Chemical Modification of Neutral Protease from Bacillus subtilis var. amylosacchariticus: Assignment of Tyrosyl Residues Iodinated

The neutral protease of Bacillus subtilis var. amylosacchariticus (B. amylosacchariticus) was iodinated with a 25-fold molar excess of iodine at pH 9.4 for 3 min at 0°C, by which treatment the proteolytic activity toward casein was markedly reduced, while the hydrolytic activity toward an N-blocked peptide substrate was rather increased. The modified enzyme was digested with Staphylococcus aureus V8 protease at pH 8.0 and the amino acid sequences of resultant peptides were compared with those obtained from the native enzyme. One of the peptides was found to have an amino acid sequence of Thr-Ala-Asn-Leu-Ile-Tyr-Glu, which corresponds to residue Nos. 153—159 of the enzyme, where Tyr-158 was identified to be mono-iodotyrosine. The other two peptides were those containing Tyr-21 which was mono- and di-iodinated, respectively. Referring to nitration experiments on the neutral protease and the active site structure of thermolysin, it was concluded that the iodination of Tyr-158 is mainly responsible for the activity changes of B. amylosacchariticus neutral protease.     

Phage-Displayed Libraries for the Selection of Optimal Affinity Peptides for Protein Purification Using Ni-Nitrilotriacetic Acid Chromatography

A random hexapeptide library, cloned in bacteriophage, was used to select affinity peptides using nickel-nitrilotriacetic acid (Ni-NTA) columns. The screening protocol was successful by isolating peptides sharing common features and, in most cases, common amino acid sequences were isolated (e.g. WHHHPH, AQHHHH). Ni-NTA chromatography of the fusion phage of the selected peptides exhibited a more homogeneous elution behavior (i.e. elution in one peak) than the most commonly used His₆peptide (elution in multiple peaks).     

Phylogenetic Analysis and Biochemical Characterization of a Thermostable Dihydropyrimidinase from Alkaliphilic Bacillus sp. TS-23

Two degenerate primers established from the alignment of highly conserved amino acid sequences of bacterial dihydropyrimidinases (DHPs) were used to amplify a 330-bp gene fragment from the genomic DNA of Bacillus sp. TS-23 and the amplified DNA was successfully used as a probe to clone a dhp gene from the strain. The open reading frame of the gene consisted of 1422 bp and was deduced to contain 472 amino acids with a molecular mass of 52 kDa. The deduced amino acid sequence exhibited greater than 45% identity with that of prokaryotic d-hydantoinases and eukaryotic DHPs. Phylogenetic analysis showed that Bacillus sp. TS-23 DHP is grouped together with Bacillus stearothermophilus d-hydantoinase and related to dihydroorotases and allantoinases from various organisms. His₆-tagged DHP was over-expressed in Escherichia coli and purified by immobilized metal affinity chromatography to a specific activity of 3.46 U mg⁻¹ protein. The optimal pH and temperature for the purified enzyme were 8.0 and 60 °C, respectively. The half-life of His₆-tagged DHP was 25 days at 50 °C. The enzyme activity was stimulated by Co²⁺ and Mn²⁺ ions. His₆-tagged DHP was most active toward dihydrouracil followed by hydantoin derivatives. The catalytic efficiencies (k_cat/K_m) of the enzyme for dihydrouracil and hydantoin were 2.58 and 0.61 s⁻¹ mM⁻¹, respectively.     

Bitter Peptides in Cow Milk Casein Digests with Bacterial Proteinase

Isolation and determination of amino acid sequence of the bitter peptides formed in the digestion of cow milk casein with alkaline proteinase of Bacillus subtilis were investigated. The casein digest with the enzyme was extracted with butanol and the extracted bitter peptides were fractionally purified by treating with several other organic solvents followed by subjecting to chromatography and gel-filtration. The amino acid sequence of one of the bitter peptides was determined as follows: Arg-Gly-Pro-Pro-Phe-Ileu-Val. Liberation of N-terminal Arg with trypsin or bacterial aminopeptidase did not affect the bitterness. Also, splitting off of Val and Ileu or Ileu-Val at the C-terminus by carboxypeptidase, or a bacterial neutral proteinase gave no influence on the bitterness. However, liberation of Arg and Gly from the peptide with bacterial aminopeptidase gave rise to a non bitter peptide.     

Development of a multifunctional tool for drug screening against plasmodial protein–protein interactions via surface plasmon resonance

We have developed an expression system capable of producing large quantities of low cost, specific peptides that are either His₁₂‐tagged, biotinylated, or unlabeled. The flexibility of this peptide system is suitable for interaction studies via surface plasmon resonance (SPR), co‐crystallization, and enzyme‐linked immunosorbent assay. Gene blocks containing peptide sequences of interest in addition to a 15 amino acid AviTag?, were cloned into a vector expressing an N‐terminal maltose binding protein. The constructs were expressed and purified, and the molecular weights of the recombinant proteins were estimated by analytical size exclusion chromatography. Successful in situ biotinylation of the AviTag was confirmed by anti‐biotin western blot and was used for coupling to the surface plasmon resonance chip. We were able to validate, as a proof of concept study, the specific protein–protein interaction of Plasmodium falciparum aldolase (PfAldolase) with three different cytoplasmic adhesin tail peptides from the family of thrombospondin‐related anonymous proteins (TRAPs), and to determine their affinities. This method of peptide production enables high yield production of peptides in a two‐day, cost effective manner. This tool will allow us to screen for protein–protein interaction inhibitors directed toward the liver stage and blood stage complexes of the glideosome in Plasmodium species. Adaptation of this tool will allow researchers to pursue their own studies of protein–protein interactions. Copyright © 2013 John Wiley & Sons, Ltd.     

Plant aldolase: cDNA and deduced amino-acid sequences of the chloroplast and cytosol enzyme from spinach

We report the sequences of full-length cDNAs for the nuclear genes encoding the chloroplastic and cytosolic fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) from spinach. A comparison of the deduced amino-acid sequences with one another and with published cytosolic aldolase sequences of other plants revealed that the two enzymes from spinach share only 54% homology on their amino acid level whereas the homology of the cytosolic enzyme of spinach with the known sequences of cytosolic aldolases of maize, rice and Arabidopsis range from 67 to 92%. The sequence of the chloroplastic enzyme includes a stroma-targeting N-terminal transit peptide of 46 amino acid residues for import into the chloroplast. The transit peptide exhibits essential features similar to other chloroplast transit peptides. Southern blot analysis implies that both spinach enzymes are encoded by single genes.     

Genetic characterization and expression of the novel fungal protease, EPg222 active in dry-cured meat products

EPg222 protease is a novel extracellular enzyme produced by Penicillium chrysogenum (Pg222) isolated from dry-cured hams that has the potential for use over a broad range of applications in industries that produce dry-cured meat products. The gene encoding EPg222 protease has been identified. Peptide sequences of EPg222 were obtained by de novo sequencing of tryptic peptides using mass spectrometry. The corresponding gene was amplified by PCR using degenerated primers based on a combination of conserved serine protease-encoding sequences and reverse translation of the peptide sequences. EPg222 is encoded as a gene of 1,361 bp interrupted by two introns. The deduced amino acid sequence indicated that the enzyme is synthesized as a preproenzyme with a putative signal sequence of 19 amino acids (aa), a prosequence of 96 aa and a mature protein of 283 aa. A cDNA encoding EPg222 has been cloned and expressed as a functionally active enzyme in Pichia pastoris. The recombinant enzyme exhibits similar activities to the native enzyme against a wide range of protein substrates including muscle myofibrillar protein. The mature sequence contains conserved aa residues characteristic of those forming the catalytic triad of serine proteases (Asp42, His76 and Ser228) but notably the food enzyme exhibits specific aa substitutions in the immunoglobulin-E recognition regions that have been identified in protein homologues that are allergenic.     

Tyr1 and Ile7 of glucose-dependent insulinotropic polypeptide (GIP) confer differential ligand selectivity toward GIP and glucagon-like peptide-1 receptors

Glucagon like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are incretin hormones released in response to food intake and potentiate insulin secretion from pancreatic β cells through their distinct yet related G protein-coupled receptors, GLP1R and GIPR. While GLP-1 and GIP exhibit similarity in their N-terminal sequence and overall α-helical structure, GLP-1 does not bind to GIPR and vice versa. To determine which amino acid residues of these peptide ligands are responsible for specific interaction with their respective receptors, we generated mutant GIP in which several GLP-1-specific amino acid residues were substituted for the original amino acids. The potency of the mutant ligands was examined using HEK293 cells transfected with GLP1R or GIPR expression plasmids together with a cAMP-responsive element-driven luciferase (CRE-luc) reporter plasmid. A mutated GIP peptide in which Tyr¹, Ile⁷, Asp¹⁵, and His¹⁸ were replaced by His, Thr, Glu, and Ala, respectively, was able to activate both GLP1R and GIPR with moderate potency. Replacing the original Tyr¹ and/or Ile⁷ in the N-terminal moiety of this mutant peptide allowed full activation of GIPR but not of GLP1R. However, reintroducing Asp¹⁵ and/or His¹⁸ in the central α-helical region did not significantly alter the ligand potency. These results suggest that Tyr/His¹ and Ile/Thr⁷ of GIP/GLP-1 peptides confer differential ligand selectivity toward GIPR and GLP1R.     

Role of Lys281 in the <Emphasis Type="Italic">Dunaliella salina</Emphasis> (6-4) Photolyase Reaction

His³⁵⁴ and His³⁵⁸, two highly conserved histidines in Xenopus laevis (6-4) photolyase [equivalent to His⁴⁰¹ and His⁴⁰⁵, in Dunaliella salina (6-4) photolyase], are critical for photoreactivation. They act as a base and an acid, respectively. However, the remaining high repair activity when the pH value is higher than the pKa of histidine suggests the involvement of other basic amino acids in photoreactivation. According to the results of in vivo enzyme assay and three-dimension structural model of Dunaliella salina (6-4) photolyase we hypothesized that Lys²⁸¹ might be involved in the photoreactivation over the pH range from 10.0 to 11.0. To test this, we generated two mutant forms of the (6-4) photolyase, K281G and K281R mutant, by overlap extension polymerase chain reaction, and performed the enzyme assay with these mutants. From these results we conclude that the Lys²⁸¹, which is highly conserved in (6-4) photolyases, participates in the photoreactivation and acts as an acid to donate a proton to His⁴⁰¹ when the environmental pH is higher than the pKa value of histidine.     

Complete Amino Acid Sequence of Neutral Protease from Bacillus subtilis var. amylosacchariticus

The neutral protease of Bacillus subtilis var. amylosacchariticus was cleaved chemically or digested with proteolytic enzymes, and the resultant peptides were separated and purified by high performance liquid chromatography. The sequence analyses of these peptides by the manual Edman procedure established the complete amino acid sequence of the enzyme. The neutral protease consisted of 300 amino acid residues with Ala and Leu as its amino- and carboxyl-termini, respectively, and the molecular weight was calculated to be 32,633. The sequence was found to be identical to that of B. subtilis 1A72 neutral protease, which was deduced from nucleotide sequencing. Comparison of the sequence with those of other Bacillus proteases revealed that the putative active site amino acid residues, Zn-binding ligands, and two Ca-binding sites were well conserved among them, as compared with those of thermolysin.     

Synthesis and characterization of β‐peptide helices as transmembrane domains in lipid model membranes

Aggregation, orientation and dynamics of transmembrane helices are of relevance for protein function and transmembrane signaling. To explore the interactions of transmembrane helices and the interdependence of peptide structure and lipid composition of the membranes, β‐peptides were explored as model transmembrane domains. Various hydrophobic β‐peptide sequences were synthesized by solid phase peptide synthesis. Conformational analyses of β‐peptide helices were performed in organic solvents (methanol and 2,2,2‐trifluoroethanol) and in large unilamellar liposomes (dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine and dioleoylphosphatidylcholine) indicating 12‐ and 14‐helix conformations, depending on β³‐amino acid sequences. The intrinsic tryptophan fluorescence of β³‐homotryptophan units inserted in the center or near the end of the sequence was used to verify the membrane insertion of the β‐peptides. A characteristic blue shift with peripheral β³‐homotryptophan compared with β‐peptides with central tryptophan served as indication for a transmembrane orientation of the β‐peptides within the lipid bilayer. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Carboxyl Proteinase from the Wood Deteriorating Basidiomycete Pycnoporus coccineus :Substrate Specificity with Oxidized Insulin Peptide B1 ~ B16 and B15 ~ B24, Angiotensin and Proangiotensin

The specificity of highly purified carboxyl proteinase from Pycnoporus coccineus (formerly designated Trametes sanguined) was investigated with oligopeptides at pH 2.7. Hydrolysis of oxidized insulin peptide Bl ~ B16 was observed at two peptide bonds (His¹⁰-Leu¹¹ and Ala¹⁴-Leu¹⁵) during 3-hr incubation. The enzyme did not hydrolyze oxidized insulin peptide B15 ~ B24. Hydrolysis of angiotensin (formerly designated angiotensin II) was observed at the Tyr⁴-Ile⁵ bond. Hydrolysis of proangiotensin (formerly designated angiotensin I) was also at the Tyr⁴-Ile⁵ bond. In conclusion, peptide bonds which have a hydrophobic amino acid in the P′₁ position (as defined by Schechter and Berger, Biochem. Biophys. Res. Commun., 27, 157 (1967)) are preferentially cleaved by the trypsinogen activating carboxyl proteinase of Pycnoporus coccineus.     

Specific cleavage of beta-amyloid peptides by a metallopeptidase from Xenopus laevis skin secretions

Dactylysin (EC 3.5.24.60) is a metalloendopeptidase first isolated from the skin granular gland secretions of Xenopus laevis. This peptidase hydrolyzes bonds on the amino-terminus of singlets and between doublets of hydrophobic amino acids and was considered to play a role in the in vivo inactivation of biologically active regulatory peptides. Here, we show that dactylysin has also the ability to cleave human β[1-40]-amyloid peptide and related peptides. Cleavage of the wild type β[1-40]-amyloid peptide form, and to a lesser extent Flemish and Dutch mutants, occurred predominantly at the His¹⁴-Glu¹⁵ bond. We demonstrate that frog skin exudate contains a full-length amyloid protein precursor detected by immunochemical cross-reactivity with monoclonal antibody against C-terminal human amyloid protein precursor. The possibility that dactylysin, might be involved in normal catabolism of β amyloid peptide of Xenopus laevis is discussed.     

Identification of mouse selenomethionine α, γ-elimination enzyme

The purpose of this study was to identify the seleno-l-methionine (l-SeMet) α,γ-elimination enzyme that catalyzes l-SeMet to generate methylselenol (CH₃SeH), a notable intermediate for the metabolism of selenium compounds, in mammalian tissues. The enzyme purified from ICR mouse liver was separated by one-dimensional gel electrophoresis, and the specific band was subjected to in-gel trypsin digestion followed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometric analysis. In the peptide mass fingerprinting search, the mass numbers of 14 peptides produced by tryptic digestion of the enzyme were consistent with the theoretical mass numbers calculated from the amino acid sequence of murine cystathionine γ-lyase (E.C. 4.4.1.1). The peptide sequence tags search was also performed to obtain the amino acid sequence data of five tryptic peptides. These peptides were significantly identical to the partial amino acid sequences of cystathionine γ-lyase. This enzyme was clearly shown to catalyze the α, γ-elimination reaction of l-cystathionine by the enzymological research. The K _m value for the catalysis of l-cystathionine was 0.81 mM and V _max was. 0.0013 unit/mg protein. These results suggested that cystathionine γ-lyase catalyzes l-SeMet to generate CH₃SeH by its α,γ-elimination reaction.     

Acid Soluble Peptides in Rat Skeletal Muscle

The acid soluble peptide fraction was prepared from rat skeletal muscle, and the amino acid composition of the fraction was analyzed. The peptide fraction was rich in glutamic acid (or glutamine) and glycine and was poor in branched chain or aromatic amino acids. Since the peptide fraction contained N^τ-methylhistidine, the fraction or at least a part of it was presumed to be composed of intermediate peptides of protein degradation in skeletal muscle. At least 31 spots were detected in the fraction by one dimensional paper electrophoresis.     

Studies on Bacterial Protease

Some physical and chemical properties and substrate specificity were investigated of the neutral protease obtained from B. amylosacchariticus, a strain of saccharogenic α-amylase producing Bacillus subtilis. The molecular weight and sedimentation coefficient of the protease were estimated to be 33,800 and 3.02, respectively, by ultracentrifugal analyses, and alanine was identified as an amino-terminal amino acid of the enzyme by the Sanger’s method. The enzyme showed more broad specificity than the neutral protease of liquefying α-amylase-producing B. subtilis, when tested with synthetic peptides, and hippuryl-l-leucinamide was the best substrate among 42 compounds tested. On a long incubation, the enzyme hydrolyzed several proteins in a degree of 10 to 25% as peptide bond cleavage.     

Primary structure and chemical modification of some amino acid residues of bifunctional alginate lyase from a marine bacterium Pseudoalteromonas sp. strain no. 272

The entire amino acid sequence of bifunctional alginate lyase from Pseudoalteromonas sp. strain No. 272 were determined by two approaches, Edman degradation of the peptides obtained from protease digestion of the enzyme protein and analysis of PCR products of the structural gene. The former resulted in incomplete amino acid sequence in the entire sequence, due to lacking of the proper peptides from the protease digestion. To compensate for this lack of sequences we applied the method of PCR of the structural gene that was initially elucidated from the primers designed from N- and C-terminal amino acid sequences of the enzyme. The results of the amino acid sequences from these two approaches showed good agreement. The enzyme consisted of 233 amino acid residues with a molecular mass of 25,549.5, including the sole W and cystine residue. The sequence homology search among the other alginate lyases from different origins indicated that they were very weakly homologous, with the exception of the sequence homology (80.3%) of Pseudoalteromonas elyakovii alginate lyase. The consensus sequence, YFKhG + Y-Q (Wong, T. Y., Preston, L. A., and Schiller, N. L. 2000. Annu. Rev. Microbiol. 54: 289–340) in the C-terminal regions was conserved. The kinetic analyses of chemical modification of some amino acid residues of the enzyme showed that W, K, and Y appeared to be important in the enzyme function.     

A Convenient Synthesis of 4-Hydroxy-3-methyl-2-(2-propynyl)-cyclopent-2-enone,an Alcohol Moiety of a Synthetic Pyrethroid Having Strong Killing and Knockdown Activity

We examined the primary structure of the α-amylase produced by Bacillus subtilis var. amylosacchariticus by attempting to isolate tryptic peptides of the enzyme. By solubilization and precipitation in buffers, the peptides were first fractionated into three. The main fraction was fractionated by ion-exchange chromatography. Twenty-seven peptides were generated from this fraction. The fraction insoluble at neutral pH was fractionated by SP-Sephadex C-25. From this fraction three peptides were obtained. The other fraction insoluble at acidic pH was fractionated by Bio-Gel P-60. Four peptides were isolated from this fraction. In total, thirty-four peptides were generated from the tryptic digest of the α-amylase. The amino acid sequences of twenty-one out of thirty-four peptides were completely determined, while those of the other thirteen peptides were partially determined. The peptides derived from the N- and C-terminal ends of the α-amylase were identified.     

NH2-terminal dodecapeptide of porcine big gastrin: Revised sequence and confirmation of structure by immunochemical analysis

A revised sequence for the NH₂-terminal dodecapeptide of porcine big gastrin is described which differs from that originally reported in the inversion of His⁷ and Pro⁹ for Pro⁷ and His⁹. The immunochemical properties of a range of synthetic peptide fragments and analogs of the original and revised sequences of porcine big gastrin were examined with an antiserum raised to the natural porcine peptide. The pattern of immunoreactivity of these peptides indicates that the antiserum has specificity for the 4–9 region of big gastrin. The dodecapeptide with the revised sequence had full immunoreactive potency relative to natural porcine big gastrin, whereas the dodecapeptide with the original sequence had about 1000-fold lower immunoreactivity. It is proposed that the synthetic peptide with the revised, but not the original, sequence is compatible with the structure of big gastrin.     

Crystal structure and site-directed mutagenesis of a nitroalkane oxidase from Streptomyces ansochromogenes

Nitroalkane oxidase (NAO) catalyzes neutral nitroalkanes to their corresponding aldehydes or ketones, hydrogen peroxide and nitrite. The crystal structure of NAO from Streptomyces ansochromogenes was determined; it consists of two domains, a TIM barrel domain bound to FMN and C-terminal domain with a novel folding pattern. Site-directed mutagenesis of His¹⁷⁹, which is spatially adjacent to FMN, resulted in the loss of enzyme activity, demonstrating that this amino acid residue is important for catalysis. The crystal structure of mutant H179D-nitroethane was also analyzed. Interestingly, Sa-NAO shows the typical function as nitroalkane oxidase but its structure is similar to that of 2-nitropropane dioxygenase. Overall, these results suggest that Sa-NAO is a novel nitroalkane oxidase with TIM barrel structure.