolic pathways leading from amino acids to vitamin B12 in Propionibacterium shermanii, and the sources of the seven methyl carbons

The metabolic pathways leading from l-[2-13C]aspartic acid, [2-13C]glycine and l-[methyl-13C]methionine to vitamin B12 were investigated, focusing on the biosynthetic pathways leading to the aminopropanol moiety of vitamin B12 and on the role of the Shemin pathway leading to delta-aminolevulinic acid (a biosynthetic intermediate of tetrapyrrole), by means of feeding experiments with Propionibacterium shermanii in combination with 13C-NMR spectroscopy. The 13C-methylene carbons of l-[2-(13)C]aspartic acid, which is transformed to [2-13C]glycine via l-[2-13C]threonine, and [2-13C]glycine added to the culture medium served mainly to enrich the seven methyl carbons of the corrin ring through C-methylation by S-adenosyl-l-[methyl-13C]methionine derived from catabolically generated l-[methyl-13C]methionine in the presence of tetrahydrofolic acid. The results indicate that the catabolism of these amino acids predominates over pathways leading to (2R)-1-amino-2-propanol or delta-aminolevulinic acid in P. shermanii. Feeding of l-[methyl-13C]methionine efficiently enriched all seven methyl carbons. In the cases of [2-13C]glycine and l-[methyl-13C]methionine, the 13C-enrichment ratio of the methyl carbon at C-25 (the site of the first C-methylation) was less than those of the other six methyl carbons, probably due to the influence of endogenous d-glucose in P. shermanii. The almost identical 13C-enrichment ratios of the other six methyl carbons indicated that these C-methylations during vitamin B12 biosynthesis were completed before the amino acids were completely consumed. However, in the case of l-[2-13C]aspartic acid, the 13C-enrichment ratios of five methyl carbons were low and similar, whereas the last two sites of C-methylation (C-53 and C-35) were not labeled, presumably because of complete consumption of the smaller amount of added label. The ratios of 13C-incorporation into the seven methyl carbons are influenced by the conditions of amino acid feeding experiments in a manner that is dependent upon the order of C-methylation in the corrin ring of vitamin B12.     

13C NMR evidence for bacteriochlorophyll c formation by the C5 pathway in green sulfur bacterium, Prosthecochloris

The 13C NMR spectra of the pheophorbide of bacteriochlorophyll c, formed in the presence of L-[1-13C]glutamate and [2-13C]glycine and [13C]bicarbonate in Prosthecochloris aestaurii, were analysed. The isotope in the glutamate was specifically incorporated into the eight carbon atoms in the tetrapyrrole macrocycle derived from the C-5 of 5-aminolevulinic acid, while no specific enrichment of these eight carbon atoms was observed in the spectrum of the pigment formed in the presence of [2-13C]glycine. These labelling patterns provide evidence for the operation of the C5 pathway of 5-aminolevulinic acid synthesis for bacteriochlorophyll c formation in the bacterium. The labelling of bacteriochlorophyll c by [13C]bicarbonate is consistent with its formation from 5-[1,4,5-13C]aminolevulinic acid formed by the C5 pathway from [1,2,5-13C]glutamic acid. It is proposed that this glutamate is the transamination product of 2-[1,2,5-13C]oxoglutaric acid, arising by carboxylation of [1,4-13C]succinyl-CoA with 13CO2 catalysed by 2-oxoglutaric acid synthase activity, and that the labelled succinyl-CoA is, in turn, derived by the fixation of 13CO2 by the reductive tricarboxylic acid cycle. The 13C chemical shifts of two sp2 quaternary carbons of bacteriopheophorbide c methyl ester (C-2 and C-4) were reassigned.     

Solid-state NMR structure determination of melittin in a lipid environment

Solid-state (13)C NMR spectroscopy was used to investigate the three-dimensional structure of melittin as lyophilized powder and in ditetradecylphosphatidylcholine (DTPC) membranes. The distance between specifically labeled carbons in analogs [1-(13)C]Gly3-[2-(13)C]Ala4, [1-(13)C]Gly3-[2-(13)C]Leu6, [1-(13)C]Leu13-[2-(13)C]Ala15, [2-(13)C]Leu13-[1-(13)C]Ala15, and [1-(13)C]Leu13-[2-(13)C]Leu16 was measured by rotational resonance. As expected, the internuclear distances measured in [1-(13)C]Gly3-[2-(13)C]Ala4 and [1-(13)C]Gly3-[2-(13)C]Leu6 were consistent with alpha-helical structure in the N-terminus irrespective of environment. The internuclear distances measured in [1-(13)C]Leu13-[2-(13)C]Ala15, [2-(13)C]Leu13-[1-(13)C]Ala15, and [1-(13)C]Leu13-[2-(13)C]Leu16 revealed, via molecular modeling, some dependence upon environment for conformation in the region of the bend in helical structure induced by Pro14. A slightly larger interhelical angle between the N- and C-terminal helices was indicated for peptide in dry or hydrated gel state DTPC (139 degrees -145 degrees ) than in lyophilized powder (121 degrees -139 degrees ) or crystals (129 degrees ). The angle, however, is not as great as deduced for melittin in aligned bilayers of DTPC in the liquid-crystalline state (approximately 160 degrees ). The study illustrates the utility of rotational resonance in determining local structure within peptide-lipid complexes.     

Synthesis of fluoroacetate from fluoride, glycerol, and beta-hydroxypyruvate by Streptomyces cattleya.

Streptomyces cattleya produces fluoroacetate and 4-fluorothreonine from inorganic fluoride added to the culture broth. We have shown by 19F nuclear magnetic resonance (NMR) spectrometry that fluoroacetate is accumulated first in the culture broth and that accumulation of 4-fluorothreonine is next. To show precursors of the carbon skeleton of fluoroacetate, we carried out tracer experiments with various 14C- and 13C-labeled compounds. Radioactivity of [U-14C]glucose, [U-14C]glycerol, [U-14C]serine, and [U-14C]beta-hydroxypyruvate was incorporated into fluoroacetate to an extent of 0.2 to 0.4%, whereas [3-14C]pyruvate, [2,3-14C]succinate, and [U-14C]aspartate were less efficiently incorporated (0.04 to 0.08%). The addition of [2-13C]glycerol to the mycelium suspension of Streptomyces cattleya caused exclusive enrichment of the carboxyl carbon of fluoroacetate with 13C; about 40% of carboxyl carbon of fluoroacetate was labeled with 13C. We studied the radioactivity incorporation of [3-14C]-, [U-14C]-, and [1-14C]beta-hydroxypyruvates to show that C-2 and C-3 of beta-hydroxypyruvate are exclusively converted to the carbon skeleton of fluoroacetate. These results suggest that the carbon skeleton of fluoroacetate derives from C-1 and C-2 of glycerol through beta-hydroxypyruvate, whose hydroxyl group is eventually replaced by fluoride.     

Erinacine Q,a new erinacine from Hericium erinaceum,and its biosynthetic route to erinacine C in the basidiomycete

Erinacines as cyathane-xylosides are known to have potent stimulating activity for nerve-growth-factor synthesis. Our search for new cyathane metabolites from a liquid culture of Hericium erinaceum YB4-6237 resulted in the isolation of a new erinacine named erinacine Q (1). NMR spectrometry and a chemical derivation from erinacine P (2) determined the compound to be a derivative in which the formyl group of erinacine P had been reduced to the hydroxymethyl group. To clarify the biosynthetic relationship between erinacine Q and the others, [1'-13C]erinacine Q ([1'-13C]-1) was chemically derived from [1'-13C]erinacine P ([1'-13C]-2) which had been prepared by feeding [1-13C]-D-glucose to the basidiomycete. The biotransformation of labeled erinacine Q into [1'-13C]erinacine C ([1'-13C]-5) via [1'-13C]erinacine P in this basidiomycete was demonstrated by NMR spectrometry.     

Recovery of (13)CO2 during rest and exercise after [1-(13)C]acetate, [2-(13)C]acetate, and NaH(13)CO3 infusions

For estimating the oxidation rates (Rox) of glucose and other substrates by use of (13)C-labeled tracers, we obtained correction factors to account for label dilution in endogenous bicarbonate pools and TCA cycle exchange reactions. Fractional recoveries of (13)C label in respiratory gases were determined during 225 min of rest and 90 min of leg cycle ergometry at 45 and 65% peak oxygen uptake (VO(2 peak)) after continuous infusions of [1-(13)C]acetate, [2-(13)C]acetate, or NaH(13)CO(3). In parallel trials, [6,6-(2)H]glucose and [1-(13)C]glucose were given. Experiments were conducted after an overnight fast with exercise commencing 12 h after the last meal. During the transition from rest to exercise, CO(2) production increased (P < 0.05) in an intensity-dependent manner. Significant differences were observed in the fractional recoveries of (13)C label as (13)CO(2) at rest (NaH(13)CO(3), 77.5 +/- 2.8%; [1-(13)C]acetate, 49.8 +/- 2.4%; [2-(13)C]acetate, 26.1 +/- 1.4%). During exercise, fractional recoveries of (13)C label from [1-(13)C]acetate, [2-(13)C]acetate, and NaH(13)CO(3) were increased compared with rest. Magnitudes of label recoveries during both exercise intensities were tracer specific (NaH(13)CO(3), 93%; [1-(13)C]acetate, 80%; [2-(13)C]acetate, 65%). Use of an acetate-derived correction factor for estimating glucose oxidation resulted in Rox values in excess (P < 0.05) of glucose rate of disappearance during hard exercise. We conclude that, after an overnight fast: 1) recovery of (13)C label as (13)CO(2) from [(13)C]acetate is decreased compared with bicarbonate; 2) the position of (13)C acetate label affects carbon dilution estimations; 3) recovery of (13)C label increases in the transition from rest to exercise in an isotope-dependent manner; and 4) application of an acetate correction factor in glucose oxidation measurements results in oxidation rates in excess of glucose disappearance during exercise at 65% of VO(2 peak). Therefore, bicarbonate, not acetate, correction factors are advocated for estimating glucose oxidation from carbon tracers in exercising men.     

Resonance Raman spectra of anionic semiquinoid form of a flavoenzyme, D-amino acid oxidase

Resonance Raman (RR) spectra of the complex of anionic semiquinoid D-amino acid oxidase (DAO) with picolinate in H2O and D2O were observed in the 300-1,750 cm-1 region. RR spectra were also measured for the complex of the semiquinoid enzyme reconstituted with isotopically labeled FAD's, i.e., [4a-13C]-, [4,10a-13C2]-, [2-13C]-, [5-15N]-, and [1,3-15N2]-FAD. On the basis of the isotope effects, tentative assignments of the observed bands of the anionic semiquinoid flavin were made. The spectra differ from those of oxidized, neutral semiquinoid, and anionic reduced flavins previously reported. The 1,602 cm-1 band was not shifted for any FAD labeled in ring II and/or ring III and was assigned to a ring I mode. The 1,516 cm-1 band underwent an isotopic shift upon [4a-13C]- or [4,10a-13C2]-labeling. The band was assigned to the mode containing C(4a)-C(10a) stretching. The 1,331 and 1,292 cm-1 bands shifted upon [4a-13C]- or [5-15N]-labeling and were assigned to the modes containing C(4a)-N(5) stretching. The 1,217 and 1,188 cm-1 bands were assigned to the skeletal vibrations of ring III coupled with the N(3)-H bending mode. The RR spectrum of the complex of anionic semiquinoid DAO with alpha-iminopropionate or N-methyl-alpha-iminopropionate was essentially identical with that of the complex with picolinate.     

In vivo effects of adenosine A(2) receptor agonist and antagonist on neuronal and astrocytic intermediary metabolism studied with ex vivo (13)C MR spectroscopy.

The effect of adenosine A(2) receptor agonist 2-[p-(2-carboxyethyl)phenylethylamino]-5'-ethylcarboxamidoadenosine (CGS 21680) and antagonist 3,7-dimethyl-1-propargylxanthine (DMPX) on [1-(13)C]glucose and [1,2-(13)C]acetate metabolism was studied in rats by (13)C magnetic resonance (MR) spectroscopy and HPLC. In the cortex a significant reduction was observed in the amounts of [2-(13)C]GABA and [3-(13)C]aspartate from [1-(13)C]glucose in CGS 21680. In the subcortex the concentration of labelled [4-(13)C]glutamate was increased in both treatment groups. The amounts of [2 + 3-(13)C]succinate and [3-(13)C]lactate were increased in the CGS 21680 group compared to control, and the DMPX group showed an increase in the total amount of [6-(13)C]N-acetyl aspartate compared to control in the subcortex. Astrocyte metabolism was only affected in the cortex as shown by a decrease in the pyruvate carboxylase/pyruvate dehydrogenase ratio in glutamate and glutamine in the treatment groups. Labelling from [1,2-(13)C]acetate was not much affected by CGS 21680 or DMPX. However, the amount of [1,2-(13)C]acetate in cortex and subcortex was reduced in the DMPX group. In the cortex a reduction in the labelling of [3-(13)C]GABA in the DMPX group compared to control and an increase in the total amount of taurine in both treatment groups was detected. The present study shows that A(2) receptor agonist and antagonist have similar effects; however, in cortex GABAergic neurones and astrocytes were affected in contrast to subcortex, where glutamatergic neurones showed the greatest changes.     

Biosynthesis of the bicycloalternarenes, mixed terpenoids of Alternaria alternata

By incorporation of [2-13C]-mevalonate, [1-13C]-acetate and [1-13C]-glucose we could reveal that the phytopathogenic fungus Alternaria alternata biosynthesized the mixed terpenoids bicycloalternarenes via the classic mevalonate pathway. The polyketid pathway does not participate in the biosynthesis of bicycloalternarenes, because there is no incorporation of [13C]-acetate into the C-ring of these compounds. The labelling pattern in this nonterpenoid part of bicycloalternarenes after feeding with [1-13C]-glucose and [U-13C6]-glucose, respectively, allows the assumption that metabolites of the shikimate pathway are involved.     

Changes in flux pattern of the central carbohydrate metabolism during kernel development in maize

Developing kernels of the inbred maize line W22 were grown in sterile culture and supplied with a mixture of [U-13C6]glucose and unlabeled glucose during three consecutive intervals (11-18, 18-25, or 25-32 days after pollination) within the linear phase of starch formation. At the end of each labeling period, glucose was prepared from starch and analyzed by 13C isotope ratio mass spectrometry and high-resolution (13)C NMR spectroscopy. The abundances of individual glucose isotopologs were calculated by computational deconvolution of the NMR data. [1,2-(13)C2]-, [5,6-(13)C2]-, [2,3-(13)C2]-, [4,5-(13)C2]-, [1,2,3-(13)C3]-, [4,5,6-(13)C3]-, [3,4,5,6-(13)C4]-, and [U-(13)C6]-isotopologs were detected as the major multiple-labeled glucose species, albeit at different normalized abundances in the three intervals. Relative flux contributions by five different pathways in the primary carbohydrate metabolism were determined by computational simulation of the isotopolog space of glucose. The relative fractions of some of these processes in the overall glucose cycling changed significantly during maize kernel development. The simulation showed that cycling via the non-oxidative pentose phosphate pathway was lowest during the middle interval of the experiment. The observed flux pattern could by explained by a low demand for amino acid precursors recruited from the pentose phosphate pathway during the middle interval of kernel development.     

Peroxisomal and mitochondrial oxidation of fatty acids in the heart, assessed from the 13C labeling of malonyl-CoA and the acetyl moiety of citrate

We previously showed that a fraction of the acetyls used to synthesize malonyl-CoA in rat heart derives from partial peroxisomal oxidation of very long and long-chain fatty acids. The 13C labeling ratio (malonyl-CoA)/(acetyl moiety of citrate) was >1.0 with 13C-fatty acids, which yields [13C]acetyl-CoA in both mitochondria and peroxisomes and < 1.0 with substrates, which yields [13C]acetyl-CoA only in mitochondria. In this study, we tested the influence of 13C-fatty acid concentration and chain length on the labeling of acetyl-CoA formed in mitochondria and/or peroxisomes. Hearts were perfused with increasing concentrations of labeled docosanoate, oleate, octanoate, hexanoate, butyrate, acetate, or dodecanedioate. In contrast to the liver, peroxisomal oxidation of 1-13C-fatty acids in heart does not form [1-13C]acetate. With [1-13C]docosanoate and [1,12-13C2]dodecanedioate, malonyl-CoA enrichment plateaued at 11 and 9%, respectively, with no detectable labeling of the acetyl moiety of citrate. Thus, in the intact rat heart, docosanoate and dodecanedioate appear to be oxidized only in peroxisomes. With [1-13C]oleate or [1-13C]octanoate, the labeling ratio >1 indicates the partial peroxisomal oxidation of oleate and octanoate. In contrast, with [3-13C]octanoate, [1-13C]hexanoate, [1-13C]butyrate, or [1,2-13C2]acetate, the labeling ratio was <0.7 at all concentrations. Therefore, in rat heart, (i) n-fatty acids shorter than 8 carbons do not undergo peroxisomal oxidation, (ii) octanoate undergoes only one cycle of peroxisomal beta-oxidation, (iii) there is no detectable transfer to the mitochondria of acetyl-CoA from the cytosol or the peroxisomes, and (iv) the capacity of C2-C18 fatty acids to generate mitochondrial acetyl-CoA decreases with chain length.     

Metabolism of alpha-D-[1,2-13C]glucose pentaacetate and alpha-D-glucose penta[2-13C]acetate in rat hepatocytes

Hepatocytes from fed rats were incubated for 120 min in the presence of alpha-D-[1,2-13C]glucose pentaacetate (1.7 mM), both D-[1,2-13C]glucose (1.7 mM) and acetate (8.5 mM), alpha-D-glucose penta[2-13C]acetate (1.7 mM), or D-[1,2-13C]glucose (8.3 mM). The amounts of 13C-enriched L-lactate and D-glucose and those of acetate and beta-hydroxybutyrate recovered in the incubation medium were comparable under the first two experimental conditions. The vast majority of D-glucose isotopomers consisted of alpha- and beta-D[1,2-13C]glucose. The less abundant single-labeled isotopomers of D-glucose were equally labeled on each C atom. The output of 13C-labeled L-lactate, mainly L-[2-13C]lactate and L-[3-13C]lactate, was 1 order of magnitude lower than that found in hepatocytes exposed to 8.3 mM D-[1,2-13C]glucose, in which case the total production of the single-labeled species of D-glucose was also increased and that of the C3- or C4-labeled hexose was lower than that of the other 13C-labeled isotopomers. In cells exposed to alpha-D-glucose penta[2-13C]acetate, the large majority of 13C atoms was recovered as [2-13C]acetate and, to a much lesser extent, beta-hydroxybutyrate labeled in position 2 and/or 4. Nevertheless, L-[2-13C]lactate, L-[3-13C]lactate, and single-labeled D-glucose isotopomers were also produced in amounts higher or comparable to those found in cells exposed to alpha-D-[1,2-13C]glucose pentaacetate. However, a modest preferential labelling of the C6-C5-C4 moiety of D-glucose, relative to its C1-C2-C3 moiety, and a lesser isotopic enrichment of the C3 (or C4), relative to that of C1 (or C6) and C2 (or C5), were now observed. These findings indicate that, despite extensive hydrolysis of alpha-D-glucose pentaacetate (1.7 mM) in the hepatocytes, the catabolism of its D-glucose moiety is not more efficient than that of unesterified D-glucose, tested at the same molar concentration (1.7 mM) in the presence of the same molar concentration of unesterified acetate (8.5 mM), and much lower than that found at a physiological concentration of the hexose (8.3 mM). The present results also argue against any significant back-and-forth interconversion of D-glucose 6-phosphate and triose phosphates, under conditions in which sizeable amounts of D-glucose are formed de novo from 13C-enriched Krebs cycle intermediates generated from either D-[1,2-13C]glucose or [2-13C]acetate.     

13C NMR studies of methylene and methine carbons of substrate bound to a 280,000-dalton protein, porphobilinogen synthase

13C NMR has been used to observe the equilibrium complex of [5,5-2H,5-13C]-5-aminolevulinate [( 5,5-2H,5-13C]ALA) bound to porphobilinogen (PBG) synthase (5-aminolevulinate dehydratase), a 280,000-dalton protein. [5,5-2H,5-13C]ALA (chemical shift 46.9 ppm in D2O) was prepared from [5-13C]ALA through enolization in deuteriated neutral potassium phosphate buffer. In the PBG synthase reaction [5,5-2H,5-13C]ALA forms [2,11,11-2H,2,11-13C]PBG (chemical shifts 116.2 ppm for C2 and 34.2 ppm for C11 in D2O). For the complex formed between [5,5-2H,5-13C]ALA and methyl methanethiosulfonate (MMTS) modified PBG synthase, which does not catalyze PBG formation but can form a Schiff base adduct, the chemical shift of 44.2 ppm (line width 92 Hz) identifies an imine structure as the predominant tautomeric form of the Schiff base. By comparison to model compounds, the stereochemistry of the imine has been deduced; however, the protonation state of the imine nitrogen remains unresolved. Reconstitution of the MMTS-modified enzyme-Schiff base complex with Zn(II) and 2-mercaptoethanol results in the holoenzyme-bound equilibrium complex; this complex contains predominantly enzyme-bound PBG, and spectra reveal two peaks from bound PBG and two from free PBG. For bound PBG, C2 is -2.8 ppm from the free signal and C11 is +2.6 ppm from the free signal; the line widths of the bound signals are 55 and 75 Hz, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Resonance Raman study on the flavin in the purple intermediates of D-amino acid oxidase

Resonance Raman (RR) spectra were measured for the purple intermediates of D-amino acid oxidase reconstituted with isotopically labelled FAD's, i.e., [4a-13C]-, [4,10a-13C2]-, [2-13C]-, [5-15N]-, and [1,3-15N2]flavin adenine dinucleotides, and compared with those with the native enzyme. The RR lines around 1605 cm-1 with D-alanine or D-proline as a substrate and at 1548 cm-1 with D-alanine undergo isotopic shifts upon [4a-13C]- and [4,10a-13C2]-labelling. These lines are assigned to the vibrational modes associated with C(10a) = C(4a) - C(4) = O moiety of reduced flavin, providing the first assignment of RR lines of reduced flavin and conclusive evidence that reduced flavin is involved in this intermediate.     

Use of 13C in biosynthetic studies. The labelling pattern in tenellin enriched from isotope-labelled acetate, methionine, and phenylalanine

The biogenetic origin of the carbon atoms in tenellin has been established by adding 13C-enriched compounds to cultures of Beauveria bassiana, and determining the isotopic distribution in the metabolite by 13C nuclear magnetic resonance spectrometry. Tenellin is formed by condensation of an acetate-derived polyketide chain with a phenylpropanoid unit that may be phenylalanine. Alternate carbon atoms of the polyketide chain were labelled with sodium [1(-13C)]- and [2-(13C]-acetate; sodium [1,2-(13C)]acetate was incorporated as intact two-carbon units, the presence of which in tenellin was apparent from coupling between adjacent 13C-enriched carbons. Substituent methyl groups of the polyketide-derived alkenyl chain were labelled with L-[Me-13C]methionine. The labelling patterns from DL-[carboxy-13C]phenylalanine and DL-[alpha-13C]phenylalanine indicated a rearrangement of the propanoid component at some stage in the synthesis. The mass spectrum of tenellin from cultures administered L-[15N]phenylalanine showed isotopic enrichment similar to that obtained with 13C- or 14C-labelled phenylalanine. During incorporation of L-[carboxy-14C, beta-3H]phenylalanine 96% of the tritium label was lost, discounting the possibility of a 1,2-hydride shift during biosynthesis of the metabolite.     

Carbon-13-enriched carbohydrates: preparation of triose, tetrose, and pentose phosphates.

Three-, four-, and five-carbon aldononitrile phosphates were prepared, purified, and catalyticlly reduced with palladium--barium sulfate (5%) to the corresponding aldose phosphates in high yields at pH 1.7 +/- 0.1 and atmopsheric pressure. DL-Glyceraldehyde 3-phosphate and the tetrose 4-phosphates were prepared with carbon-13 enrichment at C-1, while the pentose 5-phosphates were prepared with enrichment at C-1 and C-2. Preparations of glycolaldehyde phosphate and d-glyceraldehyde 3-phosphate by lead tetra-acetate oxidation of glycerol phosphate and fructose 6-phosphate, respectively, are described. The proportions of cyclic hemiacetals and linear gem-diol forms of the two- to five-carbon aldose phosphates in aqueous solution are reported. Carbon-13 chemical shifts and carbon--phosphorus and carbon--hydrogen coupling constants for the furanose phosphate ring and linear gem-diol phosphates are reported and discussed. d-[2(-13)C]Ribulose 1,5-bisphosphate and L-[3,4(-13)C]sorbose 1,6-bisphosphate were prepared enzymatically from D-[2(-13)C]ribose 5-phosphate and dl-[1(-13)C]glyceraldehyde 3-phosphate, respectively.