Recombinant horseradish peroxidase isoenzyme C: the effect of distal haem cavity mutations (His42→Leu and Arg38→Leu) on compound I formation and substrate binding

 Horseradish peroxidase isoenzyme C (HRPC) mutants were constructed in order to understand the role of two key distal haem cavity residues, histidine 42 and arginine 38, in the formation of compound I and in substrate binding. The role of these residues as general acid-base catalysts, originally proposed for cytochrome c peroxidase by Poulos and Kraut in 1980 was assessed for HRPC. Replacement of histidine 42 by leucine [(H42L)HRPC*] decreased the apparent bimolecular rate constant for the reaction with hydrogen peroxide by five orders of magnitude (k ₁ = 1.4×10² M^–1s^–1) compared with both native-glycosylated and recombinant forms of HRPC (k ₁ = 1.7×10⁷ M^–1s^–1). The first-order rate constant for the heterolytic cleavage of the oxygen-oxygen bond to form compound I was estimated to be four orders of magnitude slower for this variant. Replacement of arginine 38 by leucine [(R38L)HRPC*] decreased the observed pseudo-first-order rate constant for the reaction with hydrogen peroxide by three orders of magnitude (k ₁ = 1.1×10⁴ M^–1s^–1), while the observed rate constant of oxygen bond scission was decreased sixfold (k ₂ = 142 s^–1). These rate constants are consistent with arginine 38 having two roles in catalysing compound I formation: firstly, promotion of proton transfer to the imidazole group of histidine 42 to facilitate peroxide anion binding to the haem, and secondly, stabilisation of the transition state for the heterolytic cleavage of the oxygen-oxygen bond. These roles for arginine 38 explain, in part, why dioxygen-binding globins, which do not have an arginine in the distal cavity, are poor peroxidases. Binding studies of benzhydroxamic acid to (H42L)HRPC* and (R38L)HRPC* indicate that both histidine 42 and arginine 38 are involved in the modulation of substrate affinity. Received: 21 July 1995 / Accepted: 27 November 1995     

A Leu–Lys-rich antimicrobial peptide: activity and mechanism

To develop novel antibiotic peptides useful as therapeutic drugs, the analogues were designed to increase not only net positive charge by Lys substitution but also hydrophobic helix region by Leu substitution from cecropin A (1–8)–magainin 2 (1–12) hybrid peptide (CA–MA). In particular, CA–MA analogue P5 (P5), designed by flexible region (GIG→P) substitution, Lys (positions 4, 8, 14, 15) and Leu (positions 5, 6, 12, 13, 16, 17, 20) substitutions, showed an enhanced antimicrobial and antitumor activity without hemolysis. Confocal microscopy showed that P5 was located in the plasma membrane. The antibacterial effects of analogues were further confirmed by using 1,6-diphenyl-1,3,5-hexatriene as a plasma membrane probe. Flow cytometric analysis revealed that P5 acted in an energy-independent manner. This interaction is also independent of the ionic environment. Furthermore, P5 causes significant morphological alterations of the bacterial surfaces as shown by scanning electron microscopy and showed strong membrane disrupting activity when examined using liposomes (phosphatidyl choline/cholesterol; 10:1, w/w). Its potent antibiotic activity suggests that P5 is an excellent candidate as a lead compound for the development of novel antiinfective agents.     

Enzymatic analysis of the effect of naturally occurring Leu138Pro mutation identified in SHV β-lactamase on hydrolysis of penicillin and ampicillin

Background  

The aim of this study was to analyze the significance of leucine to proline substitution at position 138(Leu138Pro) on the hydrolysis of penicillin and ampicillin that we identified in the bla _SHV gene of clinical Escherichia coli swine isolate.     

Biosynthetic origin and longevity in vivo of α-d-mannopyranosyl-(1 → 4)-α-d-glucuronopyranosyl-(1 → 2)-myo-inositol, an unusual extracellular oligosaccharide produced by cultured rose cells

A non-reducing trisaccharide, α-D-mannopyranosyl-(1 → 4)-α-D-glucuronopyranosyl-(1 → 2)-myo-inositol (MGI) accumulated in the spent medium of cell-suspension cultures of `Paul's Scarlet' rose (Rosa sp.) predominantly during the period of rapid cell growth. This trisaccharide was also produced by cultures of sycamore (Acer pseudoplatanus L.) but not by those of the graminaceous monocots maize (Zea mays L.) and tall fescue grass (Festuca arundinacea Schreb.). When added to cultured Rosa cells, [¹⁴C]MGI was neither taken up by the cells nor bound to the cell surface and was not metabolised extracellularly. When D-[6-¹⁴C]glucuronic acid was fed to cultured Rosa cells, extracellular [¹⁴C]MGI started to appear only after a 5-h lag period, compared with a 0.5-h lag period for labelling of extracelluar polysaccharides. Furthermore, [¹⁴C]MGI continued to accumulate in the medium for at least 20 h after the accumulation of ¹⁴C-polymers had ceased. These observations indicate that extracellular MGI was produced from a slowly turning-over pool of a pre-formed intermediate. Structural considerations indicate that the intermediate could be a glucuronomannan or a phytoglycolipid (glycophosphosphingolipid). No Rosa polysaccharides could be found that generated MGI in the presence of living Rosa cells. We therefore favour phytoglycolipids as the probable biosynthetic origin of MGI. Received: 29 April 1999 / Accepted: 13 June 1999     

Crystal structure of the O intermediate of the Leu93→Ala mutant of bacteriorhodopsin

The lifetime of the O intermediate of bacteriorhodopsin (BR) is extended by a factor of ～250 in the Leu93‐to‐Ala mutant (BR_L93A). To clarify the structural changes occurring in the last stage of the proton pumping cycle of BR, we crystallized BR_L93A into a hexagonal P622 crystal. Diffraction data from the unphotolyzed state showed that the deletion of three carbon atoms from Leu93 is compensated by the insertion of four water molecules in the cytoplasmic vicinity of retinal. This insertion of water is suggested to be responsible for the blue‐shifted λ_max (540 nm) of the mutant. A long‐lived substate of O with a red‐shifted λ_max (～565 nm) was trapped when the crystal of BR_L93A was flash‐cooled after illumination with green light. This substate (O_slow) bears considerable similarity to the M intermediate of native BR; that is, it commonly shows deformation of helix C and the FG loop, downward orientation of the side chain of Arg82, and disruption of the Glu194/Glu204 pair. In O_slow, however, the main chain of Lys216 is less distorted and retinal takes on the 13‐cis/15‐syn configuration. Another significant difference is seen in the pH dependence of the structure of the proton release group, the pK_a value of which is suggested to be much lower in O_slow than in M. Proteins 2012;. © 2012 Wiley Periodicals, Inc.     

Transformation of a Leu− Mutant of Rhizopus niveus with the leuA Gene of Mucor circinelloides

The 2287-bp cryptic plasmid, pMA1, from Microcystis aeruginosa f. aeruginosa Kützing, a unicellular cyanobacterium originally derived from Kasumigaura lake, was completely sequenced and analyzed. The predicted amino acid sequence (253 residues) of an open reading frame had identities of 47%, 48%, and 53% with replication-associated proteins of Bacillus amyloliquefaciens’s plasmid, pFTB14, B. subtilis BAA1’s pBAA1, and B. coagulans’s pBC1, respectively, when conservative amino acid substitutions were included. Such high-level identities were also shown with rep proteins and ori regions in a group of Gram-positive bacterial plasmids such as Lactococci and Staphylococci that are known to replicate via single-stranded intermediates. The pMA1 does hybridize with a plasmid, pUS1-3, derived from another unicellular cyanobacterium, Synechocystis sp. PCC 6803. Novel features of pMA1 are discussed.     

Generation of a stable non-reverting Leu− mutant of Kluyveromyces lactis by gene disruption

The LEU2 gene coding for -isopropylmalate dehydrogenase of the yeast Kluyveromyces lactis strain AWJ137 was disrupted. In the resulting Leu^– strain a 0.57 × 10³-base pairs PstI/BglII fragment of the LEU2 coding region was replaced by the TRP1 gene of Saccharomyces cerevisiae. The mutant strain was characterized by stability tests and a physical map of the disrupted region was established by restriction-enzyme analysis combined with hybridization experiments. The usefulness of the mutant strain as a recipient was shown by transformation experiments.     

The effects of Leu or Val residues on cell selectivity of α-helical peptides

In this study, the peptides were designed to compare the effect of multiple Leu or Val residues as the hydrophobic side of an α-helical model on their structure, function, and interaction with model membranes. The Leu-rich peptides displayed 4- to 16-fold stronger antimicrobial activity than Val-rich peptides, while Val-containing peptides showed no haemolysis and weak cytotoxicity. The peptides LR and VR showed an α-helical-rich structure under a membranemimicking environment. Different cell selectivity for Leu- or Val-containing peptides correlated with the targeted cell membranes. The Leu-rich peptide LR(W) and Val-rich peptide VR(W) interacted preferentially with negatively charged phospholipids over zwitterionic phospholipids. VR(W) displayed no interaction with zwitterionic phospholipids, which was consistent with its lack of haemolytic activity. The ability of LR to depolarize bacterial cells was much greater than that of VR. Val- and Leu-rich peptides appeared to kill bacteria in a membrane-targeted fashion, with different modes of action. Leu-rich peptides appeared to be active via a membrane-disrupting mode, while Val-rich peptides were active via the formation of small channels.     

Backbone and sidechain methyl Ile (δ1), Leu and Val resonance assignments of the catalytic domain of the yeast mRNA decapping enzyme, Dcp2

Eukaryotic mRNA decapping by Dcp2 is the penultimate step in several mRNA decay pathways. To understand regulation of Dcp2 by ligand interactions, we have assigned the backbone and sidechain methyl Ile (δ1), Leu and Val chemical shifts of the catalytic domain of the S. Cerevisiae enzyme.     

Interactions of a synthetic Leu–Lys-rich antimicrobial peptide with phospholipid bilayers

The interaction of the synthetic antimicrobial peptide P5 (KWKKLLKKPLLKKLLKKL-NH₂) with model phospholipid membranes was studied using solid-state NMR and circular dichroism (CD) spectroscopy. P5 peptide had little secondary structure in buffer, but addition of large unilamellar vesicles (LUV) composed of dimyristoylphosphatidylcholine (DMPC) increased the β-sheet content to ~20%. Addition of negatively charged LUV, DMPC–dimyristoylphosphatidylglycerol (DMPG) 2:1, led to a substantial (~40%) increase of the α-helical conformation. The peptide structure did not change significantly above and below the phospholipid phase transition temperature. P5 peptide interacted differently with DMPC bilayers with deuterated acyl chains (d₅₄-DMPC) and mixed d₅₄-DMPC–DMPG bilayers, used to mimic eukaryotic and prokaryotic membranes, respectively. In DMPC vesicles, P5 peptide had no significant interaction apart from slightly perturbing the upper region of the lipid acyl chain with minimum effect at the terminal methyl groups. By contrast, in the DMPC–DMPG vesicles the peptide increased disorder throughout the entire acyl chain of DMPC in the mixed bilayer. P5 promoted disordering of the headgroup of neutral membranes, observed by ³¹P NMR. However, no perturbations in the T ₁ relaxation nor the T ₂- values were observed at 30°C, although a slight change in the dynamics of the headgroup at 20°C was noticeable compared with peptide-free vesicles. However, the P5 peptide caused similar perturbations of the headgroup of negatively charged vesicles at both temperatures. These data correlate with the non-haemolytic activity of the P5 peptide against red blood cells (neutral membranes) while inhibiting bacterial growth (negatively charged membranes).     

Antitumour effect of glucooligosaccharides obtained via hydrolysis of α-(1 → 3)-glucan from Fomitopsis betulina

Molecular Biology Reports - Novel α-(1?→?3)-glucooligosaccharides (α-(1?→?3)-GOS) were prepared by acid hydrolysis of α-(1→?3)-glucan...     

Galactan biosynthesis in snails: a comparative study of β-(1 → 6) galactosyltransferases from Helix pomatia and Biomphalaria glabrata

Adult snails synthesize in their albumen glands a polysaccharide which is composed exclusively of D- or D- and L-galactose (Gal) residues which are interglycosidically linked by 1 → 3 and 1 → 6 bonds. It is the only carbohydrate source for embryos and freshly hatched snails. Two galactosyltransferases are described in this study which are most likely involved in the biosynthesis of this polysaccharide. One identified in Helix pomatia acts on oligosaccharides and could be used to synthesize a tetrasaccharide when the branched trisaccharide D-Gal-β-(1 → 3)-[D-Galβ-(1 → 6)]-D-Galβ-1 → OMe was offered as acceptor. This enzyme, requiring Mg⁺⁺- and Mn⁺⁺-ions for activity, introduced a linear β-(1 → 6) linkage at the terminal non-reducing ends and was not detected in Biomphalaria glabrata. The other enzyme, which introduced β-(1 → 6) linkages at subterminal D-Gal residues, thus forming branching points in the polysaccharide, was found in H. pomatia, Arianta arbustorum and B. glabrata with comparable activities. With the enzyme preparation of H. pomatia, up to four D-Gal residues were introduced into vicinal positions, forming single-membered side chains, if a hexasaccharide with five linearly β-(1 → 3)-linked D-Gal residues was offered as a acceptor. The multiple-branched structure formed is typical for snail galactans, making this enzyme a prime candidate for the branching enzyme in galactan synthesis. The enzyme activity could be solubilized and purified by affinity chromatography. In SDS-polyacrylamide electrophoresis, the Helix- derived eluate displayed two bands (68, 37 kDa) and that of Biomphalaria five bands (68, 63, 17.5; 15; 13 kDa). The purified material showed only 8% of the total activity of the crude extracts, but it could be shown that a phosphatase present in the crude extract can degrade UDP formed in the transfer reaction and thus drive the reaction to completion. Accepted: 23 August 2000     

Protodioscin-glycosidase-1 hydrolyzing 26-O-β-d-glucoside and 3-O-(1 → 4)-α-l-rhamnoside of steroidal saponins from Aspergillus oryzae

A novel protodioscin-(steroidal saponin)-glycoside hydrolase, named protodioscin-glycosidase-1 (PGase-1), was purified and characterized from the Aspergillus oryzae strain. The molecular mass of this enzyme was determined to be about 55 kDa based on SDS-polyacrylamide gel electrophoresis. PGase-1 was able to hydrolyze the terminal 26-O-β-d-glucopyranoside of protodioscin (furostanoside) to produce dioscin (spirostanoside), and then further hydrolyze the terminal 3-O-(1?→?4)-α-l-rhamnopyranoside of dioscin to form progenin III. However, PGase-1 could hardly hydrolyze the 3-O-(1?→?2)-α-l-rhamnopyranoside of progenin III, 3-O-β-d-glucoside of trillin, and the 1-O-glycosides of ophiopogonin D (steroidal saponin). In addition, PGase-1 also could hydrolyze the α-d-galactopyranoside, β-d-glucopyranoside, and β-d-galactopyranoside of p-nitrophenyl-glycosides, but the enzyme could not hydrolyze the α-d-mannopyranoside, α-l-arabinopyranoside, α-d-glucopyranoside, β-d-xylopyranoside, and α-l-rhamnopyranoside of p-nitrophenyl-glycosides. These new properties of PGase-1 were significantly different from those of previously described steroidal saponin-glycosidases and the glycosidases currently described in Enzyme Nomenclature by the NC-IUBMB. The gene (termed as pgase-1) encoding PGase-1 was cloned, sequenced, and expressed in Pichia pastoris GS115. The complete nucleotide sequence of pgase-1 consists of 1,725 bp. The recombinant PGase-1 from recombinant P. pastoris GS115 strain also showed the activity hydrolyzing glycosides of steroidal saponins which was similar to that of the wild-type PGase-1 from A. oryzae. The PGase-1 gene is highly similar to Aspergilli α-amylase (EC 3.2.1.1), and PGase-1 should be classified as glycoside hydrolase family 13 by the method of gene sequence-based classification. But the enzyme properties of PGase-1 are different from those of α-amylase in this family.     

Investigation of tRNA<Superscript>Leu/Lys</Superscript> and ATPase 6 Genes Mutations in Huntington’s Disease

Huntington disease (HD) is a genetically dominant condition caused by expanded CAG repeats which code for glutamine in the HD gene product, huntingtin. Huntingtin is expressed in almost all tissues, so abnormalities outside the brain can also be expected. Involvement of nuclei and mitochondria in HD pathophysiology has been suggested. In fact mitochondrial dysfunction is reported in brains of patients suffering from HD. The tRNA gene mutations are one of hot spots that can cause mitochondrial disorders. In this study, possible mitochondrial DNA (mtDNA) damage was evaluated by screening for mutations in the tRNA^leu/lys and ATPase 6 genes of 20 patients with HD, using PCR and automated DNA sequencing. Mutations including an A8656G mutation in one patient were observed, which may be causal to the disease. Understanding the role of mitochondria in the pathogenesis of neurodegenerative diseases could potentially be important for the development of therapeutic strategies in HD.     

Use of “Loss-of-Contact” Substitutions to Identify Residues Involved in an Amino Acid-Base Pair Contact: Effect of Substitution of Gln18 of Lac Repressor by Gly,Ser, and Leu

Abstract

A procedure to identify which base pair of lac operator (lacO) a suspected contacting amino acid of Lac repressor (LacR) interacts with is presented. The procedure is to eliminate the ability of the amino acid under study to contact DNA, and then to determine at which base pair—if any—specificity is eliminated. To implement this procedure, four sets of Escherichia coli K-12 strains have been constructed. These strains permit: (i) the substitution of a selected amino acid of LacR by, respectively, Gly, Ser, Leu, or Gln, and (ii) the analysis of the specificity of the resulting substituted LacR with respect to base pairs 5,6,7,8,9, and 10 of lacO. This procedure has been applied to Gln18 of LacR. The preliminary data indicate that LacR(Gln18?Gly) is unable to distinguish between the O⁺ base pair G:C and the O^c base pair T:A at position 7 of lacO (K_Doc/K_DO ⁺ = 0.93). In contrast, LacR(Glnl8?Gly) discriminates O⁺ from O^c by a factor of 13 to 23 at each other position. The same qualitative pattern of results was obtained with LacR(Glnl8?Ser) and LacR(Gln18?Leu). Therefore, I propose that Gln18 contacts base pair 7 of lacO. This proposal is consistent with the contact predicted in Ebright, R. in Protein Structure, Folding, and Design. D. Oxender ed., Alan R. Liss, New York (1985), in press.     

Prognostic Potential of the Panfungal Marker (1 → 3)-β-d-Glucan in Invasive Mycoses Patients

Mycopathologia - We analyze the prognostic potential of (1?→?3)-β-d-glucan (BG) levels in predicting clinical outcomes in patients with invasive fungal infections, on a...     

Synthesis and Cytotoxicity of О- and N-Acyl Derivatives of Azepanobetulin

Russian Journal of Bioorganic Chemistry - A five-stage synthesis of azepanobetulin from betulin with a total yield of 47% has been carried out. The acylation of azepanobetulin with anhydrides or...