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1.
Phys Chem Chem Phys ; 21(42): 23338-23345, 2019 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-31617504

RESUMO

Nature exploits different strategies for enhancing the catalytic activity of enzymes, often resorting to producing beneficial mutations. The case of post-translational proline hydroxylation mutation in the active site of polysaccharide deacetylase (PDA) Bc1960 from Bacillus cereus is an interesting example of how small chemical modifications can cause significant improvements in enzymatic activity. In the present study the deacetylation mechanism promoted by both OH-proline (2Hyp) and standard proline (Pro) containing PDA is investigated using density functional theory. Although the mechanism presented for the two examined enzymes is in agreement with protease catalysis in metalloenzymes, the analysis along the potential energy surface (PES) reveals that the intermediate and product benefit energetically from the presence of the hydroxyl group on the proline. Our calculations provide evidence that for PDA-2Hyp, the hydrogen bond network established by the -OH group on the Cα of the proline with its closest neighbors stabilizes the transition states and, consequently, the reaction takes advantage of this. These results further contribute towards explaining the different catalytic activity experimentally observed for the polysaccharide deacetylase enzymes.


Assuntos
Amidoidrolases/metabolismo , Hidroxiprolina/metabolismo , Amidoidrolases/química , Bacillus cereus/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Biocatálise , Ligação de Hidrogênio , Hidroxiprolina/química , Simulação de Dinâmica Molecular , Estrutura Terciária de Proteína , Termodinâmica
2.
Food Chem ; 301: 125222, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31382108

RESUMO

Collagen hydrolysates (peptides) derived from food processing byproducts have been used to produce commercially valuable food ingredients due to their potential to trigger certain desirable physiological responses in the body. Low-molecular-weight (LMW) collagen hydrolysates are generally thought to exert better bioactivities than their larger counterparts. However, the preparation of LMW collagen hydrolysates is often impeded by their special structure, cross-linking, and hydroxyproline. This review briefly introduces the motivation of the food industry to prepare LMW collagen hydrolysate from food processing byproducts. We further summarize recent progress on the preparation of LMW collagen hydrolysates and methods to determine the molecular weight. We then discuss the challenges and then provide perspectives on future directions in preparing LMW collagen hydrolysates.


Assuntos
Colágeno/química , Indústria de Processamento de Alimentos/métodos , Hidrolisados de Proteína/química , Animais , Hidroxiprolina/química , Peso Molecular , Peptídeos/química
3.
Amino Acids ; 51(7): 991-998, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31079215

RESUMO

DMDP acetic acid [N-carboxymethyl-2,5-dideoxy-2,5-imino-D-mannitol] 5 from Stevia rebaudiana is the first isolated natural amino acid derived from iminosugars bearing an N-alkyl acid side chain; it is clear from GCMS studies that such derivatives with acetic and propionic acids are common in a broad range of plants including mulberry, Baphia, and English bluebells, but that they are very difficult to purify. Reaction of unprotected pyrrolidine iminosugars with aqueous glyoxal gives the corresponding N-acetic acids in very high yield; Michael addition of both pyrrolidine and piperidine iminosugars and that of polyhydroxylated prolines to tert-butyl acrylate give the corresponding N-propionic acids in which the amino group of ß-alanine is incorporated into the heterocyclic ring. These easy syntheses allow the identification of this new class of amino acid in plant extracts and provide pure samples for biological evaluation. DMDP N-acetic and propionic acids are potent α-galactosidase inhibitors in contrast to potent ß-galactosidase inhibition by DMDP.


Assuntos
Acetatos/síntese química , Aminoácidos/química , Glicosídeo Hidrolases/antagonistas & inibidores , Imino Açúcares/isolamento & purificação , Propionatos/síntese química , Pirrolidinas/síntese química , Stevia/química , Aminoácidos/síntese química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Cromatografia Gasosa-Espectrometria de Massas , Glicina/química , Glicosídeos/metabolismo , Hidroxiprolina/química , Imino Açúcares/química , Piperidinas/síntese química , alfa-Galactosidase/antagonistas & inibidores , beta-Alanina/química , beta-Galactosidase/antagonistas & inibidores
4.
Protein Pept Lett ; 26(9): 684-690, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30961476

RESUMO

BACKGROUND: Plant peptide hormones play a crucial role in plant growth and development. A group of these peptide hormones are signaling peptides with 5 - 23 amino acids. Flagellin peptide (flg22) also elicits an immune response in plants. The functions are expressed through recognition of the peptide hormones and flg22. This recognition relies on membrane localized receptor kinases with extracellular leucine rich repeats (LRR-RKs). The structures of plant peptide hormones - AtPep1, IDA, IDL1, RGFs 1- 3, TDIF/CLE41 - and of flg22 complexed with LRR domains of corresponding LRR-RKs and co-receptors SERKs have been determined. However, their structures are well not analyzed and characterized in detail. The structures of PIP, CEP, CIF, and HypSys are still unknown. OBJECTIVE: Our motivation is to clarify structural features of these plant, small peptides and Flg22 in their bound states. METHODS: In this article, we performed secondary structure assignments and HELFIT analyses (calculating helix axis, pitch, radius, residues per turn, and handedness) based on the atomic coordinates from the crystal structures of AtPep1, IDA, IDL1, RGFs 1- 3, TDIF/CLE41 - and of flg22. We also performed sequence analysis of the families of PIP, CEP, CIF, and HypSys in order to predict their secondary structures. RESULTS: Following AtPep1 with 23 residues adopts two left handed polyproline helices (PPIIs) with six and four residues. IDA, IDL1, RGFs 1 - 2, and TDIF/CLE41 with 12 or 13 residues adopt a four residue PPII; RGF3 adopts two PPIIs with four residues. Flg22 with 22 residues also adopts a six residue PPII. The other peptide hormones - PIP, CEP, CIF, and HypSys - that are rich in proline or hydroxyproline presumably prefer PPII. CONCLUSION: The present analysis indicates that PPII helix in the plant small peptide hormones and in flg22 is crucial for recognition of the LRR domains in receptors.


Assuntos
Flagelina/química , Hormônios Peptídicos/química , Peptídeos/química , Reguladores de Crescimento de Planta/química , Sequência de Aminoácidos , Sítios de Ligação , Hidroxiprolina/química , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína
5.
Chem Commun (Camb) ; 55(32): 4687-4690, 2019 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-30938741

RESUMO

DNP solid state NMR spectroscopy allows non-targeted analysis of wild spider silk in unprecedented detail at natural abundance, revealing hitherto unreported features across several species. A >50-fold signal enhancement for each silk, enables the detection of novel H-bonding networks and arginine conformations, and the post-translational modified amino acid, hydroxyproline.


Assuntos
Fibroínas/química , Aranhas/química , Animais , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Ligação de Hidrogênio , Hidroxiprolina/química , Isótopos de Nitrogênio , Conformação Proteica em Folha beta
6.
Org Biomol Chem ; 17(18): 4460-4464, 2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-30994683

RESUMO

An expeditious method for the synthesis of homo and heterochiral dipeptides containing l-alanine and d/l 2-methyl allo-hydroxyl prolines was developed using direct aminolysis of bicyclic lactones derived from d/l alanine. The impact of C-2 methylation and its spatial orientation on the pyrrolidine ring puckering and prolyl amide bond configuration was ascertained by solution NMR studies. The present studies reveal that C-2 methylation causes the prolyl amide bond to exist exclusively in the trans geometry in both homo- and heterochiral dipeptides. However, the spatial orientation of the C-2 methyl group and its i + 2 position in appropriately capped model dipeptides may nucleate into a turn like structure.


Assuntos
Amidas/química , Dipeptídeos/química , Hidroxiprolina/química , Pirrolidinas/química , Dipeptídeos/síntese química , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular , Conformação Proteica , Estereoisomerismo
7.
Biomater Sci ; 7(5): 2191-2199, 2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-30900708

RESUMO

Collagen plays a critical role in the structural design of the extracellular matrix (ECM) and cell signaling in mammals, which makes it one of the most promising biomaterials with versatile applications. However, there is considerable concern regarding the purity and predictability of the product performance. At present, it is mainly derived as a mixture of collagen (different types) from animal tissues, where the selective enrichment of a particular type of collagen is generally difficult and expensive. Collagen derived from bovine sources poses the risk of transmitting diseases and can cause adverse immunologic and inflammatory responses. Hence, recombinant collagen can be a good alternative. Nevertheless, the necessity of post-translational hydroxyproline (Hyp) modification limits large-scale recombinant collagen production. Here, we recombinantly expressed the collagen-like protein (CLTP) and genetically introduced the Hyp in the CLTP to form a higher order self-assembled fibril structure, similar to human collagen. During the current study, it was observed that the Hyp incorporated CLTP protein (CLTHP) formed a stable triple helical polyproline-II like structure and self-assembled to form fibrils at neutral pH, which had an initial lag phase followed by a growth phase similar to animal collagen. In contrast, the higher order fibrillar assembly was missing in the nonhydroxylated CLTP. This study demonstrated that CLTHP self-association is based on the common underlying lateral interactions between triple helical structured proteins, where the hydroxyproline forms the significantly stable hydration network. Hence, this work will be the first fundamental empirical research for flexible modifications of recombinant collagen for structural analysis and biomedical applications.


Assuntos
Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Colágeno/química , Colágeno/farmacologia , Células 3T3 , Animais , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Hidroxiprolina/química , Camundongos , Simulação de Dinâmica Molecular , Conformação Proteica em alfa-Hélice
8.
Biosci Biotechnol Biochem ; 83(6): 1146-1156, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30739561

RESUMO

Collagen hydrolysate is a well-known nutritional supplement for the improvement of healthy skin. Here, collagen peptide NS (CPNS) from fish scale was prepared, and its physicochemical properties were investigated. Gly-Pro was revealed as a representative low molecular weight peptide of CPNS, by performing prep-HPLC and LC-MS/MS. CPNS treatment attenuated matrix metalloproteinase-1 production and increased the synthesis of type 1 procollagen in HDF cells. After orally administering CPNS to rats, the plasma concentrations of Gly-Pro and Pro-Hyp increased dramatically. To examine the protective effects of CPNS against ultraviolet B (UVB)-induced photoaging in vivo, the dorsal skins of hairless mice were exposed to UVB and supplemented with CPNS for 12 weeks. The CPNS consumption significantly attenuated UVB-induced wrinkle formation, transepidermal water loss, and epidermis thickness, and increased skin hydration. Collectively, these results suggest that bioactive peptides of CPNS, Gly-Pro and Pro-Hyp, exert beneficial effects on skin health.


Assuntos
Colágeno Tipo I/química , Dipeptídeos/farmacologia , Hidroxiprolina/química , Prolina/química , Envelhecimento da Pele/efeitos dos fármacos , Envelhecimento da Pele/efeitos da radiação , Raios Ultravioleta , Administração Oral , Animais , Células Cultivadas , Cromatografia Líquida de Alta Pressão/métodos , Colágeno Tipo I/sangue , Dipeptídeos/administração & dosagem , Dipeptídeos/sangue , Dipeptídeos/química , Feminino , Humanos , Metaloproteinase 1 da Matriz/metabolismo , Camundongos , Camundongos Pelados , Peso Molecular , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas em Tandem/métodos
9.
Biomed Mater ; 14(2): 025012, 2019 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-30630147

RESUMO

Glutaraldehyde (GLUT) crosslinked bioprosthetic heart valves (BHVs) might fail within ten years due to progressive degradation and calcification. GLUT cannot stabilize glycosaminoglycans (GAGs), one important component of BHVs. In this current study we developed a new BHVs preparation strategy named as 'HPA/NT/HRP' treatment that utilized 3,4-hydroxyphenylpropionic acid (HPA)/tyramine (TRA)/neomycin trisulfate (NT) conjugated pericardiums and horseradish peroxidase (HRP)/H2O2 enzyme-oxidative-polymerization method. HPA/TRA-pericardium and HPA-NT conjugation would provide extra phenol groups for enzymatic crosslinking. HPA/TRA conjugated pericardium could be crosslinked by HRP/H2O2 enzyme-oxidative-polymerization. The feeding ratio of HPA-NT was optimized. The GAGs content, collagenase and elastase degradation in vitro, the in vivo GAGs stability and anti-calcification potential of HPA/NT/HRP treated pericardiums were characterized. We demonstrated that HPA/NT/HRP treated pericardiums had sufficiently increased GAGs stabilization and decreased calcification. This new HPA/NT/HRP enzyme-oxidative-polymerization strategy would be a promising method to make BHVs with better GAGs stability and anti-calcification properties.


Assuntos
Bioprótese , Calcinose , Glicosaminoglicanos/química , Próteses Valvulares Cardíacas , Oxigênio/química , Animais , Valva Aórtica , Ácidos Cafeicos/química , Calcificação Fisiológica , Cálcio/química , Varredura Diferencial de Calorimetria , Colagenases/química , Enzimas/química , Hexosaminas/química , Peroxidase do Rábano Silvestre/química , Humanos , Peróxido de Hidrogênio/química , Hidroxiprolina/química , Neomicina/química , Pericárdio/metabolismo , Pericárdio/patologia , Polímeros/química , Ratos , Ratos Sprague-Dawley , Estresse Mecânico , Suínos , Temperatura , Resistência à Tração , Tiramina/química
10.
Int J Mol Sci ; 19(11)2018 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-30400225

RESUMO

Herein, we describe a modified form of the antimicrobial hairpin-like peptide EcAMP1, isolated from barnyard grass (E. crusgalli) seeds, which is structurally characterized by a combination of high-pressure liquid chromatography, mass spectrometry, and automated Edman sequencing. This derivate has a single amino acid substitution (Pro19Hyp) in the second α-helical region of the molecule, which is critical for the formation of the hydrophobic core and the secondary structure elements. Comparing the antifungal activity of these two peptides, we found that the modified EcAMP1-Hyp had a significantly weaker activity towards the most-sensitive plant pathogenic fungus Fusarium solani. Molecular dynamics simulations and in vitro binding to the commercial polysaccharides allowed us to conclude that the Pro-19 residue is important for binding to carbohydrates located in the spore cell wall and it chiefly exhibits a fungistatic action representing the hyphal growth inhibition. These data are novel and significant for understanding a role of α-hairpinins in plant immunity.


Assuntos
Antifúngicos/química , Antifúngicos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Echinochloa/química , Hidroxiprolina/química , Sementes/química , Fusarium/efeitos dos fármacos , Testes de Sensibilidade Microbiana
11.
Biomater Sci ; 6(11): 3042-3052, 2018 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-30283925

RESUMO

Collagen has been widely documented as one of the most promising and competitive biomaterials for tissue engineering and medical applications. However, the properties of collagen differ from one source to another. In the present study, type I collagen (COL-I) was extracted and purified from the skins of Japanese sea bass (Lateolabrax japonicus) and Nile tilapia (Oreochromis niloticus). Ultraviolet (UV) spectroscopy, Fourier transform infrared spectroscopy (FTIR) and SDS-PAGE were performed to characterize both COL-Is. The denaturing temperature of bass collagen (BC) was observed to be 27.2 °C, and 35.3 °C for tilapia collagen (TC). The content of hydroxyproline was 13.4% in TC, which was similar to that in porcine collagen (PC, 13.6%) and higher than that in BC (10.3%), while the content of cysteine in TC (0.87%) was significantly higher than that in PC (0.04%) and BC (0.35%). After incubation at different temperatures for 9 h, more degraded collagen bands appeared in the BC hydrogel (BCH) group than in the TC hydrogel (TCH) group, indicating that TCH exhibited better thermal stability than BCH. The thermal stabilities of TCH and PC hydrogel (PCH) were similar. The compressive stress of TCH was up to 0.099 MPa, while it was 0.047 MPa for BCH and 0.003 MPa for PCH. These results demonstrated that the content of amino acids (especially hydroxyproline and cysteine) has a synergistic effect on the thermal and mechanical properties of BCH, TCH and PCH, which would be an indicator of the thermal and mechanical properties of collagen hydrogels in future studies.


Assuntos
Colágeno Tipo I/química , Cisteína/química , Hidroxiprolina/química , Animais , Bass , Materiais Biocompatíveis/química , Colágeno Tipo I/isolamento & purificação , Humanos , Hidrogéis/química , Indicadores e Reagentes/química , Teste de Materiais , Conformação Proteica , Pele/química , Solubilidade , Temperatura , Tilápia , Viscosidade
12.
Acta Biomater ; 80: 169-175, 2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30218779

RESUMO

Bacterial collagen-like proteins differ from vertebrate collagens in that they do not contain hydroxyproline, which is seen as a characteristic of the vertebrate collagens, and which provides a significant contribution to the stability of the collagen triple-helix at body temperature. Despite this difference, the bacterial collagens are stable at around body temperature through inclusion of other stabilising sequence elements. Another difference is the lack of aggregation, and certain vertebrate collagen binding domains that can be introduced into the bacterial sequence lack full function when hydroxyproline is absent. In the present study we have demonstrated that a simple method utilising co-translational incorporation during fermentation can be used to incorporate hydroxyproline into the recombinant bacterial collagen. The presence and amount of hydroxyproline incorporation was shown by amino acid analysis and by mass spectrometry. A small increase in thermal stability was observed using circular dichroism spectroscopy. STATEMENT OF SIGNIFICANCE: Recombinant bacterial collagens provide a new opportunity for biomedical materials as they are readily produced in large quantity in E. coli. Unlike animal collagens, they are stable without the need for inclusion of a secondary modification system for hydroxyproline incorporation. In animal collagens, however, introduction of hydroxyproline is essential for stability and is also important for functional molecular interactions within the mammalian extracellular matrix. The present study has shown that hydroxyproline can be readily introduced into recombinant S. pyogenes bacterial collagen through direct co-translational incorporation of this modified imino acid during expression using the codons for proline in the introduced gene construct. This hydroxylation further improves the stability of the collagen and is available to enhance any introduced molecular functions.


Assuntos
Colágeno/química , Hidroxiprolina/química , Streptococcus pyogenes/metabolismo , Sequência de Aminoácidos , Aminoácidos/análise , Proteínas de Bactérias/química , Espectrometria de Massas , Temperatura
13.
Org Biomol Chem ; 16(36): 6708-6717, 2018 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-30182115

RESUMO

Five new cyclic peptoids containing (2S,4R)-4-hydroxyproline (Hyp) residues have been designed and synthesized using a mixed "submonomer/monomer" approach. Alkali metal cation affinities and ion transport activities were assessed by experimental (NMR and HPTS assay in liposomes) and computational methods. Easy functionalization of hydroxyproline residues afforded a bouquet of cyclic oligomers showing correlation between ion transport abilities and cytotoxic activities on selected human cancer cell lines.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Hidroxiprolina/química , Peptoides/química , Peptoides/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Sódio/química
14.
Sci Rep ; 8(1): 13809, 2018 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-30218106

RESUMO

Fibrillar collagens have mechanical and biological roles, providing tissues with both tensile strength and cell binding sites which allow molecular interactions with cell-surface receptors such as integrins. A key question is: how do collagens allow tissue flexibility whilst maintaining well-defined ligand binding sites? Here we show that proline residues in collagen glycine-proline-hydroxyproline (Gly-Pro-Hyp) triplets provide local conformational flexibility, which in turn confers well-defined, low energy molecular compression-extension and bending, by employing two-dimensional 13C-13C correlation NMR spectroscopy on 13C-labelled intact ex vivo bone and in vitro osteoblast extracellular matrix. We also find that the positions of Gly-Pro-Hyp triplets are highly conserved between animal species, and are spatially clustered in the currently-accepted model of molecular ordering in collagen type I fibrils. We propose that the Gly-Pro-Hyp triplets in fibrillar collagens provide fibril "expansion joints" to maintain molecular ordering within the fibril, thereby preserving the structural integrity of ligand binding sites.


Assuntos
Colágeno/química , Colágeno/metabolismo , Prolina/metabolismo , Sequência de Aminoácidos , Aminoácidos/metabolismo , Animais , Feminino , Colágenos Fibrilares/metabolismo , Colágenos Fibrilares/fisiologia , Glicina/química , Hidroxiprolina/química , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Peptídeos/química , Prolina/fisiologia , Conformação Proteica , Ovinos
15.
Int J Mol Sci ; 19(9)2018 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-30231550

RESUMO

Protein hydroxylation is one type of post-translational modifications (PTMs) playing critical roles in human diseases. It is known that protein sequence contains many uncharacterized residues of proline and lysine. The question that needs to be answered is: which residue can be hydroxylated, and which one cannot. The answer will not only help understand the mechanism of hydroxylation but can also benefit the development of new drugs. In this paper, we proposed a novel approach for predicting hydroxylation using a hybrid deep learning model integrating the convolutional neural network (CNN) and long short-term memory network (LSTM). We employed a pseudo amino acid composition (PseAAC) method to construct valid benchmark datasets based on a sliding window strategy and used the position-specific scoring matrix (PSSM) to represent samples as inputs to the deep learning model. In addition, we compared our method with popular predictors including CNN, iHyd-PseAAC, and iHyd-PseCp. The results for 5-fold cross-validations all demonstrated that our method significantly outperforms the other methods in prediction accuracy.


Assuntos
Aprendizado Profundo , Hidroxilisina/química , Hidroxiprolina/química , Proteínas/química , Humanos , Hidroxilação , Hidroxilisina/metabolismo , Hidroxiprolina/metabolismo , Modelos Biológicos , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo
16.
PLoS One ; 13(8): e0201982, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30092047

RESUMO

In this study, we clarified the functions of three uncharacterized enzymes, XCV2724, XCV2728, and XCV2729, in Xanthomonas euvesicatoria, the causal agent of bacterial spot of tomato and pepper. The genes corresponding to the three enzymes are homologs of hypBA1, hypBA2, and hypAA from Bifidobacterium longum and are unique to Xanthomonas spp. among plant pathogenic bacteria. Functional characterization of the recombinant enzymes expressed using microbial systems revealed that they degrade the arabinofurano-oligosaccharides present on hydroxyproline (Hyp)-rich glycoproteins (HRGPs) such as extensin and solanaceous lectins in plant cell walls. These enzymes work coordinately to degrade the oligosaccharides. First, XeHypAA (XCV2728), belonging to the glycoside hydrolase (GH) 43 family, releases L-arabinose from L-arabinofuranose (Araf)-α1,3-Araf-ß1,2-Araf-ß1,2-Araf-ß-Hyp (Ara4-Hyp), cleaving its α1,3 bond; second, XeHypBA2 (XCV2729), belonging to the GH121 family, releases the disaccharide Araf-ß1,2-Araf from Araf-ß1,2-Araf-ß1,2-Araf-ß-Hyp (Ara3-Hyp); finally, XeHypBA1 (XCV2724), belonging to GH family 127, releases L-arabinose from Araf-ß-Hyp (Ara-Hyp). In summary, the main oligosaccharide structure of Ara4-Hyp on the HRGPs is degraded to Ara3-Hyp, then to Ara-Hyp, and finally to Ara monosaccharides by the action of these three enzymes. HRGPs containing oligosaccharide substrates have been reported to contribute to plant defense, and interestingly, the promoter region of the operon (xehypBA2 and xehypAA) contains the plant-inducible promoter box for binding the regulator protein HrpX involved in pathogenicity. We then analyzed the expression level of the operon gene in hrp-inducing medium and in plants and constructed gene-deletion mutants. However, although the operon was evidently upregulated by HrpX, three single-gene deletion mutants (ΔxehypBA1, ΔxehypBA2, ΔxehypAA) and even a triple-gene deletion mutant (ΔxehypBA1-BA2-AA) remained pathogenic, and had no effect on nonhost resistance, either, indicating that these three enzymes are not involved in either pathogenicity or nonhost resistance reactions. This is the first report of enzymes in plant pathogenic bacteria that catalyze the degradation of Hyp-linked-L-arabinofuranosides in plant cell walls.


Assuntos
Glicoproteínas/química , Glicosídeo Hidrolases/metabolismo , Hidroxiprolina/química , Oligossacarídeos/metabolismo , Xanthomonas/genética , Arabinose/análogos & derivados , Arabinose/metabolismo , Proteínas de Bactérias/metabolismo , Bifidobacterium/enzimologia , Catálise , Parede Celular/metabolismo , Deleção de Genes , Perfilação da Expressão Gênica , Mutação , Plantas/microbiologia , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Fatores de Transcrição/genética , Xanthomonas/enzimologia
17.
J Agric Food Chem ; 66(33): 8737-8743, 2018 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-30060651

RESUMO

Hydroxyproline (Hyp) is a collagen-specific amino acid formed by post-translational hydroxylation of Pro residues. Various Hyp-containing oligopeptides are transported into the blood at high concentrations after oral ingestion of collagen hydrolysate. Here we investigated the angiotensin-converting enzyme (ACE) inhibitory activity of X-Hyp-Gly-type tripeptides. In an in vitro assay, ginger-degraded collagen hydrolysate enriched with X-Hyp-Gly-type tripeptides dose-dependently inhibited ACE and various synthetic X-Hyp-Gly-type tripeptides showed ACE-inhibitory activity. In particular, strong inhibition was observed for Leu-Hyp-Gly, Ile-Hyp-Gly, and Val-Hyp-Gly with IC50 values of 5.5, 9.4, and 12.8 µM, respectively. Surprisingly, substitution of Hyp with Pro dramatically decreased inhibitory activity of X-Hyp-Gly, indicating that Hyp is important for ACE inhibition. This finding was supported by molecular docking experiments using Leu-Hyp-Gly/Leu-Pro-Gly. We further demonstrated that prolyl hydroxylation significantly enhanced resistance to enzymatic degradation by incubation with mouse plasma. The strong ACE-inhibitory activity and high stability of X-Hyp-Gly-type tripeptides highlight their potential for hypertension control.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/química , Colágeno/química , Peptídeos/química , Animais , Hidroxilação , Hidroxiprolina/química , Cinética , Camundongos , Simulação de Acoplamento Molecular , Peptídeos/sangue , Renina/química , Renina/metabolismo
18.
J Mech Behav Biomed Mater ; 86: 105-112, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29986285

RESUMO

Identity of the amino acids in Gly-X-Y repetitive motives governs biomechanical features of collagen such as elasticity in the extracellular matrix. Proline and hydroxyproline are the most abundant residues at the X and Y sites of collagen repetitive motives, respectively. However, their effects on the elasticity of collagen have not been identified. Here, we investigated the molecular mechanics of five different proline-rich collagens with Gly-Pro-Y repetitive motives, four hydroxyproline-rich collagens with Gly-X-Hyp repetitive motives as well as a collagen with Gly-Pro-Hyp repetitive motif, focusing on molecular stiffness and elasticity. The proteins were virtually built and stiffness, Young's modulus, cross-sectional radius, intermolecular and protein-solvent hydrogen bonds of the collagens were investigated using steered molecular dynamic simulation. Results showed a higher stiffness and Young's moduli of the proline-rich collagens compared to the hydroxyproline-rich collagens. Young's modulus of the proline-rich collagens was negatively correlated with the cross-sectional radius. There was no significant difference between the proline-rich and the hydroxyproline-rich collagen from the point of view of intermolecular and protein-water hydrogen bonds. However, a decreased and an increased number of protein-water hydrogen bonds in response to stretching was found for the proline-rich and the hydroxyproline-rich collagens respectively. Interestingly, the collagen with Gly-Pro-Hyp repetitive motif showed an intermediate stiffness, Young's moduli and cross-sectional radius. The collagen also had a lower number of intermolecular and protein -water hydrogen bonds when compared to the proline-rich and hydroxproline-rich collagens. These results suggest that the presence of proline in the structure of collagen reduces elasticity and increases stiffness, whereas presence of hydroxyproline increases elasticity of the collagen. We conclude that any codon substitution in the collagen genes causing alteration of proline and/or hydroxyproline residues may result in drastic collagen deficiency.


Assuntos
Colágeno/química , Módulo de Elasticidade , Hidroxiprolina/química , Simulação de Dinâmica Molecular , Ligação de Hidrogênio , Conformação Proteica em alfa-Hélice , Água/química
19.
Sheng Wu Gong Cheng Xue Bao ; 34(7): 1046-1056, 2018 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-30058304

RESUMO

Hydroxy amino acids, constituents of chiral pharmaceutical intermediates or precursors, have a variety of unique functions in the research fields of biotechnology and molecular biology, i.e. antifungal, antibacterial, antiviral and anticancer properties. Biosynthesis of hydroxy amino acids is preferred because of its high specificity and selectivity. The hydroxylation of hydrophobic amino acids is catalyzed by hydroxylase, which belongs to the mononuclear non-heme Fe(Ⅱ)/α-ketoglutarate-dependent dioxygenases (Fe/αKGDs). Fe/αKGDs utilize an (Fe(Ⅳ)=O) intermediate to activate diverse oxidative transformations with key biological roles in the process of catalytic reaction. Here, we review the physiological properties and synthesis of hydroxy amino acids, especially for the 4-HIL and hydroxyproline. The catalytic mechanism of Fe/αKGDs is elucidated, and the applications of hydroxy amino acids in industrial engineering are also discussed.


Assuntos
Aminoácidos/química , Hidroxilação , Ferro/química , Oxigenases de Função Mista/química , Hidroxiprolina/química , Oxirredução
20.
BMC Nephrol ; 19(1): 167, 2018 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-29980178

RESUMO

BACKGROUND: Substrate reduction therapy with analogs reduces the accumulation of substrates by inhibiting the metabolic pathways involved in their biosynthesis, providing new treatment options for patients with primary hyperoxalurias (PHs) that often progress to end-stage renal disease (ESRD). This research aims to evaluate the inhibition efficacy of Hydroxy-L-proline (HYP) analogs against calcium oxalate (CaOx) crystal formation in the Drosophila Melanogaster (D. Melanogaster) by comparing them with Pyridoxine (Vitamin B6). METHODS: Three stocks of Drosophila Melanogaster (W118, CG3926 RNAi, and Act5C-GAL4/CyO) were utilized. Two stocks (CG3926 RNAi and Act5C-GAL4 /CyO) were crossed to generate the Act5C > dAGXT RNAi recombinant line (F1 generation) of D. Melanogaster which was used to compare the efficacy of Hydroxy-L-proline (HYP) analogs inhibiting CaOx crystal formation with Vitamin B6 as the traditional therapy for primary hyperoxaluria. RESULTS: Nephrolithiasis model was successfully constructed by downregulating the function of the dAGXT gene in D. Melanogaster (P-Value = 0.0045). Furthermore, the efficacy of Hydroxy-L-proline (HYP) analogs against CaOx crystal formation was demonstrated in vivo using D. Melanogaster model; the results showed that these L-Proline analogs were better in inhibiting stone formation at very low concentrations than Vitamin B6 (IC50 = 0.6 and 1.8% for standard and dietary salt growth medium respectively) compared to N-acetyl-L-Hydroxyproline (IC50 = 0.1% for both standard and dietary salt growth medium) and Baclofen (IC50 = 0.06 and 0.1% for standard and dietary salt growth medium respectively). Analysis of variance (ANOVA) also showed that Hydroxy-L-proline (HYP) analogs were better alternatives for CaOx inhibition at very low concentration especially when both genetics and environmental factors are intertwined (p < 0.0008) for the dietary salt growth medium and (P < 0.063) for standard growth medium. CONCLUSION: Addition of Hydroxy-L-Proline analogs to growth medium resulted in the reduction of CaOx crystals formation. These analogs show promise as potential inhibitors for oxalate reduction in Primary Hyperoxaluria.


Assuntos
Oxalato de Cálcio/antagonistas & inibidores , Hidroxiprolina/química , Hidroxiprolina/uso terapêutico , Hiperoxalúria Primária/tratamento farmacológico , Cálculos Renais/tratamento farmacológico , Animais , Animais Geneticamente Modificados , Oxalato de Cálcio/toxicidade , Relação Dose-Resposta a Droga , Drosophila melanogaster , Hidroxiprolina/farmacologia , Hiperoxalúria Primária/induzido quimicamente , Hiperoxalúria Primária/genética , Hiperoxalúria Primária/patologia , Cálculos Renais/induzido quimicamente , Cálculos Renais/genética , Cálculos Renais/patologia , Transaminases/genética , Resultado do Tratamento
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