Terpenoid biosynthesis from 1-deoxy-d-xylulose in higher plants by intramolecular skeletal rearrangement

The incorporation of [1-13C]- and [2,3,4,5-13C4]1-deoxy-d-xylulose into β-carotene, lutein, phytol, and sitosterol in a cell culture of Catharanthus roseus was analyzed by NMR spectroscopy. The labeling patterns of the isoprene precursors, isopentenyl pyrophosphate and dimethylallyl pyrophosphate, were obtained from the terpenes by a retrobiosynthetic approach. 13C Enrichment and 13C13C coupling patterns showed conclusively that 1-deoxy-d-xylulose and not mevalonate is the predominant isoprenoid precursor of phytol, β-carotene, and lutein. Label from 1-deoxyxylulose was also diverted to phytosterols to a minor extent (6% relative to carotene and phytol formation). The data demonstrate that the formation of isopentenyl pyrophosphate from pentulose occurs strictly by an intramolecular rearrangement process.

Identification of a thiamin-dependent synthase in Escherichia coli required for the formation of the 1-deoxy-d-xylulose 5-phosphate precursor to isoprenoids, thiamin, and pyridoxol

In Escherichia coli, 1-deoxy-d-xylulose (or its 5-phosphate, DXP) is the biosynthetic precursor to isopentenyl diphosphate [Broers, S. T. J. (1994) Dissertation (Eidgenössische Technische Hochschule, Zürich)], thiamin, and pyridoxol [Himmeldirk, K., Kennedy, I. A., Hill, R. E., Sayer, B. G. & Spenser, I. D. (1996) Chem. Commun. 1187–1188]. Here we show that an open reading frame at 9 min on the chromosomal map of E. coli encodes an enzyme (deoxyxylulose-5-phosphate synthase, DXP synthase) that catalyzes a thiamin diphosphate-dependent acyloin condensation reaction between C atoms 2 and 3 of pyruvate and glyceraldehyde 3-phosphate to yield DXP. We have cloned and overexpressed the gene (dxs), and the enzyme was purified 17-fold to a specific activity of 0.85 unit/mg of protein. The reaction catalyzed by DXP synthase yielded exclusively DXP, which was characterized by 1H and 31P NMR spectroscopy. Although DXP synthase of E. coli shows sequence similarity to both transketolases and the E1 subunit of pyruvate dehydrogenase, it is a member of a distinct protein family, and putative DXP synthase sequences appear to be widespread in bacteria and plant chloroplasts.

Hybrid characteristics of oligonucleotides consisting of isonucleoside 2′,5′-anhydro-3′-deoxy-3′-(thymin-1-yl)-d-mannitol with different linkage modes

Oligonucleotides consisting of the isonucleoside repeating unit 2′,5′-anhydro-3′-deoxy-3′-(thymin-1-yl)-d-mannitol (4) were synthesized with the monomeric unit 4 incorporated into oligonucleotides as 1′→4′ linkage 4a (oligomer I) or 6′→4′ linkage 4b (oligomer II). The hybrid properties of the two oligonucleotides I and II with their complementary strands were investigated by thermal denaturation and CD spectra. Oligonucleotide I (4a) formed a stable duplex with d(A)14 with a slightly reduced Tm value of 36.6°C, relative to 38.2°C for the control duplex d(T)14/d(A)14, but oligomer II (4b) failed to hybridize with a DNA complementary single strand. The spectrum of the duplex oligomer I/d(A)14 showed a positive CD band at 217 nm and a negative CD band at 248 nm attributable to a B-like conformation. Molecular modeling showed that in the case of oligomer I the C6′ hydroxy group of each unit could be located in the groove area when hybridized to the DNA single strand, which might contribute additional hydrogen bonding to the stability of duplex formation.

Characterization of Recombinant Rhamnogalacturonan α-l-Rhamnopyranosyl-(1,4)-α-d-Galactopyranosyluronide Lyase from Aspergillus aculeatus1 : An Enzyme That Fragments Rhamnogalacturonan I Regions of Pectin

The four major oligomeric reaction products from saponified modified hairy regions (MHR-S) from apple, produced by recombinant rhamnogalacturonan (RG) α-l-rhamnopyranosyl-(1,4)-α-d-galactopyranosyluronide lyase (rRG-lyase) from Aspergillus aculeatus, were isolated and characterized by 1H-nuclear magnetic resonance spectroscopy. They contain an alternating RG backbone with a degree of polymerization of 4, 6, 8, and 10 and with an α-Δ-(4,5)-unsaturated d-galactopyranosyluronic acid at the nonreducing end and an l-rhamnopyranose at the reducing end. l-Rhamnopyranose units are substituted at C-4 with β-galactose. The maximum reaction rate of rRG-lyase toward MHR-S at pH 6.0 and 31°C was 28 units mg−1. rRG-lyase and RG-hydrolase cleave the same alternating RG I subunit in MHR. Both of these enzymes fragment MHR by a multiple attack mechanism. The catalytic efficiency of rRG-lyase for MHR increases with decreasing degree of acetylation. Removal of arabinose side chains improves the action of rRG-lyase toward MHR-S. In contrast, removal of galactose side chains decreased the catalytic efficiency of rRG-lyase. Native RG-lyase was purified from A. aculeatus, characterized, and found to be similar to the rRG-lyase expressed in Aspergillus oryzae.

The crystal structure of an N-terminal two-domain fragment of vascular cell adhesion molecule 1 (VCAM-1): a cyclic peptide based on the domain 1 C-D loop can inhibit VCAM-1-alpha 4 integrin interaction.

Vascular cell adhesion molecule 1 (VCAM-1) represents a structurally and functionally distinct class of immunoglobulin superfamily molecules that bind leukocyte integrins and are involved in inflammatory and immune functions. X-ray crystallography defines the three-dimensional structure of the N-terminal two-domain fragment that participates in ligand binding. Residues in domain 1 important for ligand binding reside in the C-D loop, which projects markedly from one face of the molecule near the contact between domains 1 and 2. A cyclic peptide that mimics this loop inhibits binding of alpha 4 beta 1 integrin-bearing cells to VCAM-1. These data demonstrate how crystallographic structural information can be used to design a small molecule inhibitor of biological function.

Crystal structure of 2,5-diketo-d-gluconic acid reductase A complexed with NADPH at 2.1-Å resolution

The three-dimensional structure of Corynebacterium 2,5-diketo-d-gluconic acid reductase A (2,5-DKGR A; EC 1.1.1.-), in complex with cofactor NADPH, has been solved by using x-ray crystallographic data to 2.1-Å resolution. This enzyme catalyzes stereospecific reduction of 2,5-diketo-d-gluconate (2,5-DKG) to 2-keto-l-gulonate. Thus the three-dimensional structure has now been solved for a prokaryotic example of the aldo–keto reductase superfamily. The details of the binding of the NADPH cofactor help to explain why 2,5-DKGR exhibits lower binding affinity for cofactor than the related human aldose reductase does. Furthermore, changes in the local loop structure near the cofactor suggest that 2,5-DKGR will not exhibit the biphasic cofactor binding characteristics observed in aldose reductase. Although the crystal structure does not include substrate, the two ordered water molecules present within the substrate-binding pocket are postulated to provide positional landmarks for the substrate 5-keto and 4-hydroxyl groups. The structural basis for several previously described active-site mutants of 2,5-DKGR A is also proposed. Recent research efforts have described a novel approach to the synthesis of l-ascorbate (vitamin C) by using a genetically engineered microorganism that is capable of synthesizing 2,5-DKG from glucose and subsequently is transformed with the gene for 2,5-DKGR. These modifications create a microorganism capable of direct production of 2-keto-l-gulonate from d-glucose, and the gulonate can subsequently be converted into vitamin C. In economic terms, vitamin C is the single most important specialty chemical manufactured in the world. Understanding the structural determinants of specificity, catalysis, and stability for 2,5-DKGR A is of substantial commercial interest.

Increased tricarboxylic acid cycle flux in rat brain during forepaw stimulation detected with 1H[13C]NMR.

NMR spectroscopy was used to test recent proposals that the additional energy required for brain activation is provided through nonoxidative glycolysis. Using localized NMR spectroscopic methods, the rate of C4-glutamate isotopic turnover from infused [1-(13)C]glucose was measured in the somatosensory cortex of rat brain both at rest and during forepaw stimulation. Analysis of the glutamate turnover data using a mathematical model of cerebral glucose metabolism showed that the tricarboxylic acid cycle flux [(V(TCA)] increased from 0.49 +/- 0.03 at rest to 1.48 +/- 0.82 micromol/g/min during stimulation (P < 0.01). The minimum fraction of C4-glutamate derived from C1-glucose was approximately 75%, and this fraction was found in both the resting and stimulated rats. Hence, the percentage increase in oxidative cerebral metabolic rate of glucose use (CMRglc) equals the percentage increases in V(TCA) and cerebral metabolic rate of oxygen consumption (CMRO2). Comparison with previous work for the same rat model, which measured total CMRglc [Ueki, M., Linn, F. & Hossman, K. A. (1988) J. Cereb. Blood Flow Metab. 8, 486-4941, indicates that oxidative CMRglc supplies the majority of energy during sustained brain activation.

A synthetic all d-amino acid peptide corresponding to the N-terminal sequence of HIV-1 gp41 recognizes the wild-type fusion peptide in the membrane and inhibits HIV-1 envelope glycoprotein-mediated cell fusion

Recent studies demonstrated that a synthetic fusion peptide of HIV-1 self-associates in phospholipid membranes and inhibits HIV-1 envelope glycoprotein-mediated cell fusion, presumably by interacting with the N-terminal domain of gp41 and forming inactive heteroaggregates [Kliger, Y., Aharoni, A., Rapaport, D., Jones, P., Blumenthal, R. & Shai, Y. (1997) J. Biol. Chem. 272, 13496–13505]. Here, we show that a synthetic all d-amino acid peptide corresponding to the N-terminal sequence of HIV-1 gp41 (D-WT) of HIV-1 associates with its enantiomeric wild-type fusion (WT) peptide in the membrane and inhibits cell fusion mediated by the HIV-1 envelope glycoprotein. D-WT does not inhibit cell fusion mediated by the HIV-2 envelope glycoprotein. WT and D-WT are equally potent in inducing membrane fusion. D-WT peptide but not WT peptide is resistant to proteolytic digestion. Structural analysis showed that the CD spectra of D-WT in trifluoroethanol/water is a mirror image of that of WT, and attenuated total reflectance–fourier transform infrared spectroscopy revealed similar structures and orientation for the two enantiomers in the membrane. The results reveal that the chirality of the synthetic peptide corresponding to the HIV-1 gp41 N-terminal sequence does not play a role in liposome fusion and that the peptides’ chirality is not necessarily required for peptide–peptide interaction within the membrane environment. Furthermore, studies along these lines may provide criteria to design protease-resistant therapeutic agents against HIV and other viruses.

Enhanced stability of maize endosperm ADP-glucose pyrophosphorylase is gained through mutants that alter subunit interactions

Temperature lability of ADP-glucose pyrophosphorylase (AGP; glucose-1-phosphate adenylyltransferase; ADP: α-d-glucose-1-phosphate adenylyltransferase, EC 2.7.7.27), a key starch biosynthetic enzyme, may play a significant role in the heat-induced loss in maize seed weight and yield. Here we report the isolation and characterization of heat-stable variants of maize endosperm AGP. Escherichia coli cells expressing wild type (WT) Shrunken2 (Sh2), and Brittle2 (Bt2) exhibit a reduced capacity to produce glycogen when grown at 42°C. Mutagenesis of Sh2 and coexpression with WT Bt2 led to the isolation of multiple mutants capable of synthesizing copious amounts of glycogen at this temperature. An increase in AGP stability was found in each of four mutants examined. Initial characterization revealed that the BT2 protein was elevated in two of these mutants. Yeast two-hybrid studies were conducted to determine whether the mutant SH2 proteins more efficiently recruit the BT2 subunit into tetramer assembly. These experiments showed that replacement of WT SH2 with the heat-stable SH2HS33 enhanced interaction between the SH2 and BT2 subunits. In agreement, density gradient centrifugation of heated and nonheated extracts from WT and one of the mutants, Sh2hs33, identified a greater propensity for heterotetramer dissociation in WT AGP. Sequencing of Sh2hs33 and several other mutants identified a His-to-Tyr mutation at amino acid position 333. Hence, a single point mutation in Sh2 can increase the stability of maize endosperm AGP through enhanced subunit interactions.

Adenosine diphosphate glucose pyrophosphatase: A plastidial phosphodiesterase that prevents starch biosynthesis

A distinct phosphodiesterasic activity (EC 3.1.4) was found in both mono- and dicotyledonous plants that catalyzes the hydrolytic breakdown of ADPglucose (ADPG) to produce equimolar amounts of glucose-1-phosphate and AMP. The enzyme responsible for this activity, referred to as ADPG pyrophosphatase (AGPPase), was purified over 1,100-fold from barley leaves and subjected to biochemical characterization. The calculated Keq′ (modified equilibrium constant) value for the ADPG hydrolytic reaction at pH 7.0 and 25°C is 110, and its standard-state free-energy change value (ΔG′) is −2.9 kcal/mol (1 kcal = 4.18 kJ). Kinetic analyses showed that, although AGPPase can hydrolyze several low-molecular weight phosphodiester bond-containing compounds, ADPG proved to be the best substrate (Km = 0.5 mM). Pi and phosphorylated compounds such as 3-phosphoglycerate, PPi, ATP, ADP, NADP+, and AMP are inhibitors of AGPPase. Subcellular localization studies revealed that AGPPase is localized exclusively in the plastidial compartment of cultured cells of sycamore (Acer pseudoplatanus L.), whereas it occurs both inside and outside the plastid in barley endosperm. In this paper, evidence is presented that shows that AGPPase, whose activity declines concomitantly with the accumulation of starch during development of sink organs, competes with starch synthase (ADPG:1,4-α-d-glucan 4-α-d-glucosyltransferase; EC 2.4.1.21) for ADPG, thus markedly blocking the starch biosynthesis.

Carbohydrate and Amino Acid Metabolism in the Eucalyptus globulus-Pisolithus tinctorius Ectomycorrhiza during Glucose Utilization1

The metabolism of [1-13C]glucose in Pisolithus tinctorius cv Coker & Couch, in uninoculated seedlings of Eucalyptus globulus bicostata ex Maiden cv Kirkp., and in the E. globulus-P. tinctorius ectomycorrhiza was studied using nuclear magnetic resonance spectroscopy. In roots of uninoculated seedlings, the 13C label was mainly incorporated into sucrose and glutamine. The ratio (13C3 + 13C2)/13C4 of glutamine was approximately 1.0 during the time-course experiment, indicating equivalent contributions of phosphoenolpyruvate carboxylase and pyruvate dehydrogenase to the production of α-ketoglutarate used for synthesis of this amino acid. In free-living P. tinctorius, most of the 13C label was incorporated into mannitol, trehalose, glutamine, and alanine, whereas arabitol, erythritol, and glutamate were weakly labeled. Amino acid biosynthesis was an important sink of assimilated 13C (43%), and anaplerotic CO2 fixation contributed 42% of the C flux entering the Krebs cycle. In ectomycorrhizae, sucrose accumulation was decreased in the colonized roots compared with uninoculated control plants, whereas 13C incorporation into arabitol and erythritol was nearly 4-fold higher in the symbiotic mycelium than in the free-living fungus. It appears that fungal utilization of glucose in the symbiotic state is altered and oriented toward the synthesis of short-chain polyols.

Elucidation of a PTS–Carbohydrate Chemotactic Signal Pathway in Escherichia coli Using a Time-resolved Behavioral Assay

Chemotaxis of Escherichia coli toward phosphotransferase systems (PTSs)–carbohydrates requires phosphoenolpyruvate-dependent PTSs as well as the chemotaxis response regulator CheY and its kinase, CheA. Responses initiated by flash photorelease of a PTS substrates d-glucose and its nonmetabolizable analog methyl α-d-glucopyranoside were measured with 33-ms time resolution using computer-assisted motion analysis. This, together with chemotactic mutants, has allowed us to map out and characterize the PTS chemotactic signal pathway. The responses were absent in mutants lacking the general PTS enzymes EI or HPr, elevated in PTS transport mutants, retarded in mutants lacking CheZ, a catalyst of CheY autodephosphorylation, and severely reduced in mutants with impaired methyl-accepting chemotaxis protein (MCP) signaling activity. Response kinetics were comparable to those triggered by MCP attractant ligands over most of the response range, the most rapid being 11.7 ± 3.1 s−1. The response threshold was <10 nM for glucose. Responses to methyl α-d-glucopyranoside had a higher threshold, commensurate with a lower PTS affinity, but were otherwise kinetically indistinguishable. These facts provide evidence for a single pathway in which the PTS chemotactic signal is relayed rapidly to MCP–CheW–CheA signaling complexes that effect subsequent amplification and slower CheY dephosphorylation. The high sensitivity indicates that this signal is generated by transport-induced dephosphorylation of the PTS rather than phosphoenolpyruvate consumption.

Blood glutathione synthesis rates in healthy adults receiving a sulfur amino acid-free diet

The availability of cysteine is thought to be the rate limiting factor for synthesis of the tripeptide glutathione (GSH), based on studies in rodents. GSH status is compromised in various disease states and by certain medications leading to increased morbidity and poor survival. To determine the possible importance of dietary cyst(e)ine availability for whole blood glutathione synthesis in humans, we developed a convenient mass spectrometric method for measurement of the isotopic enrichment of intact GSH and then applied it in a controlled metabolic study. Seven healthy male subjects received during two separate 10-day periods an l-amino acid based diet supplying an adequate amino acid intake or a sulfur amino acid (SAA) (methionine and cysteine) free mixture (SAA-free). On day 10, l-[1-13C]cysteine was given as a primed, constant i.v. infusion (3μmol⋅kg−1⋅h−1) for 6 h, and incorporation of label into whole blood GSH determined by GC/MS selected ion monitoring. The fractional synthesis rate (mean ± SD; day-1) of whole blood GSH was 0.65 ± 0.13 for the adequate diet and 0.49 ± 0.13 for the SAA-free diet (P < 0.01). Whole blood GSH was 1,142 ± 243 and 1,216 ± 162 μM for the adequate and SAA-free periods (P > 0.05), and the absolute rate of GSH synthesis was 747 ± 216 and 579 ± 135 μmol⋅liter−1⋅day−1, respectively (P < 0.05). Thus, a restricted dietary supply of SAA slows the rate of whole blood GSH synthesis and diminishes turnover, with maintenance of the GSH concentration in healthy subjects.

Further Studies of the Role of Cyclic β-Glucans in Symbiosis. An ndvC Mutant of Bradyrhizobium japonicum Synthesizes Cyclodecakis-(1→3)-β-Glucosyl1

The cyclic β-(1→3),β-(1→6)-d-glucan synthesis locus of Bradyrhizobium japonicum is composed of at least two genes, ndvB and ndvC. Mutation in either gene affects glucan synthesis, as well as the ability of the bacterium to establish a successful symbiotic interaction with the legume host soybean (Glycine max). B. japonicum strain AB-14 (ndvB::Tn5) does not synthesize β-glucans, and strain AB-1 (ndvC::Tn5) synthesizes a cyclic β-glucan lacking β-(1→6)-glycosidic bonds. We determined that the structure of the glucan synthesized by strain AB-1 is cyclodecakis-(1→3)-β-d-glucosyl, a cyclic β-(1→3)-linked decasaccharide in which one of the residues is substituted in the 6 position with β-laminaribiose. Cyclodecakis-(1→3)-β-d-glucosyl did not suppress the fungal β-glucan-induced plant defense response in soybean cotyledons and had much lower affinity for the putative membrane receptor protein than cyclic β-(1→3),β-(1→6)-glucans produced by wild-type B. japonicum. This is consistent with the hypothesis presented previously that the wild-type cyclic β-glucans may function as suppressors of a host defense response.

Contribution of Malic Enzyme, Pyruvate Kinase, Phosphoenolpyruvate Carboxylase, and the Krebs Cycle to Respiration and Biosynthesis and to Intracellular pH Regulation during Hypoxia in Maize Root Tips Observed by Nuclear Magnetic Resonance Imaging and Gas Chromatography-Mass Spectrometry1

In vivo pyruvate synthesis by malic enzyme (ME) and pyruvate kinase and in vivo malate synthesis by phosphoenolpyruvate carboxylase and the Krebs cycle were measured by 13C incorporation from [1-13C]glucose into glucose-6-phosphate, alanine, glutamate, aspartate, and malate. These metabolites were isolated from maize (Zea mays L.) root tips under aerobic and hypoxic conditions. 13C-Nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry were used to discern the positional isotopic distribution within each metabolite. This information was applied to a simple precursor-product model that enabled calculation of specific metabolic fluxes. In respiring root tips, ME was found to contribute only approximately 3% of the pyruvate synthesized, whereas pyruvate kinase contributed the balance. The activity of ME increased greater than 6-fold early in hypoxia, and then declined coincident with depletion of cytosolic malate and aspartate. We found that in respiring root tips, anaplerotic phosphoenolpyruvate carboxylase activity was high relative to ME, and therefore did not limit synthesis of pyruvate by ME. The significance of in vivo pyruvate synthesis by ME is discussed with respect to malate and pyruvate utilization by isolated mitochondria and intracellular pH regulation under hypoxia.