A study of l-leucine, l-phenylalanine and l-alanine transport in the perfused rat mammary gland: possible involvement of LAT1 and LAT2

The transport of l-leucine, l-phenylalanine and l-alanine by the perfused lactating rat mammary gland has been examined using a rapid, paired-tracer dilution technique. The clearances of all three amino acids by the mammary gland consisted of a rising phase followed by a rapid fall-off, respectively, reflecting influx and efflux of the radiotracers. The peak clearance of l-leucine was inhibited by BCH (65%) and d-leucine (58%) but not by l-proline. The inhibition of l-leucine clearance by BCH and d-leucine was not additive. l-leucine inhibited the peak clearance of radiolabelled l-leucine by 78%. BCH also inhibited the peak clearance of l-phenylalanine (66%) and l-alanine (33%) by the perfused mammary gland. Lactating rat mammary tissue was found to express both LAT1 and LAT2 mRNA. The results suggest that system L is situated in the basolateral aspect of the lactating rat mammary epithelium and thus probably plays a central role in neutral amino acid uptake from blood. The finding that l-alanine uptake by the gland was inhibited by BCH suggests that LAT2 may make a significant contribution to neutral amino acid uptake by the mammary epithelium.     

Structural and functional characterization of enantiomeric glutamic acid derivatives as potential transition state analogue inhibitors of MurD ligase

Mur ligases play an essential role in the intracellular biosynthesis of bacterial peptidoglycan, the main component of the bacterial cell wall, and represent attractive targets for the design of novel antibacterials. UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase (MurD) catalyses the addition of D-glutamic acid to the cytoplasmic intermediate UDP-N-acetylmuramoyl-L-alanine (UMA) and is the second in the series of Mur ligases. MurD ligase is highly stereospecific for its substrate, D-glutamic acid (D-Glu). Here, we report the high resolution crystal structures of MurD in complexes with two novel inhibitors designed to mimic the transition state of the reaction, which contain either the D-Glu or the L-Glu moiety. The binding modes of N-sulfonyl-D-Glu and N-sulfonyl-L-Glu derivatives were also characterised kinetically. The results of this study represent an excellent starting point for further development of novel inhibitors of this enzyme.     

Base catalysed isomerisation of aldoses of the arabino and lyxo series in the presence of aluminate

Base-catalysed isomerisation of aldoses of the arabino and lyxo series in aluminate solution has been investigated. L-Arabinose and D-galactose give L-erythro-2-pentulose (L-ribulose) and D-lyxo-2-hexulose (D-tagatose), respectively, in good yields, whereas lower reactivity is observed for 6-deoxy-D-galactose (D-fucose). From D-lyxose, D-mannose and 6-deoxy-L-mannose (L-rhamnose) are obtained mixtures of ketoses and C-2 epimeric aldoses. Small amounts of the 3-epimers of the ketoses were also formed. 6-Deoxy-L-arabino-2-hexulose (6-deoxy-L-fructose) and 6-deoxy-L-glucose (L-quinovose) were formed in low yields from 6-deoxy-L-mannose and isolated as their O-isopropylidene derivatives. Explanations of the differences in reactivity and course of the reaction have been suggested on the basis of steric effects.     

Synergistic production of L-arabinose from arabinan by the combined use of thermostable endo- and exo-arabinanases from Caldicellulosiruptor saccharolyticus

The optimum conditions for the production of l-arabinose from debranched arabinan were determined to be pH 6.5, 75 °C, 20 g l⁻¹ debranched arabinan, 42 U ml⁻¹ endo-1,5-α-l-arabinanase, and 14 U ml⁻¹ α-l-arabinofuranosidase from Caldicellulosiruptor saccharolyticus and the conditions for sugar beet arabinan were pH 6.0, 75 °C, 20 g l⁻¹ sugar beet arabinan, 3 U ml⁻¹ endo-1,5-α-l-arabinanase, and 24 U ml⁻¹ α-l-arabinofuranosidase. Under the optimum conditions, 16 g l⁻¹l-arabinose was obtained from 20 g l⁻¹ debranched arabinan or sugar beet arabinan after 120 min, with a hydrolysis yield of 80% and a productivity of 8 g l⁻¹ h⁻¹. This is the first reported trial for the production of l-arabinose from the hemicellulose arabinan by the combined use of endo- and exo-arabinanases.     

Effects of L- or D-Pro incorporation into hydrophobic or hydrophilic helix face of amphipathic alpha-helical model peptide on structure and cell selectivity

A synthetic amphipathic alpha-helical model peptide, KLW, displays non-cell selective cytotoxicity. To investigate the effects of L- or D-Pro kink incorporation into hydrophobic or hydrophilic helix face of KLW on structure, cell selectivity, and membrane-binding affinity, we designed a series of four peptides, in which Leu(9) and Lys(11) in the hydrophobic and hydrophilic helix face of KLW, respectively, are substituted with L- or D-Pro. A L- or D-Pro substitution (KLW-L9P or KLW-L9p) of Leu(9) at the hydrophobic helix face of KLW induced a more significant reduction in hemolytic activity with improved antibacterial activity than that (KLW-K11P or KLW-K11p) of Lys(11) in the hydrophilic helix face. In addition, D-Pro-containing peptides (KLW-L9p and KLW-K11p) displayed less hemolytic activity than L-Pro-containing peptides (KLW-L9P and KLW-K11P). Tryptophan fluorescence studies revealed that bacterial cell selectivity of KLW-L9P, KLW-L9p, and KLW-K11p is closely related to selective interactions with negatively charged phospholipids. CD analysis revealed that L- or D-Pro incorporation into KLW reduces the alpha-helicity of the peptide and D-Pro incorporation induces more significant disruption in alpha-helical structure than L-Pro incorporation. Our results collectively suggest that D-Pro incorporation into the hydrophobic helix face of non-cell selective amphipathic alpha-helical peptides may be useful for the design of novel antimicrobial peptides possessing high bacterial cell selectivity without hemolytic activity.     

Phosphatidylglycerol lipids enhance folding of an alpha helical membrane protein

Membrane lipids are increasingly being recognised as active participants in biological events. The precise roles that individual lipids or global properties of the lipid bilayer play in the folding of membrane proteins remain to be elucidated, Here, we find a significant effect of phosphatidylglycerol (PG) on the folding of a trimeric α helical membrane protein from Escherichia coli diacylglycerol kinase. Both the rate and the yield of folding are increased by increasing the amount of PG in lipid vesicles. Moreover, there is a direct correlation between the increase in yield and the increase in rate; thus, folding becomes more efficient in terms of speed and productivity. This effect of PG seems to be a specific requirement for this lipid, rather than a charge effect. We also find an effect of single-chain lyso lipids in decreasing the rate and yield of folding. We compare this to our previous work in which lyso lipids increased the rate and yield of another membrane protein, bacteriorhodopsin. The contrasting effect of lyso lipids on the two proteins can be explained by the different folding reaction mechanisms and key folding steps involved. Our findings provide information on the lipid determinants of membrane protein folding.     

Thermodynamic determination of the binding constants of angiotensin-converting enzyme inhibitors by a displacement method

Somatic angiotensin I-converting enzyme (s-ACE) plays a central role in blood pressure regulation and has been the target of most antihypertensive drugs. A displacement isothermal titration calorimetry method has been used to accurately determine the binding constant of three strong s-ACE inhibitors. Under the experimental conditions studied in this work, the relative potency of the inhibitors was determined to be enalaprilat>lisinopril>captopril. We analyze the thermodynamic behaviour of the binding process using the new structural information provided by the ACE structures, as well as the conformational changes that occur upon binding.     

Substitution of the leucine zipper sequence in melittin with peptoid residues affects self-association, cell selectivity, and mode of action

Melittin (ME), a non-cell-selective antimicrobial peptide, contains the leucine zipper motif, wherein every seventh amino acid is leucine or isolucine. Here, we attempted to generate novel cell-selective peptides by substituting amino acids in the leucine zipper sequence of ME with peptoid residues. We generated a series of ME analogues by replacing Leu-6, Lue-13 and Ile-20 with Nala, Nleu, Nphe, or Nlys, and we examined their secondary structure, self-association activity, cell selectivity and mode of action. Circular dichroism spectroscopy indicated that the substitutions disrupt the α-helical structure of ME in micelles of sodium dodecyl sulfate and on negatively charged and zwitterionic phospholipid vesicles. Substitution by Nleu, Nphe, or Nlys but not Nala disturbed the self-association in an aqueous environment, interaction with zwitterionic membranes, and toxicity to mammalian cells of ME but did not affect the interaction with negatively charged membranes or antibacterial activity. Notably, peptides with Nphe or Nlys substitution had the highest therapeutic indices, consistent with their lipid selectivity. In addition, all of peptoid residue-containing ME analogues had little or no ability to induce membrane disruption, membrane depolarization and lipid flip-flop. Taken together, our studies indicate that substitution of the leucine zipper motif in ME with peptoid residues increases its selectivity against bacterial cells by impairing self-association activity and changes its mode of antibacterial action from membrane-targeting mechanism to possible intracellular targeting mechanism. Furthermore, our ME analogues especially those with Nleu, Nphe, or Nlys substitutions, may be therapeutically useful antimicrobial peptides.     

Structural Basis for Catalysis by the Mono- and Dimetalated Forms of the dapE-Encoded N-succinyl-l,l-Diaminopimelic Acid Desuccinylase

Biosynthesis of lysine and meso-diaminopimelic acid in bacteria provides essential components for protein synthesis and construction of the bacterial peptidoglycan cell wall. The dapE operon enzymes synthesize both meso-diaminopimelic acid and lysine and, therefore, represent potential targets for novel antibacterials. The dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase functions in a late step of the pathway and converts N-succinyl-l,l-diaminopimelic acid to l,l-diaminopimelic acid and succinate. Deletion of the dapE gene is lethal to Helicobacter pylori and Mycobacterium smegmatis, indicating that DapE's are essential for cell growth and proliferation. Since there are no similar pathways in humans, inhibitors that target DapE may have selective toxicity against only bacteria. A major limitation in developing antimicrobial agents that target DapE has been the lack of structural information. Herein, we report the high-resolution X-ray crystal structures of the DapE from Haemophilus influenzae with one and two zinc ions bound in the active site, respectively. These two forms show different activity. Based on these newly determined structures, we propose a revised catalytic mechanism of peptide bond cleavage by DapE enzymes. These structures provide important insight into catalytic mechanism of DapE enzymes as well as a structural foundation that is critical for the rational design of DapE inhibitors.     

Structural enzymology of sulphur metabolism in Mycobacterium tuberculosis

The emergence of multidrug-resistant strains of Mycobacterium tuberculosis poses a serious threat to human health and has led to world-wide efforts focusing on the development of novel vaccines and antibiotics against this pathogen. Sulphur metabolism in this organism has been linked to essential processes such as virulence and redox defence. The cysteine biosynthetic pathway is up-regulated in models of persistent M. tuberculosis infections and provides potential targets for novel anti-mycobacterial agents, directed specifically toward the pathogen in its persistent phase. Functional and structural characterization of enzymes from sulfur metabolism establishes a necessary framework for the design of strong binding inhibitors that might be developed into new drugs. This review summarizes recent progress in the elucidation of the structural enzymology of the sulphate reduction and cysteine biosynthesis pathways.     

L-Glutamate in the extracellular space regulates endogenous D-aspartate homeostasis in rat pheochromocytoma MPT1 cells

In previous studies [FEBS Lett. 434 (1998) 231, Arch. Biochem. Biophys. 404 (2002) 92], we demonstrated for the first time that D-aspartate (D-Asp) is synthesized in cultured mammalian cell lines, such as pheochromocytoma 12 (PC12) and its subclone, MPT1. Our current focus is analysis of the dynamics of D-Asp homeostasis in these cells. In this communication, we show that L-glutamate (Glu) and L-Glu transporter substrates in the extracellular space regulate the homeostasis of endogenous D-Asp in MPT1 cells. D-Asp is apparently in dynamic homeostasis, whereby endogenous D-Asp is constantly released into the extracellular space by an undefined mechanism, and continuously and intensively taken up into cells by an L-Glu transporter. Under these conditions, L-Glu and its transporter substrates in the medium may competitively inhibit the uptake of D-Asp via the transporter, resulting in accumulation of the amino acid in the extracellular space. We additionally demonstrate that DL-TBOA, a well-established L-Glu transporter inhibitor, is taken up by the transporter during long time intervals, but not on a short time-scale.     

Synergism between hydrogen sulfide (H(2)S) and nitric oxide (NO) in vasorelaxation induced by stonustoxin (SNTX), a lethal and hypotensive protein factor isolated from stonefish Synanceja horrida venom

Stonustoxin (SNTX) is a 148 kDa, dimeric, hypotensive and lethal protein factor isolated from the venom of the stonefish Synanceja horrida. SNTX (10-320 ng/ml) progressively causes relaxation of endothelium-intact, phenylephrine (PE)-precontracted rat thoracic aortic rings. The SNTX-induced vasorelaxation was inhibited by L-N(G)-nitro arginine methyl ester (L-NAME), suggesting that nitric oxide (NO) contributes to the SNTX-induced response. Interestingly, D, L-proparglyglycine (PAG) and beta-cyano-L-alanine (BCA), irreversible and competitive inhibitors of cystathionine-gamma-lyase (CSE) respectively, also inhibited SNTX-induced vasorelaxation, indicating that H(2)S may also play a part in the effect of SNTX. The combined use of L-NAME with PAG or BCA showed that H(2)S and NO act synergistically in effecting SNTX-induced vasorelaxation.     

First structures of an active bacterial tyrosinase reveal copper plasticity

Tyrosinase is a member of the type 3 copper enzyme family that is involved in the production of melanin in a wide range of organisms. The crystal structures of a tyrosinase from Bacillus megaterium were determined at a resolution of 2.0-2.3 Å. The enzyme crystallized as a dimer in the asymmetric unit and was shown to be active in crystal. The overall monomeric structure is similar to that of the monomer of the previously determined tyrosinase from Streptomyces castaneoglobisporus, but it does not contain an accessory Cu-binding “caddie” protein. Two Cu(II) ions, serving as the major cofactors within the active site, are coordinated by six conserved histidine residues. However, determination of structures under different conditions shows varying occupancies and positions of the copper ions. This apparent mobility in copper binding modes indicates that there is a pathway by which copper is accumulated or lost by the enzyme. Additionally, we suggest that residues R209 and V218, situated in a second shell of residues surrounding the active site, play a role in substrate binding orientation based on their flexibility and position. The determination of a structure with the inhibitor kojic acid, the first tyrosinase structure with a bound ligand, revealed additional residues involved in the positioning of substrates in the active site. Comparison of wild-type structures with the structure of the site-specific variant R209H, which possesses a higher monophenolase/diphenolase activity ratio, lends further support to a previously suggested mechanism by which monophenolic substrates dock mainly to CuA.     

Total synthesis and identification of two diastereoisomers of 1-methyl-2-[N-(p-tolylsulfonyl)-2-pyrrolidinyl]ethyl beta-L-fucopyranoside

The reaction of a racemic mixture of (2R,2'S)- and (2S,2'R)-N-(p-tolylsulfonyl)-2-pyrrolidinyl-2-propanol, prepared from (S)-proline, with 2,3,4-tri-O-acetyl-alpha-L-fucopyranosyl trichloroacetimidate led to both diastereoisomers of the title compound after O-deacetylation.     

Synthesis of the 6-deoxytalose-containing tri- and hexasaccharides of the O-antigen polysaccharide from Mesorhizobium huakuii IFO15243T

The synthesis of a trisaccharide and a hexasaccharide, the monomer and dimer of the repeating unit of O-antigen polysaccharide from Mesorhizobium huakuii IFO15243, has been accomplished through suitable protecting group manipulations and stereoselective glycosylation reactions starting from commercially available l-rhamnose. The target oligosaccharides in the form of their p-methoxyphenyl glycosides are suitable for further glycoconjugate formation via selective cleavage of this group.     

Low levels of mammalian TGF-beta1 are protective against malaria parasite infection, a paradox clarified in the mosquito host

Nitric oxide (NO), derived from catalysis of inducible NO synthase (iNOS), limits malaria parasite growth in mammals. Transforming growth factor (TGF)-beta1 suppresses iNOS in cells in vitro as well as in vivo in mice, but paradoxically severe malaria in humans is associated with low levels of TGF-beta1. We hypothesized that this paradox is a universal feature of infection and occurs in the mosquito Anopheles stephensi, an invertebrate host for Plasmodium that also regulates parasite development with inducible NO synthase (AsNOS). We show that exogenous human TGF-beta1 dose-dependently regulates mosquito AsNOS expression and that parasite killing by low dose TGF-beta1 depends on AsNOS catalysis. Furthermore, induction of AsNOS expression by TGF-beta1 is regulated by NO synthesis. These results suggest that TGF-beta1 plays similar roles during parasite infection in mammals and mosquitoes and that this role is linked to the effects of TGF-beta1 on inducible NO synthesis.     

Synthesis and intramolecular reactions of Tyr-Gly and Tyr-Gly-Gly related 6-O-glucopyranose esters

6-O-(L-Tyrosylglycyl)- and 6-O-(L-tyrosylglycylglycyl)-D-glucopyranose were synthesized by condensation of the pentachlorophenyl esters of the respective di- and tripeptide with fully unprotected D-glucose. The intramolecular reactivity of the sugar conjugates was studied in pyridine-acetic acid and in dry methanol, at various temperatures and for various incubation times. The composition of the incubation mixtures was monitored by a reversed-phase HPLC method that permits simultaneous analysis of the disappearance of the starting material and the appearance of rearrangement and degradation products. To determine the influence of esterification of the peptide carboxy group on its amino group reactivity, parallel experiments were done in which free peptides were, under identical reaction conditions, incubated with D-glucose (molar ratios 1:1 and 1:5). Depending on the starting compound, different types of Amadori products (cyclic and bicyclic form), methyl ester of peptides, and Tyr-Gly-diketopiperazine were obtained.     

Efficient synthesis of 2-deoxy-L-erythro-pentose (2-deoxy-L-ribose) from L-arabinose

An efficient and practical route for the large-scale synthesis of 2-deoxy-L-erythro-pentose (2-deoxy-L-ribose) starting from L-arabinose was developed using Barton-type free-radical deoxygenation reaction as a key step. The radical precursor, a phenoxythiocarbonyl ester, was prepared in situ, and the most efficient deoxygenation was achieved by slow addition of tributyltin hydride to the reaction mixture.