Characterization of the catalytic activity of the γ-phage lysin, PlyG, specific for Bacillus anthracis

Bacillus anthracis causes anthrax, a lethal disease affecting humans that has attracted attention due to its bioterrorism potential. PlyG is a lysin of γ-phage, which specifically infects B. anthracis and lyses its cell wall. PlyG contains a T7 lysozyme-like amidase domain, which appears to be the catalytic domain, in the N-terminal region and has a high degree of sequence similarity with PlyL, which is an N -acetylmuramoyl- l -alanine amidase encoded by the B. anthracis genome. Here, we demonstrated that two amino acid residues of PlyG, H29 and E90, are necessary for its catalytic activity in B. anthracis . These residues are structurally analogous to residues whose mutation in T7 lysozyme abolished its catalytic activity. A C-terminal deletion mutant of PlyG lacking the core sequence for binding to B. anthracis showed completely abolished binding activity, unlike PlyL, despite high sequence similarity with PlyL in the N-terminal region. This suggests that the C-terminal binding domain, as well as the N-terminal catalytic domain, is essential for the catalytic activity of PlyG. Our observations provide new insights into the mechanism of specific catalysis of PlyG in B. anthracis and may contribute to the establishment of new methods for anthrax therapy.     

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.     

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.     

Identification of human L-fucose kinase amino acid sequence

Fucose is a major component of complex carbohydrates. L-Fucose kinase (fucokinase) takes part in the salvage pathway for reutilization of fucose from the degradation of oligosaccharides. The amino acid sequence of human fucokinase was derived from a cDNA encoding a protein of hitherto unidentified function. Human fucokinase polypeptide chain consists of 990 amino acids with a predicted molecular mass of 107 kDa. The C-terminal part of its amino acid sequence showed sequence motifs typical for sugar kinases. Fucokinase full-length protein and a deletion mutant lacking the first 363 amino acids of the N-terminus were expressed in Escherichia coli BL21 cells. Both proteins displayed fucokinase activity. These results reveal that the discovered cDNA encodes the fucokinase protein and they confirm that a functional kinase domain is located in the C-terminal part of the enzyme.     

Structure of the O-polysaccharide of Proteus mirabilis CCUG 10705 (OF) containing an amide of D-galacturonic acid with L-alanine

The structure of the O-polysaccharide of Proteus mirabilis CCUG 10705 (OF) was determined by chemical analyses along with one- and two-dimensional (1)H and (13)C NMR spectroscopy. The polysaccharide was found to contain an amide of D-galacturonic acid with L-alanine and based on the uniqueness of the O-polysaccharide structure and serological data, it was suggested to classify P. mirabilis OF into a new separate Proteus serogroup, O74. A weak cross-reactivity of P. mirabilis OF and P. mirabilis O5 was observed and accounted for by a similarity of their O-repeating units. The following structure of the polysaccharide of P. mirabilis OF was established: [chemical structure: see text]     

Structure of the O-polysaccharide of Proteus vulgaris O44: a new O-antigen that contains an amide of D-glucuronic acid with L-alanine

The O-polysaccharide of Proteus vulgaris O44, strain PrK 67/57 was studied by 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, ROESY, H-detected 1H, 13C HMQC, HMQC-TOCSY and HMBC experiments. The polysaccharide was found to contain an amide of D-glucuronic acid with L-alanine [D-GlcA6(L-Ala)], and the following structure of the linear pentasaccharide repeating unit was established: [structure: see text]. The structural data of the O-polysaccharide and the results of serological studies with P. vulgaris O44 O-antiserum showed that the strain studied is unique among Proteus bacteria, which is in agreement with its classification in a separate Proteus serogroup, O44.     

Distinct metal dependence for catalytic and structural functions in the L-arabinose isomerases from the mesophilic Bacillus halodurans and the thermophilic Geobacillus stearothermophilus

L-Arabinose isomerase (AI) catalyzes the isomerization of L-arabinose to L-ribulose. It can also convert d-galactose to d-tagatose at elevated temperatures in the presence of divalent metal ions. The araA genes, encoding AI, from the mesophilic bacterium Bacillus halodurans and the thermophilic Geobacillus stearothermophilus were cloned and overexpressed in Escherichia coli, and the recombinant enzymes were purified to homogeneity. The purified enzymes are homotetramers with a molecular mass of 232 kDa and close amino acid sequence identity (67%). However, they exhibit quite different temperature dependence and metal requirements. B. halodurans AI has maximal activity at 50 degrees C under the assay conditions used and is not dependent on divalent metal ions. Its apparent K(m) values are 36 mM for L-arabinose and 167 mM for d-galactose, and the catalytic efficiencies (k(cat)/K(m)) of the enzyme were 51.4 mM(-1)min(-1) (L-arabinose) and 0.4 mM(-1)min(-1) (d-galactose). Unlike B. halodurans AI, G. stearothermophilus AI has maximal activity at 65-70 degrees C, and is strongly activated by Mn(2+). It also has a much higher catalytic efficiency of 4.3 mM(-1)min(-1) for d-galactose and 32.5 mM(-1)min(-1)for L-arabinose, with apparent K(m) values of 117 and 63 mM, respectively. Irreversible thermal denaturation experiments using circular dichroism (CD) spectroscopy showed that the apparent melting temperature of B. halodurans AI (T(m)=65-67 degrees C) was unaffected by the presence of metal ions, whereas EDTA-treated G. stearothermophilus AI had a lower T(m) (72 degrees C) than the holoenzyme (78 degrees C). CD studies of both enzymes demonstrated that metal-mediated significant conformational changes were found in holo G. stearothermophilus AI, and there is an active tertiary structure for G. stearothermophilus AI at elevated temperatures for its catalytic activity. This is in marked contrast to the mesophilic B. halodurans AI where cofactor coordination is not necessary for proper protein folding. The metal dependence of G. stearothermophilus AI seems to be correlated with their catalytic and structural functions. We therefore propose that the metal ion requirement of the thermophilic G. stearothermophilus AI reflects the need to adopt the correct substrate-binding conformation and the structural stability at elevated temperatures.     

Accessibility of N-acyl-D-mannosamines to N-acetyl-D-neuraminic acid aldolase

N-Acetyl-D-neuraminic acid (NeuNAc) aldolase is an important enzyme for the metabolic engineering of cell-surface NeuNAc using chemically modified D-mannosamines. To explore the optimal substrates for this application, eight N-acyl derivatives of D-mannosamine were prepared, and their accessibility to NeuNAc aldolase was quantitatively investigated. The N-propionyl-, N-butanoyl-, N-iso-butanoyl-, N-pivaloyl-, and N-phenylacetyl-D-mannosamines proved to be as good substrates as, or even better than, the natural N-acetyl-D-mannosamine, while the N-trifluoropropionyl and benzoyl derivatives were poor. It was proposed that the electronic effects might have a significant influence on the enzymatic aldol condensation reaction of D-mannosamine derivatives, with electron-deficient acyl groups having a negative impact. The results suggest that N-propionyl-, N-butanoyl-, N-iso-butanoyl-, and N-phenylacetyl-D-mannosamines may be employed to bioengineer NeuNAc on cells.     

Structure of the O-polysaccharide from the lipopolysaccharide of Providencia stuartii O43 containing an amide of D-galacturonic acid with L-serine

The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Providencia stuartii O43:H28 and studied by sugar and methylation analyses, Smith degradation and 1H and 13C NMR spectroscopy, including 2D ROESY, and H-detected 1H, 13C HSQC and HMBC experiments, as well as a NOESY experiment in a 9:1 H2O/D2O mixture to reveal correlations for NH protons. It was found that the polysaccharide is built up of linear tetrasaccharide repeating units containing an amide of D-galacturonic acid with L-serine [D-GalA6(L-Ser)] and has the following structure:[3)-beta-D-GalpA6(L-Ser)-(1-->3)-beta-D-GlcpNAc-(1-->2)-alpha-D-Rhap4NAc-(1-->4)-beta-D-GlcpA-(1-->]n.     

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.     

A specific enzymatic high-performance liquid chromatography method to determine N-methyl-D-aspartic acid in biological tissues

Recently we demonstrated that N-methyl-D-aspartic acid (NMDA) is present as an endogenous compound in the nervous tissues and endocrine glands of the rat where it plays a role in the regulation of the luteinizing hormone, growth hormone, and prolactin (FASEB J. 14 (2000) 699; Endocrinology 141 (2000) 3861). Based on the prediction that NMDA could have future importance in neuroendocrinology, we have devised an improved method for the specific and routine determination of NMDA in biological tissue. This method is based on the detection by HPLC of methylamine (CH(3)NH(2)) which comes from the oxidation of NMDA by D-aspartate oxidase, an enzyme which specifically oxidizes NMDA, yielding CH(3)NH(2) as one of the oxidative products of the reaction. The sensitivity of the method permits the accurate determination of NMDA in the supernatant of a tissue homogenate at levels of about 5-10 picomol/assay. However, for those tissues in which the concentration of NMDA is less than 1nmol/g, the sample must be further purified by treatment with o-phthaldialdehyde in order to separate the NMDA from the other amino acids and amino compounds and then concentrated and analyzed by HPLC. Using this method we have conducted a comparative study in order to measure the amount of NMDA in neuroendocrine and other tissues of various animal phyla from mollusks to mammals.     

Cloning of apoB intrabodies: specific knockdown of apoB in HepG2 cells

Apolipoprotein (apo) B is essential for the assembly and secretion of triglyceride-rich lipoproteins made by the liver. As the sole protein component in LDL, apoB is an important determinant of atherosclerosis susceptibility and a potential pharmaceutical target. Single-chain antibodies (sFvs) are the smallest fragment of an IgG molecule capable of maintaining the antigen binding specificity of the parental antibody. In the present study, we describe the cloning and construction of two intracellular antibodies (intrabodies) to human apoB. We targeted these intrabodies to the endoplasmic reticulum for the purpose of retaining nascent apoB within the ER, thereby preventing its secretion. Expression of the 1D1 intrabody in the apoB-secreting human hepatoma cell line HepG2 resulted in marked reduction of apoB secretion. This study demonstrates the utility of an intrabody to specifically block the secretion of a protein determinant of plasma LDL as a therapeutic strategy for the treatment of hyperlipidemia.     

A mammalian peptidoglycan recognition protein with N-acetylmuramoyl-L-alanine amidase activity

The family of peptidoglycan recognition proteins (PGRPs) is conserved from insects to mammals. Recently, Drosophila PGRP-SC1B was demonstrated to be an N-acetylmuramoyl-L-alanine amidase (NAMLAA), an enzyme that cleaves the lactylamide bond between muramic acid and the peptide chain in peptidoglycan (PGN). We now show an M x mPGRP-L mRNA to be expressed in the liver. The recombinant M x mPGRP-L protein has NAMLAA activity and degrades PGN from both Escherichia coli and Staphylococcus aureus; however, the Gram-positive PGN was a better substrate after lysozyme treatment. The activity of M x mPGRP-L was further analysed using Bordetella pertussis tracheal toxin as a substrate. Cleavage products were separated on HPLC and identified using mass spectrometry. From these results we conclude that M x mPGRP-L has activity and other properties identifying it as the NAMLAA protein present in mammalian sera.     

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.     

Crystal structure and functional characterization of a D-stereospecific amino acid amidase from Ochrobactrum anthropi SV3, a new member of the penicillin-recognizing proteins

D-amino acid amidase (DAA) from Ochrobactrum anthropi SV3, which catalyzes the stereospecific hydrolysis of D-amino acid amides to yield the D-amino acid and ammonia, has attracted increasing attention as a catalyst for the stereospecific production of D-amino acids. In order to clarify the structure-function relationships of DAA, the crystal structures of native DAA, and of the D-phenylalanine/DAA complex, were determined at 2.1 and at 2.4 A resolution, respectively. Both crystals contain six subunits (A-F) in the asymmetric unit. The fold of DAA is similar to that of the penicillin-recognizing proteins, especially D-alanyl-D-alanine-carboxypeptidase from Streptomyces R61, and class C beta-lactamase from Enterobacter cloacae strain GC1. The catalytic residues of DAA and the nucleophilic water molecule for deacylation were assigned based on these structures. DAA has a flexible Omega-loop, similar to class C beta-lactamase. DAA forms a pseudo acyl-enzyme intermediate between Ser60 O(gamma) and the carbonyl moiety of d-phenylalanine in subunits A, B, C, D, and E, but not in subunit F. The difference between subunit F and the other subunits (A, B, C, D and E) might be attributed to the order/disorder structure of the Omega-loop: the structure of this loop cannot assigned in subunit F. Deacylation of subunit F may be facilitated by the relative movement of deprotonated His307 toward Tyr149. His307 N(epsilon2) extracts the proton from Tyr149 O(eta), then Tyr149 O(eta) attacks a nucleophilic water molecule as a general base. Gln214 on the Omega-loop is essential for forming a network of water molecules that contains the nucleophilic water needed for deacylation. Although peptidase activity is found in almost all penicillin-recognizing proteins, DAA lacks peptidase activity. The lack of transpeptidase and carboxypeptidase activities may be attributed to steric hindrance of the substrate-binding pocket by a loop comprised of residues 278-290 and the Omega-loop.     

Mechanisms of the protective effect of L-alanine to D-galactosamine-induced hepatocellular injury: comparative studies of L-alanine and pyruvate

The addition of L-alanine reduced lactate dehydrogenase leakage from primary cultured rat hepatocytes treated with galactosamine (D-gal), while D-alanine and other amino acids did not. However, the mechanisms have not yet been entirely clarified. In this study, we used various inhibitors of metabolism, i.e., aminooxyacetate, oligomycin, and quinolinic acid, to examine the relation between this protective effect and the metabolism of L-alanine. Quinolinic acid (10 mM) did not affect the hepatoprotective effect of L-alanine, while oligomycin (0.1 mug/ml) and aminooxyacetate (1 mM) eliminated the hepatoprotective effect of L-alanine. L-Alanine also increased the albumin secretion by cultured hepatocytes treated with D-gal, while pyruvate had little effect. It was revealed that the intracellular content of pyruvate did not increase as a result of addition of L-alanine. These results are consistent with the hypothesis that L-alanine metabolism is important for hepatoprotection, but pyruvate cannot be used as a substitute for L-alanine.     

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.     

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.     

Metabolic engineering of Escherichia coli and Corynebacterium glutamicum for the production of l-threonine

l-threonine is an essential amino acid for mammals and as such has a wide and expanding application in industry with a fast growing market demand. The major method of production of l-threonine is microbial fermentation. To optimize l-threonine production the fundamental solution is to develop robust microbial strains with high productivity and stability. Metabolic engineering provides an effective alternative to the random mutation for strain development. In this review, the updated information on genetics and molecular mechanisms for regulation of l-threonine pathways in Escherichia coli and Corynebacterium glutamicum are summarized, including l-threonine biosynthesis, intracellular consumption and trans-membrane export. Upon such knowledge, genetically defined l-threonine producing strains have been successfully constructed, some of which have already achieved the productivity of industrial producing strains. Furthermore, strategies for strain construction are proposed and potential problems are identified and discussed. Finally, the outlook for future strategies to construct industrially advantageous strains with respect to recent advances in biology has been considered.     

Role of substrate functional groups in binding to nitric oxide synthase

The interactions between the heme CO ligand in the oxygenase domain of nitric oxide synthase and a set of substrate analogues were determined by measuring the resonance Raman spectra of the Fe-C-O vibrational modes. Substrates were selected that have variations in all the functional units: the guanidino group, the amino acid site and the number of methylene units connecting the two ends. In comparison to the substrate free form of the enzyme, Interactions of the analogues with the CO moiety caused the Fe-CO stretching and the Fe-C-O bending modes to shift in frequency due to the electrostatic environment. An unmodified guanidino group interacted with the CO in a similar fashion despite changes in the amino acid end. However, an unmodified amino acid end is required for catalysis owing to the H-bonding network involving the substrate, the heme and the pterin cofactor.