Determination of the anomeric specificity of the Escherichia coli CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase by 13C NMR spectroscopy

[99%, 1-13C]- and [90%, 2-13C]3-deoxy-D-manno-octulosonic acid (KDO) were prepared enzymatically and used to determine the anomeric specificity of the CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyl transferase (CMP-KDO synthetase) by 13C NMR spectroscopy. Addition of CMP-KDO synthetase to reaction mixtures containing either 1-13C- or 2-13C-labeled KDO resulted in rapid CMP-KDO formation which was accompanied by a substantial decrease in the 13C-enriched resonances of the beta-pyranose form of KDO relative to the resonances of other KDO species in solution, demonstrating that the beta-pyranose is the preferred substrate. Concomitant with the production of CMP-KDO was the appearance of peaks at 174.3 and 101.4 ppm when [1-13C]- and [2-13C]KDO, respectively, were used as substrates. The correspondence of these resonances to the enriched carbons in CMP-KDO was confirmed by the expected 3-bond (3JP,C-1 = 6.9 Hz) and 2-bond coupling (2JP,C-2 = 8.3 Hz) between the labeled carbons and the ketosidically linked phosphoryl group. A large coupling (3J = 5.7 Hz) was observed in proton-coupled spectra of CMP-[1-13C]KDO between carbon 1 and the axial proton at carbon 3 of KDO. The magnitude of this coupling constant supports a diaxial relationship between these two groups and, along with chemical shift data, indicates that KDO retains the beta-configuration when linked in CMP-KDO.     

Inhibition of exogenous 3-deoxy-D-manno-octulosonate incorporation into lipid A precursor of toluene-treated Salmonella typhimurium cells.

Analogs of 3-deoxy-D-manno-octulosonate (KDO) were designed to inhibit CTP:CMP-KDO cytidylyltransferase (CMP-KDO synthetase). Since these analogs lacked whole-cell antibacterial activity, a permeabilized-cell method was developed to measure intracellular compound activity directly. The method employed a mutant of Salmonella typhimurium defective in KDO-8-phosphate synthetase (kdsA), which accumulated lipid A precursor at 42 degrees C. Cells permeabilized with 1% toluene were used to evaluate inhibitor effect on [3H]KDO incorporation into preformed lipid A precursor. KDO incorporation proceeded through the enzymes CMP-KDO synthetase and CMP-KDO:lipid A KDO transferase. Optimum KDO incorporation occurred between pH 8 and 9 and required CTP, prior lipid A precursor accumulation, and a functional kdsB gene product, CMP-KDO synthetase. The apparent Km for KDO in this coupled system at pH 7.6 was 1.38 mM. The reaction products isolated and characterized contained 1 and 2 KDO residues per lipid A precursor molecule. Several KDO analogs produced concentration-related reductions of KDO incorporation in toluenized cells with 50% inhibitory concentrations comparable to those obtained in purified CMP-KDO synthetase systems. Two compounds, 8-amino-2-deoxy-KDO (A-60478) and 8-aminomethyl-2-deoxy-KDO (A-60821), competitively inhibited KDO incorporation, displaying Kis of 4.2 microM for A-60478 and 2.5 microM for A-60821. These data indicated that the inactivity of the KDO analogs on intact bacteria was the result of poor permeation into cells rather than intracellular inactivation.     

Lipid A precursor from Pseudomonas aeruginosa is completely acylated prior to addition of 3-deoxy-D-manno-octulosonate

Inhibition of lipopolysaccharide (LPS) synthesis in Pseudomonas aeruginosa at the stage of incorporation of 3-deoxy-D-manno-octulosonate (KDO) caused accumulation of a lipid A precursor which contained all of the fatty acids present on the lipid A of mature LPS. The enzyme CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase) from P. aeruginosa is inhibited by the KDO analog alpha-C-[1,5-anhydro-8-amino-2,7,8-trideoxy-D-manno-octopyranosyl] carboxylate (I), and I is effectively delivered to P. aeruginosa following attachment by amide linkage to the carboxyl terminus of alanylalanine. Intracellular hydrolysis releases the free inhibitor (I) which then inhibits activation of KDO by CMP-KDO synthetase causing accumulation of lipid A precursor and subsequent growth stasis. The major lipid A precursor species accumulated was purified and found to contain glucosamine, phosphate, C12:O, 2OH-C12:O and 3OH-C10:0 (in ester linkage), and 3OH-C12:0 (in amide linkage) in molar ratios of 1:1:0.5:0.5:1:1. Analysis of precursor by fast atom bombardment mass spectroscopy yielded a major ion (M - H)- of mass 1616 and fragments which were consistent with the structure of lipid A from P. aeruginosa. In contrast, Salmonella typhimurium, Escherichia coli, Citrobacter sp., Serratia marcescens, Enterobacter aerogenes, and Enterobacter cloacae all accumulated underacylated lipid A precursors which only contained 3-OH-C14:0, glucosamine, and phosphate. This difference and species-specific patterns of major and minor precursor species show that early steps in the assembly of lipid A are similar, but not identical in enteric and nonenteric Gram-negative bacteria.     

Purification and characterization of cytidine 5''-triphosphate:cytidine 5''-monophosphate-3-deoxy-D-manno-octulosonate cytidylyltransferase

Cytidine 5'-triphosphate:cytidine 5'-monophosphate-3-deoxy-D-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase) was purified 2,300-fold from frozen Escherichia coli B cells. The enzyme catalyzed the formation of CMP-KDO, a very labile product, from CTP and KDO. No other sugar tested could replace KDO as an alternate substrate. Uridine 5'-triphosphate at pH 9.5 and deoxycytidine 5'-triphosphate at pH 8.0 and 9.5 could be used as alternate substrates in place of CTP. CMP-KDO synthetase required Mg2+ at a concentration of 10.0 mM for optimal activity. The pH optimum was determined to be between 9.6 and 9.3 in tris(hydroxymethyl)aminomethane-acetate or sodium-glycine buffer. This enzyme had an isoelectric point between pH 4.15 and 4.4 and appeared to be a single polypeptide chain with a molecular weight of 36,000 to 40,000. The apparent Km values for CTP and KDO in the presence of 10.0 mM Mg2+ were determined to be 2.0 X 10(-4) and 2.9 X 10(-4) M, respectively, at pH 9.5. Uridine 5'-triphosphate and deoxycytidine 5'-triphosphate had apparent Km values of 8.8 X 10(-4) and 3.4 X 10(-4) M. respectively, at pH 9.5.     

Biosynthesis of lipid A in Escherichia coli. Acyl carrier protein-dependent incorporation of laurate and myristate

In previous studies we described enzyme(s) from Escherichia coli that transfer two 3-deoxy-D-manno-octulosonate (KDO) residues from two CMP-KDO molecules to a tetraacyldisaccharide-1,4'-bis-phosphate precursor of lipid A, termed lipid IVA (Brozek, K. A., Hosaka, K., Robertson, A. D., and Raetz, C. R. H. (1989) J. Biol. Chem. 264, 6956-6966). The product, designated (KDO)2-IVA, can be prepared in milligram quantities and/or radiolabeled with 32P at position 4' of the IVA moiety. We now demonstrate the presence of enzymes in E. coli extracts that transfer laurate and/or myristate residues from lauroyl or myristoyl-acyl carrier protein (ACP) to (KDO)2-IVA. Thioesters of coenzyme A are not substrates. The cytosolic fraction catalyzes rapid acylation with lauroyl-ACP, but not with myristoyl, R-3-hydroxymyristoyl, palmitoyl, or palmitoleoyl-ACP. The membrane fraction transfers both laurate and myristate to (KDO)2-IVA. Evidence for the enzymatic acylation of (KDO)2-IVA is provided by (a) conversion of [4'-32P](KDO)2-IVA to more rapidly migrating products in the presence of the appropriate acyl-ACP, (b) incorporation of [1-14C]laurate or [1-14C]myristate into these metabolites in the presence of (KDO)2-IVA, (c) fast atom bombardment-mass spectrometry, and (d) 1H NMR spectroscopy. At protein concentrations less than 0.5 mg/ml, the acylation of (KDO)2-IVA by the cytoplasmic fraction is absolutely dependent upon the addition of exogenous acyl-ACP. These acyltransferases cannot utilize lipid IVA as a substrate, demonstrating that they possess novel KDO recognition domains. The unusual substrate specificity of these enzymes provides compelling evidence for their involvement in lipid A biosynthesis. Depending on the conditions it is possible to acylate (KDO)2-IVA with 1 or 2 lauroyl residues, with 1 or 2 myristoyl residues, or with 1 of each.     

Local structural features around the C-terminal segment of Streptomyces subtilisin inhibitor studied by carbonyl carbon nuclear magnetic resonances three phenylalanyl residues

The carbonyl carbon NMR signals of the Phe residues in Streptomyces subtilisin inhibitor (SSI) were selectively observed for [F]SSI, in which all phenylalanines were uniformly labeled with [1-13C]Phe. The three enhanced resonances in the spectrum of [F]SSI were unambiguously assigned to the specific sites in the amino acid sequence by means of 15N,13C double-labeling techniques. Namely, the resonances at 174.9 and 172.6 ppm (in D2O, pH 7.3, 50 degrees C) showed the satellite peaks due to 13C-15N spin coupling in the spectra of [F,GS]SSI and [F,A]SSI, in which Ser/Gly and Ala residues were labeled with [15N]Gly/Ser and [15N]Ala, respectively, together with [1-13C]Phe. The carbonyl groups of Phe-97 and Phe-111 are involved in peptide bonds with the amino nitrogens of Ser-98 and Ala-112, respectively. These results clearly indicate that the signals at 174.5 and 172.6 ppm are due to Phe-97 and Phe-111, respectively. The signal at the lowest field (177.1 ppm) was thus assigned to the carboxyl carbon of the C-terminal Phe-113. The lifetimes of the amide hydrogens of the three Phe residues and their C-terminal-side neighbors (Ser-98 and Ala-112) were investigated by using the effect of deuterium-hydrogen exchange of amide on the line shapes (DEALS) for the Phe carbonyl carbon resonances. In this method, the NMR spectra of [F]SSI dissolved in 50% D2O (pH 7.3) were measured at various temperatures, and the line shape changes caused by deuteriation isotope shifts were analyzed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of Salmonella typhimurium strains that utilize exogenous 3-deoxy-D-manno-octulosonate for synthesis of lipopolysaccharide.

Spontaneous mutants of Salmonella typhimurium LT2 were selected for the ability to accumulate exogenous 3-deoxy-D-manno-octulosonate (KDO). Bacteria containing a gene (kdsA) which codes for a temperature-sensitive KDO-8-phosphate synthetase were plated at the restrictive temperature of 42 degrees C on medium containing 5 mM KDO. Since bacteria containing the kdsA lesion are unable to grow at 42 degrees C due to inhibition of lipopolysaccharide (LPS) synthesis and accumulation of lipid A precursor, this method allowed direct, positive selection of mutants capable of utilizing exogenous KDO for LPS synthesis. Spontaneous mutants, selected at a frequency of about 10(-6), required exogenous KDO for growth at 42 degrees C. The growth rate at 42 degrees C was nearly normal in the presence of 20 mM KDO and was directly proportional to KDO concentrations below 20 mM. Exogenous KDO also suppressed accumulation of lipid A precursor. The apparent Km for KDO accumulation was 23 mM, and the maximum rate of transport was calculated to be 505 pmol of KDO per min per 10(8) cells. Bacteria incorporated exogenous [3H]KDO exclusively into LPS, with less than 10% dilution in specific activity due to residual endogenous KDO synthesis. The mutation giving rise to the ability to accumulate exogenous KDO was extremely useful in the direct screening for new mutations in the kdsA gene after localized mutagenesis. Five mutations in kdsA were isolated, four of which were new alleles as determined by on fine-structure analysis. The ability to introduce labeled (3H, 13C, and 14C) KDO in vivo should simplify and extend the analysis of this critical metabolic pathway in gram-negative bacteria.     

Purine nucleoside phosphorylase cleaves the C--O bond of ribose 1-phosphate. Evidence from the 18O shift in 31P NMR.

An equilibrium mixture of highly enriched [18(O)]Pi (represents the mixture of [[18(O)4]Pi, [[18(O)3]Pi, [18(O)2]Pi as represented in the figures, unless otherwise specified), alpha-D-ribose 1-[16(O)]phosphate, and hypoxanthine plus inosine was equilibrated with calf spleen purine-nucleoside phosphorylase (EC 2.4.2.1). The 31P NMR spectrum clearly indicated the formation of alpha-D-ribose 1-[18(O)4]-phosphate and of [16(O)]Pi. Incubation for the same time span in the absence of alpha-D-ribose 1-phosphate left the [18(O)4]Pi isotopic distribution unchanged. The results clearly demonstrated that the C--O bond of alpha-D-ribose 1-phosphate is cleaved in the enzymatic reaction. It is unlikely that the enzyme catalyzes the exchange of oxygen between Pi and H2O. Several possible mechanistic pathways are ruled out by the results, which demand attack by a phosphate oxygen at the anomeric C-1' atom.     

Biosynthetic studies on the tropane ring system of the tropane alkaloids from Datura stramonium

Isotopic labelling experiments have been carried out in Datura stramonium root cultures with the following isotopically labelled precursors; [2H3]- [2-13C, 2H3]-, [1-13C, 18O2]-acetates, 2H2O, [2H3-methyl]-methionine, [2-13C]-phenyllactate, [3-2H]-tropine and [2'-13C, 3-2H]-littorine. The study explored the incorporation of isotope into the tropane ring system of littorine 1 and hyoscyamine 2 and revealed that deuterium from acetate is incorporated only into C-6 and C-7, and not into C-2 and C-4 as previously reported. Oxygen-18 was not retained at a detectable level into the C(3)-O bond from [1-13C, 18O2]-acetate. The intramolecular nature of the rearrangement of littorine 1 to hyoscyamine 2 is revealed again by a labelling study using [2'-13C, 3-2H]-littorine, [2-13C]-phenyllactate and [3-2H]-tropine.     

3-hydroxy-3-methylglutaryl-coenzyme A synthase reaction intermediates: detection of a covalent tetrahedral adduct by differential isotope shift 13C nuclear magnetic resonance spectroscopy

Binding of [1,2-(13)C]acetyl-CoA to wild-type 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase is characterized by large upfield shifts for C1 (184 ppm, Deltadelta = 20 ppm) and C2 (26 ppm, Deltadelta = 7 ppm) resonances that are attributable to formation of the covalent [1,2 -(13)C]acetyl-S-enzyme reaction intermediate. NMR spectra of [1, 2-(13)C]acetyl-S-enzyme prepared in H(2)(16)O versus H(2)(18)O indicate a 0.055 ppm upfield shift of the C1 resonance in the presence of the heavier isotope. The magnitude of this (18)O-induced (13)C shift suggests that the 184 ppm resonance is attributable to a reaction intermediate in which C1 exhibits substantial carbonyl character. No significant shift of the C2 resonance occurs. These observations suggest that, in the absence of second substrate (acetoacetyl-CoA), enzymatic addition of H(2)(18)O to the C1 carbonyl of acetyl-S-enzyme occurs to transiently produce a tetrahedral species. This tetrahedral adduct exchanges oxygen upon backward collapse to re-form the sp(2)-hybridized thioester carbonyl. In contrast with HMG-CoA synthase, C378G Zoogloea ramigera beta-ketothiolase, which also forms a (13)C NMR-observable covalent acetyl-enzyme species, exhibits no (18)O-induced shift. Formation of the [(13)C]acetyl-S-enzyme reaction intermediate of HMG-CoA synthase in D(2)O versus H(2)O is characterized by a time-dependent isotope-induced upfield shift of the C1 resonance (maximal shift = 0. 185 ppm) in the presence of the heavier isotope. A more modest upfield shift (0.080 ppm) is observed for C378G Z. ramigera beta-ketothiolase in similar experiments. The slow kinetics for the development of the deuterium-induced (13)C shift in the HMG-CoA synthase experiments suggest a specific interaction (hydrogen bond) with a slowly exchangeable proton (deuteron) of a side chain/backbone of an amino acid residue at the active site.     

3-Deoxy-D-manno-octulosonate-8-phosphate synthase catalyzes the C-O bond cleavage of phosphoenolpyruvate

The mechanism of 3-deoxy-D-manno-octulosonate-8-phosphate (KDO8P) synthase was investigated. When [18O]-PEP specifically labeled in the enolic oxygen is a substrate for KDO8P synthase, the 18O is recovered in Pi. This indicates that the KDO8P synthase reaction proceeds with C-O bond cleavage of PEP similar to that observed in the 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase catalyzed condensation of PEP and erythrose-4-phosphate (1). No evidence for a covalent enzyme-PEP intermediate could be obtained. No [32P]-Pi exchange into PEP nor scrambling of bridge 18O to non-bridging positions in [18O]-PEP was observed in the presence or absence of arabinose-5-phosphate or its analog ribose-5-phosphate. Bromopyruvate inactivated KDO8P synthase in a time dependent process. It is likely that bromopyruvate reacts with a functional group at the PEP binding site since PEP, but not arabinose-5-phosphate, protects against inactivation.     

Mechanistic insight into 3-deoxy-D-manno-octulosonate-8-phosphate synthase and 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase utilizing phosphorylated monosaccharide analogues

Escherichia coli 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8-P) synthase is able to utilize the five-carbon phosphorylated monosaccharide, 2-deoxyribose 5-phosphate (2dR5P), as an alternate substrate, but not D-ribose 5-phosphate (R5P) nor the four carbon analogue D-erythrose 4-phosphate (E4P). However, E. coli KDO8-P synthase in the presence of either R5P or E4P catalyzes the rapid consumption of approximately 1 mol of PEP per active site, after which consumption of PEP slows to a negligible but measurable rate. The mechanism of this abortive utilization of PEP was investigated using [2,3-(13)C(2)]-PEP and [3-F]-PEP, and the reaction products were determined by (13)C, (31)P, and (19)F NMR to be pyruvate, phosphate, and 2-phosphoglyceric acid (2-PGA). The formation of pyruvate and 2-PGA suggests that the reaction catalyzed by KDO8-P synthase may be initiated via a nucleophilic attack to PEP by a water molecule. In experiments in which the homologous enzyme, 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7-P) synthase was incubated with D,L-glyceraldehyde 3-phosphate (G3P) and [2,3-(13)C(2)]-PEP, pyruvate and phosphate were the predominant species formed, suggesting that the reaction catalyzed by DAH7-P synthase starts with a nucleophilic attack by water onto PEP as observed in E. coli KDO8-P synthase.     

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)     

Adenosine deaminase converts purine riboside into an analogue of a reactive intermediate: a 13C NMR and kinetic study

The 13C NMR spectra of [2-13C]- and [6-13C]purine ribosides have been obtained free in solution and bound to the active site of adenosine deaminase. The positions of the resonances of the bound ligand are shifted relative to those of the free ligand as follows: C-2, -3.7 ppm; C-6, -73.1 ppm. The binary complexes are in slow exchange with free purine riboside on the NMR time scale, and the dissociation rate constant is estimated to be 13.5 s-1 from the slow exchange broadening of the free signal. In aqueous solution, protonation of purine riboside at N-1 results in changes in 13C chemical shift relative to those of the free base as follows: C-2, -4.9 ppm; C-6, -7.9 ppm. The changes in chemical shift that occur when purine riboside binds to the enzyme indicate that the hybridization of C-6 changes from sp2 to sp3 in the binary complex with formation of a new bond to oxygen or sulfur. A change in C-2 hybridization can be eliminated as can protonation at N-1 as the sole cause of the chemical shift changes. The kinetic constants for the adenosine deaminase catalyzed hydrolysis of 6-chloro- and 6-fluoropurine riboside have been compared, and the reactivity order implies that carbon-halogen bond breaking does not occur in the rate-determining step. These observations support a mechanism for the enzyme in which formation of a tetrahedral intermediate is the most difficult chemical step. Enzymic stabilization of this intermediate may be an important catalytic strategy used by the enzyme to lower the standard free energy of the preceding transition state.     

Carbon-13 Nuclear Magnetic Resonance Study of Metabolism of Propionate by Escherichia coli

We have evaluated the use of [1,2-13C2]propionate for the analysis of propionic acid metabolism, based on the ability to distinguish between the methylcitrate and methylmalonate pathways. Studies using propionate-adapted Escherichia coli MG1655 cells were performed. Preservation of the 13C-13C-12C carbon skeleton in labeled alanine and alanine-containing peptides involved in cell wall recycling is indicative of the direct formation of pyruvate from propionate via the methylcitrate cycle, the enzymes of which have recently been demonstrated in E. coli. Additionally, formation of 13C-labeled formate from pyruvate by the action of pyruvate-formate lyase is also consistent with the labeling of pyruvate C-1. Carboxylation of the labeled pyruvate leads to formation of [1,2-13C2]oxaloacetate and to multiply labeled glutamate and succinate isotopomers, also consistent with the flux through the methylcitrate pathway, followed by the tricarboxylic acid (TCA) cycle. Additional labeling of TCA intermediates arises due to the formation of [1-13C]acetyl coenzyme A from the labeled pyruvate, formed via pyruvate-formate lyase. Labeling patterns in trehalose and glycine are also interpreted in terms of the above pathways. The information derived from the [1, 2-13C2]propionate label is contrasted with information which can be derived from singly or triply labeled propionate and shown to be more useful for distinguishing the different propionate utilization pathways via nuclear magnetic resonance analysis.     

Early biosynthetic pathway to abscisic acid in Cercospora cruenta

A new biosynthetic intermediate of ABA, (2Z,4E)-gamma-ionylideneacetaldehyde, was isolated from young mycelia of Cercospora cruenta. Under an (18)O2 atmosphere, an oxygen atom of this endogenous aldehyde was exclusively labeled. Similarly, three (18)O atoms were incorporated into the ABA molecule recovered after prolonged incubation; selectively labeled were one of the carboxyl oxygen atoms and the two on the ring portion of ABA. A feeding experiment with [1-(13)C]glucose proved the exclusive operation of the mevalonate pathway for the formation of both ABA and beta-carotene. These results suggest that (2Z,4E)-gamma-ionylideneacetaldehyde can be a key ABA biosynthetic intermediate formed by the oxidative cleavage of a carotenoid precursor.     

A 13C NMR study of [5,8-13C2]spermidine binding to tRNA and to Escherichia coli macromolecules

[5,8-13C2]Spermidine was prepared by synthesis, and its binding to macromolecular structures of Escherichia coli was studied. When added to E. coli cells, the two signals of [13C]spermidine (C-5, 47.8 ppm, and C-8, 39.6 ppm; JC-C = 5.8 Hz) were strongly broadened due to binding to macromolecules. When [13C]spermidine was added to E. coli tRNA, the C-5 resonance broadened to v1/2 = 4.7 Hz, whereas the C-8 resonance broadened to v1/2 = 2.7 Hz. tRNA-bound [13C]spermidine could be chased by [12C]spermidine or spermine, but not by putrescine or cadaverine. By using mixtures of [5-13C]- and [8-13C]spermidines (where 13C-13C coupling was avoided), it was possible to estimate a dissociation constant (Kd) of 3 x 10(-3) M using the C-5 v1/2obs values and a Kd of 2.10(-3) M using the C-8 v1/2obs values. The number of spermidine-binding sites (n) could also be estimated by fitting the bound spermidine molar fraction versus tRNA concentration. Values of n = 12 +/- 2 and 14 +/- 3 were obtained for C-5 and C-8, respectively. Measurements of line narrowing at increasing Mg2+ concentrations indicated that approximately 11 spermidines (of the 12-14 bound ones) could be displaced by the former, whereas 3 spermidines remain strongly bound to the tRNA backbone. Measurements of free and bound T1 allowed the determination of a correlation time of 10(-10)s for tRNA-bound spermidine.     

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.     

GTP hydrolysis by tubulin occurs with water oxygen incorporation into inorganic phosphate

Tubulin assembly was conducted in [18O]H2O and the resulting mixture of GTP, GDP and Pi was examined by 31P-NMR. Two Pi signals, separated by about 0.02 ppm, were observed. By combining this mixture with a solution of Pi containing all five possible 16O and 18O isotopomers of Pi, it was shown that the two signals were due to [16O4]- and [16O3, 18O]Pi. The amount of 18O incorporated into the Pi was that expected if the hydrolysis of GTP during tubulin assembly occurs with cleavage of the gamma-phosphorus-bridge oxygen bond.     

Surface dynamics of bacteriorhodopsin as revealed by (13)C NMR studies on [(13)C]Ala-labeled proteins: detection of millisecond or microsecond motions in interhelical loops and C-terminal alpha-helix

We have recorded (13)C NMR spectra of [2-(13)C]-, [1-(13)C]-, [3-(13)C],- and [1,2,3-(13)C(3)]Ala-labeled bacteriorhodopsin (bR), and its mutants, A196G, A160G, and A103C, by means of cross polarization-magic angle spinning (CP-MAS) and dipolar decoupled-magic angle spinning (DD-MAS) techniques, to reveal the conformation and dynamics of bR, with emphasis on the loop and C-terminus structures. The (13)C NMR signals of the loop (C-D, E-F, and F-G) regions were almost completely suppressed from [2-(13)C]-, [1-(13)C]Ala-, and [1-(13)C]Gly-labeled bR, due to the presence of conformational fluctuation with correlation times of 10(-4) s that interfered with the peak-narrowing by magic angle spinning. The observation of such suppressed peaks for specific residues provides a unique means of detecting intermediate frequency motions on the time scale of ms or micros in the surface loops of membrane proteins. Instead, the three well-resolved (13)C CP-MAS NMR signals of [2-(13)C]Ala-bR, at 50.38, 49.90, and 47.96 ppm, were ascribed to the C-terminal alpha-helix previously proposed from the data for [3-(13)C]Ala-bR: the former two peaks were assigned to Ala 232 and 238, in view of the results of successive proteolysis experiments, while the highest-field peak was ascribed to Ala 235 prior to Pro 236. Even such (13)C NMR signals were substantially broadened when (13)C NMR spectra of fully labeled [1,2,3-(13)C]Ala-bR were recorded, because the broadening and splitting of peaks due to the accelerated transverse relaxation rate caused by the increased number of relaxation pathways through a number of (13)C-(13)C homo-nuclear dipolar interactions and scalar J couplings, respectively, are dominant among (13)C-labeled nuclei. In addition, approximate correlation times for local conformational fluctuations of different domains, including the C-terminal tail, C-terminal alpha-helix, loops, and transmembrane alpha-helices, were estimated by measurement of the spin-lattice relaxation times in the laboratory frame and spin-spin relaxation times under the conditions of cross-polarization-magic angle spinning, and comparative study of suppressed specific peaks between the CP-MAS and DD-MAS experiments.