Aspartate Group Research Articles

Ab initio molecular dynamics-based assignment of the protonation state of pepstatin A/HIV-1 protease cleavage site.

A recent 13C NMR experiment (Smith et al. Nature Struct. Biol. 1996, 3, 946-950) on the Asp 25-Asp25' dyad in pepstatin A/HIV-1 protease measured two separate resonance lines, which were interpreted as being a singly protonated dyad. We address this issue by performing ab initio molecular dynamics calculations on models for this site accompanied by calculations of 13C NMR chemical shifts and isotopic shifts. We find that already on the picosecond time-scale the model proposed by Smith et al. is not stable and evolves toward a different monoprotonated form whose NMR pattern differs from the experimental one. We suggest, instead, a different protonation state in which both aspartic groups are protonated. Despite the symmetric protonation state, the calculated 13C NMR properties are in good agreement with the experiment. We rationalize this result using a simple valence bond model, which explains the chemical inequality of the two C sites. The model calculations, together with our calculations on the complex, allow also the rationalization of 13C NMR properties on other HIV-1 PR/inhibitor complexes. Both putative binding of the substrate to the free enzyme, which has the dyad singly protonated (Piana, S.; Carloni, P. Proteins: Struct., Funct., Genet. 2000, 39, 26-36), and pepstatin A binding to the diprotonated form are consistent with the inverse solvent isotope effect on the onset of inhibition of pepsin by pepstatin and the kinetic iso-mechanism proposed for aspartic proteases (Cho, T.-K.; Rebholz, K.; Northrop, D.B. Biochemistry 1994, 33, 9637-9642).

Characterization and mutagenesis of the recombinant N-acetylneuraminate lyase from Clostridium perfringens: insights into the reaction mechanism.

The N-acetylneuraminate lyase from Clostridium perfringens was expressed in Escherichia coli as a fusion protein with a His-tag and purified to homogeneity using metal chelate affinity and anion exchange chromatography. The purified enzyme has a pH optimum of 7.6 and a temperature optimum of 65-70 degrees C. In kinetic studies the lyase exhibits a Km of 3.2 mM for Neu5Ac and a Vmax of 27.5 U x mg(-1). To clarify the functional role of some putative active site residues, site-directed mutagenesis was performed. Lysine 161 was identified as the residue forming the Schiff base intermediate with the substrate. Tyrosine 133 was shown to be also a catalytically important residue; it seems to function as an acceptor for the proton of the C4 hydroxyl group, as already suggested by other groups. Furthermore, it is involved in stabilizing the Schiff base intermediate. Mutations of aspartate 187 and glutamate 188 indicate that both residues are involved in substrate binding. In this respect the carboxy group of aspartate 187 seems to be particularly important. Based on the results of these studies, a model of the reaction mechanism is discussed.

Carbon sources affect metabolic capacities of Bacillus species for the production of industrial enzymes: theoretical analyses for serine and neutral proteases and α-amylase

The metabolic fluxes through the central carbon pathways were calculated for the genus Bacillus separately for the enzymes serine alkaline protease (SAP), neutral protease (NP) and α-amylase (AMY) on five carbon sources that have different reduction degrees ( γ), to determine the theoretical ultimate limits of the production capacities of Bacillus species and to predict the selective substrate for the media design. Glucose ( γ=4.0), acetate ( γ=4.0), and the TCA cycle organic-acids succinate ( γ=3.5), malate ( γ=3.0), and citrate ( γ=3.0) were selected for the theoretical analyses and comparisons. A detailed mass flux balance-based general stoichiometric model based on the proposed metabolic reaction network starting with the alternative five carbon sources for the synthesis of each enzyme in Bacillus licheniformis that simulates the behaviour of the metabolic pathways with 107 metabolites and 150 reaction fluxes is developed. Highest and lowest specific cell growth rates ( μ) were calculated as 1.142 and 0.766 h −1, respectively, when glucose that has the highest degree of reduction and citrate that has the lowest degree of reduction were used as the carbon sources. Highest and lowest SAP, NP and AMY synthesis rates were also obtained, respectively, when glucose and citrate were used. Metabolic capacity analyses showed that the maximum SAP, NP, and AMY synthesis rates were, respectively, 0.0483, 0.0215 and 0.0191 mmol g −1 DW h −1 when glucose uptake rate was 10 mmol g −1 DW h −1 and specific growth rate was zero. The amino acid compositions and the molecular weights of the enzyme influence the production yield and selectivity. For SAP and NP oxaloacetate and pyruvate, for AMY oxaloacetate appear to be the critical main branch points. Consequently, for SAP and NP syntheses the fluxes towards the alanine group and aspartate group, and for AMY synthesis the flux towards the aspartate group amino acids need to be high. The results encourage the discussion of the potential strategies for improving productions of SAP, NP and AMY.

Imidazole-Ligated Compound I Intermediates: The Effects of Hydrogen Bonding

An unresolved issue of peroxidase activity is the role played by the hydrogen bond between the proximal histidine ligand and a highly conserved aspartate group. It has been postulated that this hydrogen bond imparts imidazolate character to the axial ligand, increasing its donating ability and stabilizing the high valent compound I intermediate. A general feature of biological compound I intermediates is that they display weaker spin couplings than model systems. To understand how the Asp-His hydrogen bond affects the electronic structure of the compound I intermediate and to establish a connection between it and the decreased coupling strengths observed in peroxidases, we have perfomed density functional calculations on imidazole- and imidazolate-ligated perferryl porphyrin species. It was found that the imidazolate compound I intermediate possesses an imidazolate-based radical, while the imidazole compound I intermediate possesses a porphyrin-based radical. Delocalizaton of the radical onto the axial ligand was found to reduce the strength of the spin coupling.

Role of Pro-297 in the catalytic mechanism of sheep liver serine hydroxymethyltransferase

Serine hydroxymethyltransferase belongs to the α class of pyridoxal-5´-phosphate enzymes along with aspartate aminotransferase. Recent reports on the three-dimensional structure of human liver cytosolic serine hydroxymethyltransferase had suggested a high degree of similarity between the active-site geometries of the two enzymes. A comparison of the sequences of serine hydroxymethyltransferases revealed the presence of several highly conserved residues, including Pro-297. This residue is equivalent to residue Arg-292 of aspartate aminotransferase, which binds the γ-carboxy group of aspartate. In an attempt to change the reaction specificity of the hydroxymethyltransferase to that of an aminotransferase and to assign a possible reason for the conserved nature of Pro-297, it was mutated to Arg. The mutation decreased the hydroxymethyltransferase activity significantly (by 85–90%) and abolished the ability to catalyse alternative reactions, without alteration in the oligomeric structure, pyridoxal 5´-phosphate content or substrate binding. However, the concentration of the quinonoid intermediate and the extent of proton exchange was decreased considerably (by approx. 85%) corresponding to the decrease in catalytic activity. Interestingly, mutant Pro-297 Arg was unable to perform the transamination reaction with l-aspartate. All these results suggest that although Pro-297 is indirectly involved in catalysis, it might not have any role in imparting substrate specificity, unlike the similarly positioned Arg-292 in aspartate aminotransferase.

Role of Pro-297 in the catalytic mechanism of sheep liver serine hydroxymethyltransferase

Serine hydroxymethyltransferase belongs to the alpha class of pyridoxal-5'-phosphate enzymes along with aspartate aminotransferase. Recent reports on the three-dimensional structure of human liver cytosolic serine hydroxymethyltransferase had suggested a high degree of similarity between the active-site geometries of the two enzymes. A comparison of the sequences of serine hydroxymethyltransferases revealed the presence of several highly conserved residues, including Pro-297. This residue is equivalent to residue Arg-292 of aspartate aminotransferase, which binds the gamma-carboxy group of aspartate. In an attempt to change the reaction specificity of the hydroxymethyltransferase to that of an aminotransferase and to assign a possible reason for the conserved nature of Pro-297, it was mutated to Arg. The mutation decreased the hydroxymethyltransferase activity significantly (by 85-90%) and abolished the ability to catalyse alternative reactions, without alteration in the oligomeric structure, pyridoxal 5'-phosphate content or substrate binding. However, the concentration of the quinonoid intermediate and the extent of proton exchange was decreased considerably (by approx. 85%) corresponding to the decrease in catalytic activity. Interestingly, mutant Pro-297 Arg was unable to perform the transamination reaction with L-aspartate. All these results suggest that although Pro-297 is indirectly involved in catalysis, it might not have any role in imparting substrate specificity, unlike the similarly positioned Arg-292 in aspartate aminotransferase.

The dielectric effect of the environment on the pKa of the retinal Schiff base and on the stabilization of the ion pair in bacteriorhodopsin

We have performed DFT calculations, using the DMOL implementation of the COSMO (Conductor like Solvent Model), to investigate the effect of different dielectric responses of the environment on the structure, electronic configuration and proton affinity (PA) of a series of conjugated Schiff base models. The studied molecules include conjugated Schiff base models containing different number of double bonds and methyl substitution(s), as well as the complete structure of the retinal Schiff base. The COSMO implementation of the DMOL package program was used to implicitly simulate the solvent effects. The calculations were performed using different dielectric constants, in order to simulate gas phase (ϵ=1.0), protein environment (ϵ=4.0) and the aqueous solution (ϵ=78.4). The effect of the solvent model on the geometry and the PA of a series of conjugated Schiff base structure has been studied. Simulating the environment as a continuum model suggests that the protein environment may, very efficiently, adjust the pKa of the chromophore by modifying local screening effects in the vicinity of the retinal Schiff base and, in this way, control the process of the proton transfer. The results show that slightly modifying the dielectric response of the microenvironment can significantly influence the protonation state of the Schiff base group. Comparison of the PA of the retinal Schiff base and a model for the aspartate side chain shows that the ion pair cannot be found stable within gas phase calculations. Considering the environment in the form of a continuum model significantly influences the potential energy surface of the proton transfer between a protonated Schiff base group and the aspartate side chain. In gas phase calculations, the calculated PA of the aspartate group is much larger than that of the studied model Schiff bases. After inclusion of the solvent model in the calculations, the PA of the retinal Schiff base and aspartic acid become very close to each other. The explicit inclusion of the protein environment, however, seems to be essential for obtaining a stable ion pair. Without inclusion of further stabilization effects, for example explicit consideration of the monopole/dipole effects of the environment and/or inclusion of implicit solvent models, one cannot approach any stable ion pair of the protonated Schiff base group and negatively charged aspartate, which is believed to exist in bacteriorhodopsin (bR) as the starting configuration before the photocycle. With this respect, the effect of water molecules is also discussed.

The aspartate aminotransferase-catalysed exchange of the α-protons of aspartate and glutamate: the effects of the R386A and R292V mutations on this exchange reaction

1H-NMR was used to follow the aspartate aminotransferase-catalysed exchange of the α-protons of aspartate and glutamate. The effect of the concentrations of both the amino acids and the cognate keto acids on exchange rates was determined for wild-type and the R386A and R292V mutant forms of aspartate aminotransferase. The wild-type enzyme is found to be highly stereospecific for the exchange of the α-protons of L-aspartate and L-glutamate. The R386A mutation which removes the interaction of Arg-386 with the α-carboxylate group of aspartate causes an ∼10 000-fold decrease in the first order exchange rate of the α-proton of L-aspartate. The R292V mutation which removes the interaction of Arg-292 with the β-carboxylate group of L-aspartate and the γ-carboxylate group of L-glutamate causes even larger decreases of 25 000- and 100 000-fold in the first order exchange rate of the α-proton of L-aspartate and L-glutamate respectively. Apparently both Arg-386 and Arg-292 must be present for optimal catalysis of the exchange of the α-protons of L-aspartate and L-glutamate, perhaps because the interaction of both these residues with the substrate is essential for inducing the closed conformation of the active site.

Implication of arginine-131 and arginine-303 in the substrate site of adenylosuccinate synthetase of Escherichia coli by affinity labeling with 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-monophosphate.

Adenylosuccinate synthetase from Escherichia coli is inactivated in a biphasic reaction by 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-monophosphate (6-BDB-TAMP) at pH 7.0 and 25 degrees C. The initial fast-phase inactivation is not affected by the presence of active-site ligands and can be completely eliminated by blocking Cys291 of the enzyme with N-ethylmaleimide (NEM). Reaction of the NEM-treated enzyme with 6-BDB-[32P]TAMP results in 2 mol of reagent incorporated/mol of enzyme subunit. The inactivation kinetics of the slow-phase exhibit an apparent KI of 40.6 microM and kmax of 0.0228 min-1. Active-site ligands, either adenylosuccinate or IMP and GTP, completely prevent inactivation of the enzyme by 6-BDB-TAMP, whereas IMP or IMP and aspartate is much less effective in protection. 6-BDB-TAMP-inactivated enzyme has a 3-fold increase in Km for aspartate with no change in Km for IMP or GTP. Protease digestion of 6-BDB-[32P]TAMP inactivated enzyme reveals that both Arg131 and Arg303 are modified by the affinity-labeling reagent. The crystal structure [Poland, B. W., Fromm, H. J., and Honzatko, R. B. (1996) J. Mol. Biol. 264, 1013-1027] and site-directed mutagenesis [Kang, C., Sun, N., Poland, B. W., Gorrell, A., and Fromm, H. J. (1997) J. Biol. Chem. 272, 11881-11885] of E. coli adenylosuccinate synthetase show that Arg303 interacts with the carboxyl group of aspartate and the 2'-OH of the ribose of IMP and Arg131 is involved in stabilizing aspartate in the active site of the enzyme. We conclude that 6-BDB-TAMP functions as a reactive adenylosuccinate analogue in modifying both Arg131 and Arg303 in the active site of adenylosuccinate synthetase.

The effect of the protein environment on the structure and charge distribution of the retinal Schiff base in bacteriorhodopsin

The effects of the amino acid side chains of the binding pocket of bacteriorhodopsin (bR) and of a water molecule on the structure of the retinal Schiff base have been studied using Becke3LYP/6-31G* level of density functional theory. A model protonated Schiff base structure including six conjugated double bonds and methyl substituents was optimized in the presence of several amino acid side chains and of a water molecule, separately. The Schiff base structure was also calculated in the form of a neutral species. At each optimized complex geometry the atomic charges of the model Schiff base were calculated using Mulliken population analysis. In agreement with previously proposed counterion(s) of the protonated retinal Schiff base in bR, the results show that Asp85 and Asp212, which are present in the form of negatively charged groups, have significantly large effects on the structure and electronic configuration of both unprotonated and protonated model Schiff bases. The presence of a water molecule in the vicinity of the Schiff base demonstrates significant effects which are comparable to those of aspartate groups. Other side chains studied did not show any significant effect in this direction. Apart from the aspartate groups and the water molecule, in none of the other complexes studied are the atomic charges and the bond alternation of the model Schiff base significantly influenced by the presence of the neighboring amino acids.

Aspartate improves recovery of the recently infarcted rat heart after cardioplegic arrest.

We have previously shown that aspartate improves the tolerance of normal hearts to cardioplegia. The aim of this study was to investigate whether aspartate is also beneficial in the recently infarcted heart. Myocardial infarction was produced in rats by left coronary artery ligation. Twenty hours later their hearts were perfused on an isolated working rat heart apparatus and underwent cardioplegic arrest for 30 min at 37 degrees C with or without 20 mM aspartate in the cardioplegic solution (n = 11 per group). Functional recovery and myocardial high energy phosphate levels were measured at the end of arrest and after 30 min of reperfusion. There was no difference in pre-arrest pump function between the untreated and aspartate-treated groups. However, after reperfusion the aspartate group generated more power (3.4 +/- 0.2 mJ/s per g) than the untreated group (2.5 +/- 0.3 mJ/s per g; P < 0.05) such that the percentage recovery of pre-arrest power in the aspartate group (67.7 +/- 3.5%) was greater than in the untreated group (53.6 +/- 4.9%; P < 0.05). The aspartate group also showed increases in aortic flow and myocardial oxygen consumption compared to the untreated group (P < 0.05). There were no between-group differences in high energy phosphate levels at the end of arrest or after reperfusion. Aspartate improves functional recovery of the recently infarcted heart during cardioplegic arrest, and therefore has potential as a useful adjunct to myocardial protection in patients with recent myocardial infarction undergoing cardiac surgery.

KNI-577, a potent small-sized HIV protease inhibitor based on the dipeptide containing the hydroxymethylcarbonyl isostere as an ideal transition-state mimic.

The development of an effective therapeutic agent for the treatment of AIDS continues to be a challenging problem. Since the discovery that the virally encoded HIV protease is vital for propagation, inhibition of this enzyme has become a major target for AIDS chemotherapy. Consequently, numerous efforts aimed at the development of potent and selective inhibitors have been undertaken[2]. Based on the substrate transition state, we designed and synthesized a novel class of HIV protease inhibitors containing allophenylnorstatine [Apns; (2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid] with a hydroxymethylcarbonyl (HMC) isostere. Among them, the tripeptide KNI-272 was a highly selective and superpotent HIV protease inhibitor (Ki=5.5 pM)[3]. KNI-272 exhibited potent in vitro and in vivo antiviral activities with low cytotoxicity[4]. The NMR, X-ray crystallography, and molecular modeling studies showed that the HMC group in KNI-272 interacted excellently with the aspartic acid carboxyl groups of the HIV protease active site[5].

Alkylation of a catalytic aspartate group of the SIV protease by an epoxide inhibitor

Specific irreversible inhibition of the SIV protease by FMOC-protected piperidine epoxide 1 involves alkylation of the protein. Tryptic digestion of the alkylated protein and mass spectrometric analysis of the peptides identify an active site aspartic acid (Asp-25) as the single residue that is alkylated. Computer modeling of 1 bound in the crystal structure of the SIV protease using DOCK 3.5 indicates that 1 has appropriate access to the active site. It is able to align in an orientation that allows a proton to be transferred to the epoxide from one of the catalytic aspartic acid groups in conjunction with nucleophilic attack on the epoxide of the carboxylate moiety of the second catalytic aspartic acid residue. Hydrophobic interactions are not optimal for this process due, in part, to the rigidity of the inhibitor ring system and the planar conformation of the amide. The combination of modeling with protein alkylation can provide insights into structural modifications of the inhibitor that may lead to improved inhibitory activity.

P Ka calculations along a bacteriorhodopsin molecular dynamics trajectory

Electrostatic calculations of p K a-values are reported along a 400 ps molecular dynamics trajectory of bacteriorhodopsin. The sensitivity of calculated p K a values to a number of structural factors and factors related to the modelling of the electrostatics are also studied. The results are very sensitive to the choice of internal dielectric constant of the protein (in the interval 2–4). Moreover it is important to include internal water molecules and to average over a long enough portion (∼ 100 ps) of an equilibrium molecular dynamics trajectory. The internal waters are necessary to get an ion-counter ion complex with the Schiff base and Arg 82 protonated and the aspartic groups (85 and 212) deprotonated. The fluctuations along the MD-trajectory do not change the protonation state of internal residues at neutral pH. However, at other pH values the averaging along a trajectory maybe crucial to get correct protonation states. A relationship is found between the arginine group 82, the aspartic group 85 and the glutamate group 204. Glu 204 is protonated in the ground state but the p K a value decreases towards deprotonation when the chromophore isomerizes into the cis state.

Use of 1H-15N heteronuclear multiple-quantum coherence NMR spectroscopy to study the active site of aspartate aminotransferase.

Aspartate aminotransferase from Escherichia coli, an 88 kDa enzyme, was uniformly and selectively enriched with 15N and was studied by heteronuclear multiple-quantum coherence NMR spectroscopy in H2O. Good resolution was obtained for the downfield region (above 9.5 ppm chemical shift in the 1H dimension) for NH protons in the amide, indole, imidazole, and guanidinium group regions and several resonances were tentatively assigned. Two downfield resonances, at 12.6 and 11.36 ppm, appear to belong to oxygen- or sulfur-bound protons. The most downfield amide resonance at 11.78 ppm was assigned to the active site cysteine 192 whose peptide proton is 2.9 A away from the negatively charged carboxyl group of aspartate 199. Large downfield shifts (up to 1.15 ppm) of the indole NH resonance of the active site tryptophan 140 were observed upon binding of dicarboxylic inhibitors to the pyridoxal 5'-phosphate (PLP) form and of inorganic dianions to the pyridoxamine 5'-phosphate (PMP) form of the enzyme. We discuss these striking differences in the light of the available crystallographic data. Active sites of proteins, as well as specific inhibitory molecules, often contain negatively charged groups. These may be able to form hydrogen-bonds to NH groups and to shift the NH resonances downfield into a less crowded and therefore more readily observable region for many large proteins. Our approach, which makes use of both HMQC spectroscopy and NOE observations, should be widely applicable.

ObservingN-Acetyl Aspartate via Both ItsN-Acetyl and Its Strongly Coupled Aspartate Groups inin VivoProton Magnetic Resonance Spectroscopy

The ∼2.6 ppm aspartate multiplet ofN-acetyl aspartate (NAA) is considered a potential source of additional information onN-acetyl aspartatein vivo.Because the aspartate multiplet is the AB part of a strongly coupled ABX system it gives rise, as is shown in the analysis presented, to a significant field-strength dependence in the echo-time-dependent modulations of the response to typical spatial-localization sequences. The echo-time dependence of this response is developed analytically, not only for the STEAM and the PRESS localization sequences, but also for a spin-echo sequence. It is then verified experimentally at 2.35 T. The field-strength dependence of the response is demonstrated by evaluating the changes in the echo-time-dependent responses to each of the three sequences at field strengths of 1.5, 2.35, and 4.0 T. By means of these results, the preferred sequence (PRESS) can be optimized for the NAA aspartate multiplet at each field strength, as is illustrated with the human brain spectra obtainedin vivoat 1.5 T. Thesein vivospectra compare the optimal, long TE timing (163 ms) with a suboptimal TE (70 ms), for the observation of the ∼2.6 ppm aspartate resonances of NAA.

Irreversible Inhibition of the HIV-1 Protease: Targeting Alkylating Agents to the Catalytic Aspartate Groups

Irreversible inhibition of the HIV-1 protease by agents that specifically alkylate its catalytic aspartate residues is a potentially useful approach for circumventing the evolution of HIV strains that are resistant to protease inhibitors. Five haloperidol- and two FMOC-based epoxides of differing reactivities have been synthesized and tested as irreversible inhibitors of the HIV-1 protease (HIV-1 PR). Of these, two trisubstituted epoxides, a cis-1,2-disubstituted epoxide, a 1,1-disubstituted epoxide, and a monosubstituted epoxide function as irreversible inhibitors, but two trans-1,2-disubstituted epoxides do not. The most effective of the epoxides (6) inactivates HIV-1 PR with K(inact) = 65 μM and V(inact) = 0.009 min. 1,2-Epoxy-3-(p-nitrophenoxy)propane (EPNP), a nonspecific inactivating agent for aspartyl proteases, has been used to validate a protocol for establishing the stoichiometry and site of protein alkylation. Mass spectrometric analysis of the inactivated enzyme shows that one molecule of either EPNP or the cyclic 1,2-disubstituted epoxide 6 is covalently bound per HIV-1 PR dimer. Mass spectrometric sequencing of labeled proteolytic peptides shows that both inhibitors are covalently bound to a catalytic aspartate residue. The covalent binding of three α,β-unsaturated ketone derivatives of haloperidol has been similarly examined. Analysis of HIV-1 PR inactivated by these agents establishes that they bind covalently to the two cysteines and the N-terminal amino group but not detectably to the catalytic aspartate residues. The results indicate that aspartate-targeted inactivation of HIV-1 PR depends on (a) matching the reactivity of the alkylating functionality to that of the aspartates, preferably by exploiting the two-aspartate catalytic motif of the protease to activate the alkylating agent, and (b) appropriate positioning of the alkylating functionality within the active site. These requirements are readily met by a monosubstituted, 1,1-disubstituted, or cyclic cis-1,2-disubstituted epoxide but not by trans-1,2-disubstituted epoxides or α,β-unsaturated ketones.

Molecular basis of the inhibition of gentamicin nephrotoxicity by daptomycin; an infrared spectroscopic investigation

The lipopeptide daptomycin has been reported to reduce in vivo the nephrotoxicity of aminoglycoside antibiotics (Wood et al. (1989) Antimicrob. Agents Chemother. 33, 1280–1285; Beauchamp et al. (1990) Antimicrob. Agents Chemother. 34, 139–147). A recent dialysis study confirmed the existence of an electrostatic interaction between daptomycin and tobramycin (Couture et al. (1994) Antimicrob. Agents Chemother. 38, 742–749). The interaction of gentamicin with daptomycin and phosphatidylinositol (PI) dispersions was investigated by FTIR spectroscopy. We found no evidence of a direct interaction involving the neutralization of the aspartate groups of daptomycin by gentamicin and the amide I band of daptomycin did not reveal significant conformational changes of its peptidic moiety. On the other hand, daptomycin readily inserts within bilayers of PI, dimyristoylphosphatidylglycerol or dipalmitoyl-phosphatidylcholine, as judged from its influence on the fluidity of these bilayers. The incorporation of daptomycin into PI bilayers has no significant effect on the lipopeptide amide I band. Gentamicin also binds to PI bilayers and the associated modifications of the lipid bands are consistent with a tightening of the lipid network resulting from head group neutralization by gentamicin. The affinity of the aminoglycoside for PI is slightly increased in the presence of daptomycin, in agreement with the results of the dialysis study mentioned above. The lipid features indicate that its head group is still affected by gentamicin charges, but the thermotropic behavior of the hydrophobic portion becomes similar to that of the pure lipid. It is proposed that the contribution of daptomycin to the membrane charge density and its effect on the lipid packing both combine to counteract the inhibition of phospholipase activity due to aminoglycosides. Further work will attempt to determine how the peptide rings and gentamicin molecules are organized at the bilayer surface, how specific these interactions are and to confirm the influence of daptomycin on the phospholipid catabolism.

A Novel Structure of (L-Aspartato)(1,10-phenanthroline)copper(II) Hydrate

The title compound, [Cu(C 4 H 5 NO 4 )(C 12 H 8 N 2 )].3.5H 2 O, consists of two types of different pentacoordinate copper complex: one monomeric, with the aspartate group acting as a tridentate ligand, and the other polymeric, with the aspartate group bridging two different copper centers. The whole structure is held together by a complex hydrogen-bonding scheme involving the water molecules of solvation

Biosynthesis of 5-hydroxy-4-oxo-L-norvaline in Streptomyces akiyoshiensis

The biosynthesis of 5-hydroxy-4-oxo-L-norvaline (HON) in Streptomyces akiyoshiensis has been investigated using 13C-labelled substrates. Incorporations of 13C label from sodium [1-13C]-, [2-13C]-, and [1,2-13C2]acetate indicated that HON was formed from a four-carbon compound derived from the citric acid cycle and the methyl carbon of acetate. Feeding experiments using DL-[4-13C]- and DL-[2-13C,15N]aspartate demonstrated that aspartate served as the four-carbon precursor to HON. Both enantiomers of aspartate were metabolized by S. akiyoshiensis, but the D isomer was consumed at a slower rate. The distribution of 13C label in the intracellular L-glutamic acid isolated in these feeding experiments is consistent with the operation of the citric acid cycle in S. akiyoshiensis. A biosynthetic hypothesis that involves a condensation reaction between acetyl or malonyl CoA and the β-carboxyl group of aspartate, and subsequent oxidative decarboxylation, is proposed to account for the incorporation results. An analogous condensation step has been proposed for the biosynthesis of other natural products, including the carbapenem antibiotics. DL-[2-13C,15N]Aspartate was synthesized from [2-13C]diethylmalonate and potassium [15N]phthalimide via diethyl [2-13C,15N]phthalimidomalonate.