Synthesis of the carboxy-terminal octapeptide of cholecystokinin (CKK-8) based on incorporation of O4-sulfotyrosine by enzymatically catalyzed formation of peptide bonds

Ribulose-1,5-bisphosphate carboxylase/oxygense (RuBisCO) is present in plants and autotrophic organisms like microalgae. The aim of this study was to perform an in silico evaluation of RuBisCo protein in microalgae and cyanobacteria as potential precursors of bioactive peptides, as well as to determine whether such peptides can be released by selected proteolytic enzymes. Fourteen RuBioCo sequences of microalgae and cyanobacteria were analysed by using the BIOPEP server amd database. The biological activity, enzyme action and calculation of active peptide tools were used to determine the frequency of occurrence of fragments, proteolysis, and the frequency of release of fragments with given activity by selected enzymes. The physio-chemical parameters of the selected sequences were performed with Protpram tool. Amongst the RuBisCo proteins of selected algae, Chaetoceros. calcitrans exhibits the best prospect as a source of DPP-IV inhibiting peptides, Chlorella pyrenoidosa for ACE inhibitor and Aphanizomenon flos-aquae for antioxidative, activating ubiquitin, and antiamnestic activities. High number of bioactive fragments in Aphanizomenon flos-aquae, Dunaliella salina, Chlorella pyrenoidosa, and Chlorella vulgaris are associated with a high content of glycine and proline amino acids that are most rich in biologically active fragments. Papain and Proteinase K, an enzyme with wide specificity, can release considerably more biologically active fragments than bromealin and chymotrpsin. These findings will contribute towards consumption of microalgal and cyanobacterial RuBisCO as alternative sources of bioactive peptide fragments based nutraceuticals for human.

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Computational screening of microalgae and cyanobacteria RuBisCO as potential precursor for bioactive peptides

10.7287/peerj.preprints.2875v1 ◽

2017 ◽

Author(s):

Gurudeeban Selvaraj ◽

Satyavani Kaliamurthi ◽

Zeynep Elibol Cakmak ◽

Turgay Cakmak

Keyword(s):

Bioactive Peptides ◽

Proteolytic Enzymes ◽

Chlorella Pyrenoidosa ◽

Biologically Active ◽

Proteinase K ◽

Peptide Fragments ◽

Bisphosphate Carboxylase ◽

Computational Screening ◽

Active Fragments ◽

Chaetoceros Calcitrans

Ribulose-1,5-bisphosphate carboxylase/oxygense (RuBisCO) is present in plants and autotrophic organisms like microalgae. The aim of this study was to perform an in silico evaluation of RuBisCo protein in microalgae and cyanobacteria as potential precursors of bioactive peptides, as well as to determine whether such peptides can be released by selected proteolytic enzymes. Fourteen RuBioCo sequences of microalgae and cyanobacteria were analysed by using the BIOPEP server amd database. The biological activity, enzyme action and calculation of active peptide tools were used to determine the frequency of occurrence of fragments, proteolysis, and the frequency of release of fragments with given activity by selected enzymes. The physio-chemical parameters of the selected sequences were performed with Protpram tool. Amongst the RuBisCo proteins of selected algae, Chaetoceros. calcitrans exhibits the best prospect as a source of DPP-IV inhibiting peptides, Chlorella pyrenoidosa for ACE inhibitor and Aphanizomenon flos-aquae for antioxidative, activating ubiquitin, and antiamnestic activities. High number of bioactive fragments in Aphanizomenon flos-aquae, Dunaliella salina, Chlorella pyrenoidosa, and Chlorella vulgaris are associated with a high content of glycine and proline amino acids that are most rich in biologically active fragments. Papain and Proteinase K, an enzyme with wide specificity, can release considerably more biologically active fragments than bromealin and chymotrpsin. These findings will contribute towards consumption of microalgal and cyanobacterial RuBisCO as alternative sources of bioactive peptide fragments based nutraceuticals for human.

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The Use of Carbohydrate Protein Conjugates of Proteases [CPC(Proteases)] for the Catalytic Formation of Peptide Bonds

MRS Proceedings ◽

10.1557/proc-218-23 ◽

1990 ◽

Vol 218 ◽

Author(s):

Peng Wang ◽

Tara G. Hill ◽

Mark D. Bednarski ◽

Matthew R. Callstrom

Keyword(s):

Proteolytic Enzymes ◽

Peptide Bond ◽

Peptide Sequence ◽

Covalent Attachment ◽

General Strategy ◽

Protein Chemistry ◽

Peptide Fragments ◽

Peptide Bonds ◽

Protein Conjugates ◽

The Stability

The discovery of catalysts that can selectively couple unprotected peptide fragments would revolutionize protein chemistry by allowing convergent polypeptide synthesis. Proteolytic enzymes have the capability to perform this chemistry because the protein can specifically recognize and bind to C-terminal and N-terminal peptide sequences, activate the C-terminal peptide sequence by forming an acyl-enzyme intermediate, and couple the two peptide fragments together. However, barriers that limit the use of proteases as catalysts for convergent peptide synthesis include (i) the stability of proteolytic enzymes in organic solvent systems; (ii) a simple and effective C-erminal and N-terminal protecting group strategy; and (iii) the isolation of the polypeptide product from the reaction mixture. In the previous paper we reported the stabilization of enzymes by the covalent attachment of proteins through their ο-lysine residues to a series of carbohydrate-based macromolecules. In this paper we report the use of carbohydrate protein conjugates of proteases [CPC(proteases)] as catalysts for peptide bond synthesis and a general strategy for convergent oligopeptide synthesis.

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Synthesis and biological properties of cholecystokinin heptapeptide analogues containing D- and L-forms of tert-leucine or neopentylglycine in position 5

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19912209 ◽

1991 ◽

Vol 56 (10) ◽

pp. 2209-2217 ◽

Cited By ~ 8

Author(s):

Jan Hlaváček ◽

Jana Pírková ◽

Jan Pospíšek ◽

Jiřina Slaninová ◽

Lenka Maletínská

Keyword(s):

Gall Bladder ◽

Solid Phase ◽

Biological Activities ◽

Biological Properties ◽

Phase Synthesis ◽

Structural Modifications ◽

Bladder Contraction ◽

Anorectic Effect ◽

Carboxy Terminal ◽

Anorectic Activity

Using solution or solid-phase synthesis we prepared the cholecystokinin fragment Boc-CCK-7 (Boc-Tyr-(SO3-.Na+)-Met-Gly-Trp-Met-Asp-PheNH2) and its four analogues in which the methionine moiety (Met) in the carboxy-terminal part is replaced by tert-leucine (Tle) or neopentylglycine (Neo) residue or D-enantiomers of these non-coded amino acids. These structural modifications led to reduction of the studied biological activities (gall bladder contraction, anorectic activity, analgetic and sedation activity) of all prepared analogues except Boc[Neo5]-CCK-7 which, being less analgetically active, retains full gall bladder and sedation activity of CCK-8. Moreover, its anorectic activity is substantially higher (400%). This analogue is very interesting particularly for its selectively increased (4x) anorectic effect compared with that of CCK-8.

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Analogues of the cholecystokinin octapeptide modified in the central part of the molecule

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19911963 ◽

1991 ◽

Vol 56 (9) ◽

pp. 1963-1970 ◽

Cited By ~ 3

Author(s):

Jan Hlaváček ◽

Václav Čeřovský ◽

Jana Pírková ◽

Pavel Majer ◽

Lenka Maletínská ◽

...

Keyword(s):

Amino Acid ◽

Biological Activities ◽

Point Of View ◽

Biologically Active ◽

Side Chain ◽

Amino Acid Residues ◽

Cholecystokinin Octapeptide ◽

Biological Tests ◽

Biological Potency ◽

Biologically Active Conformation

In a series of analogues of the cholecystokinin octapeptide (CCK-8) the amino acid residues were gradually modified by substituting Gly by Pro in position 4, Trp by His in position 5, Met by Cle in position 6, or the Gly residue was inserted between Tyr and Met in positions 2 and 3 of the peptide chain, and in the case of the cholecystokinin heptapeptide (CCK-7) the Met residues were substituted by Nle or Aib. These peptides were investigated from the point of view of their biological potency in the peripheral and central region. From the results of the biological tests it follows that the modifications carried out in these analogues and in their Nα-Boc derivatives mean a suppression of the investigated biological activities by 2-3 orders of magnitude (at a maximum dose of the tested substance of 2 . 10-2 mg per animal).This means that a disturbance of the assumed biologically active conformation of CCK-8, connected with a considerable decrease of the biological potency of the molecule, takes place not only after introduction of the side chain into its centre (substitution of Gly4), but also after the modification of the side chains of the amino acids or by extension of the backbone in further positions around this central amino acid.

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Endo-fucoidan hydrolases from glycoside hydrolase family 107 (GH107) display structural and mechanistic similarities to α-l-fucosidases from GH29

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.005134 ◽

2018 ◽

Vol 293 (47) ◽

pp. 18296-18308 ◽

Cited By ~ 10

Author(s):

Chelsea Vickers ◽

Feng Liu ◽

Kento Abe ◽

Orly Salama-Alber ◽

Meredith Jenkins ◽

...

Keyword(s):

Glycoside Hydrolase ◽

Biological Activities ◽

Bacterial Species ◽

Structural Data ◽

Side Chain ◽

Glycoside Hydrolase Family ◽

X Ray Crystallography ◽

Catalytic Mechanisms ◽

Thickening Agents ◽

Hydrolase Family

Fucoidans are chemically complex and highly heterogeneous sulfated marine fucans from brown macro algae. Possessing a variety of physicochemical and biological activities, fucoidans are used as gelling and thickening agents in the food industry and have anticoagulant, antiviral, antitumor, antibacterial, and immune activities. Although fucoidan-depolymerizing enzymes have been identified, the molecular basis of their activity on these chemically complex polysaccharides remains largely uninvestigated. In this study, we focused on three glycoside hydrolase family 107 (GH107) enzymes: MfFcnA and two newly identified members, P5AFcnA and P19DFcnA, from a bacterial species of the genus Psychromonas. Using carbohydrate-PAGE, we show that P5AFcnA and P19DFcnA are active on fucoidans that differ from those depolymerized by MfFcnA, revealing differential substrate specificity within the GH107 family. Using a combination of X-ray crystallography and NMR analyses, we further show that GH107 family enzymes share features of their structures and catalytic mechanisms with GH29 α-l-fucosidases. However, we found that GH107 enzymes have the distinction of utilizing a histidine side chain as the proposed acid/base catalyst in its retaining mechanism. Further interpretation of the structural data indicated that the active-site architectures within this family are highly variable, likely reflecting the specificity of GH107 enzymes for different fucoidan substructures. Together, these findings begin to illuminate the molecular details underpinning the biological processing of fucoidans.

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“Newton’s cradle” proton relay with amide–imidic acid tautomerization in inverting cellulase visualized by neutron crystallography

Science Advances ◽

10.1126/sciadv.1500263 ◽

2015 ◽

Vol 1 (7) ◽

pp. e1500263 ◽

Cited By ~ 53

Author(s):

Akihiko Nakamura ◽

Takuya Ishida ◽

Katsuhiro Kusaka ◽

Taro Yamada ◽

Shinya Fushinobu ◽

...

Keyword(s):

Glycoside Hydrolases ◽

Side Chain ◽

Enzymatic Reactions ◽

X Ray Diffraction ◽

Peptide Bonds ◽

Proton Relay ◽

Newton’S Cradle ◽

Dynamics Simulations ◽

Hydrolysis Of

Hydrolysis of carbohydrates is a major bioreaction in nature, catalyzed by glycoside hydrolases (GHs). We used neutron diffraction and high-resolution x-ray diffraction analyses to investigate the hydrogen bond network in inverting cellulase PcCel45A, which is an endoglucanase belonging to subfamily C of GH family 45, isolated from the basidiomycete Phanerochaete chrysosporium. Examination of the enzyme and enzyme-ligand structures indicates a key role of multiple tautomerizations of asparagine residues and peptide bonds, which are finally connected to the other catalytic residue via typical side-chain hydrogen bonds, in forming the “Newton’s cradle”–like proton relay pathway of the catalytic cycle. Amide–imidic acid tautomerization of asparagine has not been taken into account in recent molecular dynamics simulations of not only cellulases but also general enzyme catalysis, and it may be necessary to reconsider our interpretation of many enzymatic reactions.

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Weissellicin 110, a Newly Discovered Bacteriocin from Weissella cibaria 110, Isolated from Plaa-Som, a Fermented Fish Product from Thailand

Applied and Environmental Microbiology ◽

10.1128/aem.02484-06 ◽

2007 ◽

Vol 73 (7) ◽

pp. 2247-2250 ◽

Cited By ~ 75

Author(s):

Sirinat Srionnual ◽

Fujitoshi Yanagida ◽

Li-Hsiu Lin ◽

Kuang-Nan Hsiao ◽

Yi-sheng Chen

Keyword(s):

Proteolytic Enzymes ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Protein Band ◽

Proteinase K ◽

Mass Spectrometry Analysis ◽

Fermented Fish ◽

Spectrometry Analysis ◽

Weissella Cibaria ◽

Fish Product

ABSTRACT Weissella cibaria 110, isolated from the Thai fermented fish product plaa-som, was found to produce a bacteriocin active against some gram-positive bacteria. Bacteriocin activity was not eliminated by exposure to high temperatures or catalase but was destroyed by exposure to the proteolytic enzymes proteinase K and trypsin. The bacteriocin from W. cibaria 110 was purified, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified bacteriocin contained one protein band that was approximately 2.5 kDa in size. Mass spectrometry analysis showed the mass of the peptide to be approximately 3,487.8 Da. N-terminal amino acid sequence analysis was performed, and 27 amino acids were identified. Because it has no similarity to other known bacteriocins, this bacteriocin was defined as a new bacteriocin and termed weissellicin 110.

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Characterization of a novel 23-kilodalton protein of unactive progesterone receptor complexes

Molecular and Cellular Biology ◽

10.1128/mcb.14.3.1956-1963.1994 ◽

1994 ◽

Vol 14 (3) ◽

pp. 1956-1963

Author(s):

J L Johnson ◽

T G Beito ◽

C J Krco ◽

D O Toft

Keyword(s):

Progesterone Receptor ◽

Cdna Clone ◽

Peptide Fragments ◽

Receptor Complexes ◽

Human Cdna ◽

Partial Clone ◽

Chicken Oviduct ◽

Brain Cdna Library ◽

Carboxy Terminal ◽

And Function

Immunoprecipitation of unactivated avian progesterone receptor results in the copurification of hsp90, hsp70, and three additional proteins, p54, p50, and p23. p23 is also present in immunoaffinity-purified hsp90 complexes along with hsp70 and another protein, p60. Antibody and cDNA probes for p23 were prepared in an effort to elucidate the significance and function of this protein. Antibodies to p23 detect similar levels of p23 in all tissues tested and cross-react with a protein of the same size in mice, rabbits, guinea pigs, humans, and Saccharomyces cerevisiae, indicating that p23 is a conserved protein of broad tissue distribution. These antibodies were used to screen a chicken brain cDNA library, resulting in the isolation of a 468-bp partial cDNA clone encoding a sequence containing four sequences corresponding to peptide fragments isolated from chicken p23. This partial clone was subsequently used to isolate a full-length human cDNA clone. The human cDNA encodes a protein of 160 amino acids that does not show homology to previously identified proteins. The chicken and human cDNAs are 88% identical at the DNA level and 96.3% identical at the protein level. p23 is a highly acidic phosphoprotein with an aspartic acid-rich carboxy-terminal domain. Bacterially overexpressed human p23 was used to raise several monoclonal antibodies to p23. These antibodies specifically immunoprecipitate p23 in complex with hsp90 in all tissues tested and can be used to immunoaffinity isolate progesterone receptor complexes from chicken oviduct cytosol.

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