- Development of a fluorogenic small substrate for dipeptidyl peptidase-4
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A series of aniline and m-phenylenediamine derivatives with electron-withdrawing 3,3,3-trifluoropropenyl substituents were synthesized as small and chemically stable fluorescent organic compounds. Their fluorescence performances were evaluated by converting 2,4-disubstituted aniline 1 to the non-fluorescent dipeptide analogue H-Gly-Pro-1 for the use as a fluorogenic substrate for dipeptidyl peptidase-4 (DPP-4). The progress of the enzymatic hydrolysis of H-Gly-Pro-1 with DPP-4 was monitored by fluorometric determination of 1 released into the reaction medium. The results suggest that 1 could be used as fluorophore in OFF-ON-type fluorogenic probes.
- Ogawa, Futa,Takeda, Masanori,Miyanaga, Kanae,Tani, Keita,Yamazawa, Ryuji,Ito, Kiyoshi,Tarui, Atsushi,Sato, Kazuyuki,Omote, Masaaki
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Read Online
- Identification and characterization of prokaryotic dipeptidyl-peptidase 5 from porphyromonas gingivalis
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Porphyromonas gingivalis, a Gram-negative asaccharolytic anaerobe, is a major causative organism of chronic periodontitis. Because the bacterium utilizes amino acids as energy and carbon sources and incorporates them mainly as dipeptides, a wide variety of dipeptide production processes mediated by dipeptidyl-peptidases (DPPs) should be beneficial for the organism. In the present study, we identified the fourth P. gingivalis enzyme, DPP5. In a dpp4-7-11-disrupted P. gingivalis ATCC 33277, a DPP7-like activity still remained. PGN-0756 possessed an activity indistinguishable from that of the mutant, and was identified as a bacterial orthologue of fungal DPP5, because of its substrate specificity and 28.5% amino acid sequence identity with an Aspergillus fumigatus entity. P. gingivalis DPP5 was composed of 684 amino acids with a molecular mass of 77,453, and existed as a dimer while migrating at 66 kDa on SDS-PAGE. It preferred Ala and hydrophobic residues, had no activity toward Pro at the P1 position, and no preference for hydrophobic P2 residues, showed an optimal pH of 6.7 in the presence of NaCl, demonstrated Km and kcat/Km values for Lys-Ala-MCA of 688 μM and 11.02 μM-1 s-1, respectively, and was localized in the periplasm. DPP5 elaborately complemented DPP7 in liberation of dipeptides with hydrophobic P1 residues. Examinations of DPP- and gingipain gene-disrupted mutants indicated that DPP4, DPP5, DPP7, and DPP11 together with Arg- and Lys-gingipains cooperatively liberate most dipeptides from nutrient oligopeptides. This is the first study to report that DPP5 is expressed not only in eukaryotes, but also widely distributed in bacteria and archaea.
- Ohara-Nemoto, Yuko,Rouf, Shakh M. A.,Naito, Mariko,Yanase, Amie,Tetsuo, Fumi,Ono, Toshio,Kobayakawa, Takeshi,Shimoyama, Yu,Kimura, Shigenobu,Nakayama, Koji,Saiki, Keitarou,Konishi, Kiyoshi,Nemoto, Takayuki K.
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p. 5436 - 5448
(2014/03/21)
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- Zinc(II) complexes of Pro-Gly and Pro-Leu dipeptides: Synthesis, characterization, in vitro DNA binding and cleavage studies
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Dipeptide (Pro-Gly and Pro-Leu) Zinc(II) complexes 1 and 2 were designed and synthesized for potential use as cancer chemotherapeutic agents. In order to augment the DNA recognition of metallonuclease activity, zinc metal ion was tethered to peptide motif to carry out DNA site specific hydrolytic cleavage. The structural formulation of the complexes 1 and 2 was done by elemental analysis, spectroscopic methods (IR, NMR, electronic) and molar conductance measurements. Their in vitro DNA binding profile was investigated by UV-vis titrations, fluorescence titrations and circular dichroism which revealed that these complexes bind to CT DNA by electrostatic interactions via groove binding mode. Zn(II) Pro-Gly complex 1 showed greater binding affinity to CT DNA as compared to the Zn(II) Pro-Leu complex 2 due to steric constraints in the latter. The supercoiled pBR322 DNA cleavage activity of complex 1, ascertained by gel electrophoresis demonstrated efficient DNA cleaving ability via hydrolytic mechanistic pathway. Further, the molecular docking studies confirmed that complex 1 bind to the minor groove of DNA having AT-rich sequences with relative binding energy of -196.72 kJ mol-1.
- Parveen, Shazia,Arjmand, Farukh,Mohapatra
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- Functional identification and structure determination of two novel prolidases from cog1228 in the amidohydrolase superfamily
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Two uncharacterized enzymes from the amidohydrolase superfamily belonging to cog1228 were cloned, expressed, and purified to homogeneity. The two proteins, Sgx9260c (gi|44242006) and Sgx9260b (gi|44479596), were derived from environmental DNA samples originating from the Sargasso Sea. The catalytic function and substrate profiles for Sgx9260c and Sgx9260b were determined using a comprehensive library of dipeptides and N-acyl derivative of l-amino acids. Sgx9260c catalyzes the hydrolysis of Gly-l-Pro, l-Ala-l-Pro, and N-acyl derivatives of l-Pro. The best substrate identified to date is N-acetyl-l-Pro with a value of kcat/Km of 3 × 105 M -1 s-1. Sgx9260b catalyzes the hydrolysis of l-hydrophobic l-Pro dipeptides and N-acyl derivatives of l-Pro. The best substrate identified to date is N-propionyl-l-Pro with a value of kcat/Km of 1 × 105 M-1 s-1. Three-dimensional structures of both proteins were determined by X-ray diffraction methods (PDB codes 3MKV and 3FEQ). These proteins fold as distorted (β/α) 8-barrels with two divalent cations in the active site. The structure of Sgx9260c was also determined as a complex with the N-methylphosphonate derivative of l-Pro (PDB code 3N2C). In this structure the phosphonate moiety bridges the binuclear metal center, and one oxygen atom interacts with His-140. The α-carboxylate of the inhibitor interacts with Tyr-231. The proline side chain occupies a small substrate binding cavity formed by residues contributed from the loop that follows β-strand 7 within the (β/α)8-barrel. A total of 38 other proteins from cog1228 are predicted to have the same substrate profile based on conservation of the substrate binding residues. The structure of an evolutionarily related protein, Cc2672 from Caulobacter crecentus, was determined as a complex with the N-methylphosphonate derivative of l-arginine (PDB code 3MTW).
- Xiang, Dao Feng,Patskovsky, Yury,Xu, Chengfu,Fedorov, Alexander A.,Fedorov, Elena V.,Sisco, Abby A.,Sauder, J. Michael,Burley, Stephen K.,Almo, Steven C.,Raushel, Frank M.
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experimental part
p. 6791 - 6803
(2011/05/05)
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- Identification of novel L-amino acid α-ligases through hidden markov model-based profile analysis
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L-Amino acid α-ligase (Lal), catalyzing the formation of α-dipeptides from unprotected l-amino acids in an ATP-dependent manner, is used in cost-effective fermentative production of dipeptides. We searched for novel Lals by in silico screening using Hidden Markov Model-based profile analysis, and identified five novel Lals that showed low similarity and different substrate specificity from known Lals.
- Senoo, Akihiro,Tabata, Kazuhiko,Yonetani, Yoshiyuki,Yagasaki, Makoto
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body text
p. 415 - 418
(2010/09/30)
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- Peptides based on the sequence of human lactoferrin and their use
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The present invention relates to new peptides designed based on the sequence of human lactoferrin and to use thereof, in particular for treatment and/or prevention of infections, inflammations, tumours, pain, wounds and/or scars.
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- Development of a dual fluorogenic and chromogenic dipeptidyl peptidase IV substrate
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A new far-red dual fluorogenic and chromogenic substrate, 5-glycylprolylglycylprolyl-9-di-3-sulfonyl-propylaminobenza[a]phenoxazonium perchlorate (GPGP-2SBPO), was developed for dipeptidyl peptidase IV (DPP-IV) sensing. The glycylprolylglycylprolyl tetrapeptide was chosen as the recognition sequence due to its stability under physiological conditions. In contrast, the truncated substrate, GP-2SBPO, containing only a glycylprolyl peptide, is unstable. Proteolysis of GPGP-2SBPO was assayed by monitoring the absorbance and fluorescence signals from the released fluorochrome, 2SBPO, at 625 and 670 nm, respectively.
- Ho, Nan-Hui,Weissleder, Ralph,Tung, Ching-Hsuan
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p. 2599 - 2602
(2007/10/03)
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- Dipeptidyl peptidase IV: Regional and subcellular distribution in goat brain
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Dipeptidyl peptidase IV, an important serine proteinase, has been identified in the goat brain tissue. It shows a pH optimum of 7.45 in crude extracts. The regional and subcellular distribution of this proteinase is established in goat brain. The cerebrum portion of the brain has maximum percent activity followed by cerebellum, medulla oblongata, pons-varolli, thalamus, pituitary body and hypothalamus. In terms of activity per gram wet tissue weight, pituitary body is found to have the highest activity. The subcellular localization studies indicate its cytosolic origin in the brain.
- Mittal, Ashwani,Khurana, Shiwani,Sadana, Rachna,Singh, Hari,Kamboj, Ramesh C.
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p. 605 - 608
(2007/10/03)
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- Chromatographic method for the determination of conditional equilibrium constants for the carbamate formation reaction from amino acids and peptides in aqueous solution
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A novel and sensitive method has been developed and evaluated for the study of carbamate formation equilibria of amino acids and peptides in aqueous solution. The method is based on reversed-phase liquid chromatography with cetyltrimethylammonium bromide. The reliability of the method was established by comparing the results determined from the present study with the few data in the literature. The relaxation rate of the carbamate reaction was shown to be faster than the chromatographic distribution relaxation rate (seconds). As a result, the retention time of amine solutes is increased in the presence of CO2. Carbamate formation constants and mole fractions of carbamates at physiological pH of eleven L-α-amino acids and peptides were determined. No correlation between the formation constant and the ammonium pKa was found. There is significant dependence of the amount of a particular amino acid or peptide that exists as carbamate at pH 7.4 on the pKa of the ammonium group, however. This is due to mass action rather than reflecting the influence of pKa on the propensity of the amine to react with CO2. It is suggested that amino acids and peptides with ammonium pKa greater than 9.5 do not form significant amounts of carbamates in aqueous solution near neutral pH.
- Chen, Jian-Ge,Sandberg, Mats,Weber, Stephen G.
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p. 7343 - 7350
(2007/10/02)
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- Catalytic properties of X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis subsp. cremoris nTR.
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An X-prolyl dipeptidyl aminopeptidase (X-PDAP; EC 3.4.14.5) was identified to be loosely bound on the inner cell membrane fraction of Lactococcus lactis subsp. cremoris nTR. The biosynthesis of X-PDAP was continuously increased before the late-log growth phase of the bacteria. Both Gly-Pro-pNA and Ala-Ala-pNA were hydrolyzed by X-PDAP; the kcat/Km value of the former was about 10-fold that of the latter. The Ki of X-Pro and Pro-X were more specific to X-PDAP than those of X-Ala. The enzyme splitting a dipeptide sequentially from beta-casomorphin as a model catalytic pattern was identified and some properties of the enzyme were further characterized.
- Yan,Ho,Hou
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p. 704 - 707
(2007/10/02)
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- SYNTHESIS OF A NEW CYCLIC ANALOGUE OF LULIBERIN
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A new cyclic analogue of luliberin possessing the capacity for stimulating ovulation in sexually mature and infantile rate and also exhibiting a pronounced prolongation of its influence on a number of behavioral reactions of animals has been synthesized.
- Nikolaev, S. V.,Burov, S. V.,Bakharev, V. D.,Makusheva, V. P.,Korkhov, V. V.
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p. 686 - 690
(2007/10/02)
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- PHOSPHONAMIDATE COMPOUNDS
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Phosphonamidates of the formula STR1 wherein X is a substituted or unsubstituted imino or amino acid or ester. These compounds possess angiotensin converting enzyme activity and are thus useful as hypotensive agents.
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- An Excursion into the Synthesis of Potential Angiotensin Converting Enzyme Inhibitors
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An attempt to prepare dehydro analogues of 1 gave the expected tripeptide 2a, but rearrangement of a thiazolone derivative of ΔZ-Phe made only a thiazolinecarboxylic acid (8) available.The latter was also converted into a tripeptide (11) and both compounds, 2a and 11, showed moderate angiotensin converting enzyme inhibition.
- Nitz, Theodore J.,Lindsey, John,Stammer, Charles H.
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p. 4029 - 4032
(2007/10/02)
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