Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 14 de 14
Filter
Add more filters










Publication year range
1.
Biotechnol Appl Biochem ; 69(6): 2580-2591, 2022 Dec.
Article in English | MEDLINE | ID: mdl-34967474

ABSTRACT

The investigation of a plant glycosylated serine protease (EuRP-61) isolated from Euphorbia resinifera latex for potential antiplatelet and anticoagulation activities has been previously reported. In the present study, the protein sequence and native crystal structure of EuRP-61 were characterized. The structure was identified using single-wavelength anomalous diffraction with a refinement resolution of 1.7 Å (PDB ID: 7EOX). The main structural components of EuRP-61 were composed of three domains: catalytic, protease-associated (PA), and fibronectin type III (Fn3)-like domains. The crystal structure revealed that some loops in the PA and catalytic domains of EuRP-61 were different from the other subtilisin-like proteases (cucumisin and SBT3). These different loops might be involved in the general monomer formation of EuRP-61, substrate specificity, and maintenance of the catalytic domain. The Fn3-like domain may provide flexibility to the enzyme to bind with various substrates and cell receptors. Additionally, the active site of EuRP-61 consisted of the catalytic triad of Ser434, His106, and Asp32, similar to other serine proteases. The present study provides additional information and insight into the protease and antithrombotic activities of EuRP-61, which could contribute to further development of this enzyme for biomedical treatment.


Subject(s)
Euphorbia , Latex/chemistry , Serine Proteases/chemistry , Amino Acid Sequence , Sequence Analysis
2.
FEBS J ; 289(11): 3217-3240, 2022 06.
Article in English | MEDLINE | ID: mdl-34967505

ABSTRACT

HpaR is a transcription regulator in the MarR family that controls the expression of the gene cluster responsible for conversion of p-hydroxyphenylacetate to pyruvate and succinate for cellular metabolism. Here, we report the biochemical and structural characterization of Acinetobacter baumannii HpaR (AbHpaR) and its complex with cognate DNA. Our study revealed that AbHpaR binds upstream of the divergently transcribed hpaA gene and the meta-cleavage operon, as well as the hpaR gene, thereby repressing their transcription by blocking access of RNA polymerase. Structural analysis of AbHpaR-DNA complex revealed that the DNA binding specificity can be achieved via a combination of both direct and indirect DNA sequence readouts. DNA binding of AbHpaR is weakened by 3,4-dihydroxyphenylacetate (DHPA), which is the substrate of the meta-cleavage reactions; this likely leads to expression of the target genes. Based on our findings, we propose a model for how A. baumannii controls transcription of HPA-metabolizing genes, which highlights the independence of global catabolite repression and could be beneficial for metabolic engineering toward bioremediation applications. DATABASES: The structural data that support these findings are openly available in the wwPDB at https://doi.org/10.2210/pdb7EL2/pdb and https://doi.org/10.2210/pdb7EL3/pdb for AbHpaR and AbHpaR-DNA complex, respectively.


Subject(s)
Acinetobacter baumannii , Acinetobacter baumannii/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Base Sequence , Gene Expression Regulation, Bacterial , Operon , Protein Binding
3.
Acta Crystallogr D Struct Biol ; 77(Pt 11): 1425-1436, 2021 Nov 01.
Article in English | MEDLINE | ID: mdl-34726170

ABSTRACT

ß-Mannanase (EC 3.2.1.78) is an enzyme that cleaves within the backbone of mannan-based polysaccharides at ß-1,4-linked D-mannose residues, resulting in the formation of mannooligosaccharides (MOS), which are potential prebiotics. The GH26 ß-mannanase KMAN from Klebsiella oxytoca KUB-CW2-3 shares 49-72% amino-acid sequence similarity with ß-mannanases from other sources. The crystal structure of KMAN at a resolution of 2.57 Šrevealed an open cleft-shaped active site. The enzyme structure is based on a (ß/α)8-barrel architecture, which is a typical characteristic of clan A glycoside hydrolase enzymes. The putative catalytic residues Glu183 and Glu282 are located on the loop connected to ß-strand 4 and at the end of ß-strand 7, respectively. KMAN digests linear MOS with a degree of polymerization (DP) of between 4 and 6, with high catalytic efficiency (kcat/Km) towards DP6 (2571.26 min-1 mM-1). The predominant end products from the hydrolysis of locust bean gum, konjac glucomannan and linear MOS are mannobiose and mannotriose. It was observed that KMAN requires at least four binding sites for the binding of substrate molecules and hydrolysis. Molecular docking of mannotriose and galactosyl-mannotetraose to KMAN confirmed its mode of action, which prefers linear substrates to branched substrates.


Subject(s)
Bacterial Proteins/chemistry , Klebsiella oxytoca/chemistry , beta-Mannosidase/chemistry , Bacterial Proteins/metabolism , Crystallography, X-Ray , Humans , Kinetics , Klebsiella Infections/microbiology , Klebsiella oxytoca/metabolism , Models, Molecular , Protein Conformation , Substrate Specificity , beta-Mannosidase/metabolism
4.
Chem Commun (Camb) ; 57(98): 13373-13376, 2021 Dec 09.
Article in English | MEDLINE | ID: mdl-34821892

ABSTRACT

We designed and synthesized a fatty aldehyde surrogate containing a formyl thioester group, which can be reduced by fatty aldehyde reductase (FALR) with stoichiometric formaldehyde generation. It can be rapidly visualized and quantified using the Purpald assay. We demonstrated its successful application in the high throughput screening of FALR engineering.


Subject(s)
Aldehyde Oxidoreductases/chemistry , Aldehyde Oxidoreductases/metabolism , Aldehydes/chemistry , Chemical Engineering/methods , Fatty Acids/chemistry , High-Throughput Screening Assays/methods
5.
J Biol Chem ; 295(1): 170-180, 2020 01 03.
Article in English | MEDLINE | ID: mdl-31776187

ABSTRACT

Calcium (Ca2+) and redox signaling enable cells to quickly adapt to changing environments. The signaling protein calredoxin (CRX) from the green alga Chlamydomonas reinhardtii is a chloroplast-resident thioredoxin having Ca2+-dependent activity and harboring a unique combination of an EF-hand domain connected to a typical thioredoxin-fold. Using small-angle X-ray scattering (SAXS), FRET, and NMR techniques, we found that Ca2+-binding not only induces a conformational change in the EF-hand domain, but also in the thioredoxin domain, translating into the onset of thioredoxin redox activity. Functional analyses of CRX with genetically altered EF-hands revealed that EF-hand 4 is important for mediating the communication between the two domains. Moreover, we crystallized a variant (C174S) of the CRX target protein peroxiredoxin 1 (PRX1) at 2.4 Å resolution, modeled the interaction complex of the two proteins, and analyzed it by cross-linking and MS analyses, revealing that the interaction interface is located close to the active sites of both proteins. Our findings shed light on the Ca2+ binding-induced changes in CRX structure in solution at the level of the overall protein and individual domains and residues.


Subject(s)
Calcium Signaling , Calcium-Binding Proteins/metabolism , Chloroplast Thioredoxins/metabolism , EF Hand Motifs , Calcium-Binding Proteins/chemistry , Chlamydomonas reinhardtii , Chloroplast Thioredoxins/chemistry , Molecular Dynamics Simulation , Protein Binding
6.
Int J Biol Macromol ; 145: 998-1007, 2020 Feb 15.
Article in English | MEDLINE | ID: mdl-31678105

ABSTRACT

A serine protease designated as EuRP-61 was purified from Euphorbia resinifera latex. The N-terminal sequence of 15 amino acids of EuRP-61 supported the conclusion that the enzyme was a serine protease because its amino acid sequence had homology (between 50 and 70% identities) with the subtilisin-like proteases of other plants. EuRP-61 had a molecular weight estimated at 61 kDa analyzed by MALDI-TOF MS. The enzyme could cleave human fibrinogen with optimal conditions at pH 5.0 and 45 °C. The enzyme had a broad range of pH stability from 1 to 14 and tolerance to denaturation up to a temperature of approximately 65-66 °C. EuRP-61 hydrolyzed fibrinogen with a Michaelis constant (Km) of 4.95 ±â€¯0.1 µM; a maximal velocity (Vmax) of 578.1 ±â€¯11.81 ng min-1; and a catalytic efficiency (Vmax/Km) of 116.8 ±â€¯1 ng µM-1 min-1. EuRP-61was crystallized under the condition of sodium iodide (0.2 M), Bis-Tris propane (0.1 M, pH 8.5) and PEG3350 (20%) by the sitting-drop method. The crystal belonged to space group P212121, with unit cell dimension a = 109.91, b = 67.38 and c = 199.45 Šand diffracted X-ray to 2.53 Šresolution. The crystal structure of EuRP-61 will be explored further by special phase solving techniques.


Subject(s)
Euphorbia/chemistry , Euphorbia/enzymology , Latex/chemistry , Peptide Hydrolases/chemistry , Peptide Hydrolases/isolation & purification , Amino Acid Sequence , Catalysis , Crystallography, X-Ray , Enzyme Stability , Fibrinogen/metabolism , Fibrinolytic Agents/chemistry , Glycoproteins/isolation & purification , Humans , Hydrogen-Ion Concentration , Hydrolysis , Kinetics , Molecular Weight , Sequence Analysis, Protein , Sequence Homology , Serine Endopeptidases/chemistry , Serine Proteases/chemistry , Substrate Specificity , Temperature , Trace Elements/analysis
7.
BMC Med Genet ; 20(1): 156, 2019 09 11.
Article in English | MEDLINE | ID: mdl-31510962

ABSTRACT

BACKGROUND: Pompe disease is a lysosomal storage disorder caused by the deficiency of acid alpha-glucosidase (EC. 3.2.1.20) due to mutations in human GAA gene. The objective of the present study was to examine clinical and molecular characteristics of infantile-onset Pompe disease (IOPD) in Thailand. METHODS: Twelve patients with infantile-onset Pompe disease (IOPD) including 10 Thai and two other Asian ethnicities were enrolled. To examine the molecular characteristics of Pompe patients, GAA gene was analyzed by PCR amplification and direct Sanger-sequencing of 20 exons coding region. The novel mutations were transiently transfected in COS-7 cells for functional verification. The severity of the mutation was rated by study of the GAA enzyme activity detected in transfected cells and culture media, as well as the quantity and quality of the proper sized GAA protein demonstrated by western blot analysis. The GAA three dimensional structures were visualized by PyMol software tool. RESULTS: All patients had hypertrophic cardiomyopathy, generalized muscle weakness, and undetectable or < 1% of GAA normal activity. Three patients received enzyme replacement therapy with variable outcome depending on the age of the start of enzyme replacement therapy (ERT). Seventeen pathogenic mutations including four novel variants: c.876C > G (p.Tyr292X), c.1226insG (p.Asp409GlyfsX95), c.1538G > A (p.Asp513Gly), c.1895 T > G (p.Leu632Arg), and a previously reported rare allele of unknown significance: c.781G > A (p.Ala261Thr) were identified. The rating system ranked p.Tyr292X, p. Asp513Gly and p. Leu632Arg as class "B" and p. Ala261Thr as class "D" or "E". These novel mutations were located in the N-terminal beta-sheet domain and the catalytic domain. CONCLUSIONS: The present study provides useful information on the mutations of GAA gene in the underrepresented population of Asia which are more diverse than previously described and showing the hotspots in exons 14 and 5, accounting for 62% of mutant alleles. Almost all mutations identified are in class A/B. These data can benefit rapid molecular diagnosis of IOPD and severity rating of the mutations can serve as a partial substitute for cross reactive immunological material (CRIM) study.


Subject(s)
Genetic Predisposition to Disease/genetics , Glycogen Storage Disease Type II/genetics , Mutation , alpha-Glucosidases/genetics , Alleles , Animals , Asian People/genetics , Base Sequence , COS Cells , Cardiomyopathy, Hypertrophic/genetics , Chlorocebus aethiops , Enzyme Replacement Therapy , Female , Glycogen Storage Disease Type II/diagnosis , Humans , Infant , Male , Models, Molecular , Pathology, Molecular , Sequence Analysis, Protein , Thailand , alpha-Glucosidases/chemistry
8.
Acta Crystallogr F Struct Biol Commun ; 74(Pt 2): 86-91, 2018 Feb 01.
Article in English | MEDLINE | ID: mdl-29400317

ABSTRACT

Peroxiredoxins (PRXs) are a group of antioxidant enzymes that are found in all organisms, including plants and green algae. The 2-Cys PRX from Chlamydomonas reinhardtii (CrPRX1) is a chloroplast-localized protein that is critical for clearing reactive oxygen species in chloroplasts. CrPRX1 is reduced by thioredoxins or calredoxin (CrCRX), a recently identified calcium-dependent redox protein. The molecular interaction between PRXs and thioredoxin/CrCRX is functionally important, but discussion has been limited owing to a lack of structural information on CrPRX1, especially regarding its oligomeric state. In this study, high-speed atomic force microscopy (HS-AFM) images of CrPRX1 and an X-ray crystallographic analysis have enabled examination of the oligomeric state of CrPRX1. Diffraction data from a crystal of the Cys174Ser mutant of CrPRX1 indicate the existence of noncrystallographic fivefold symmetry. HS-AFM images of CrPRX1 further show that CrPRX1 particles form rings with pentagonal rotational symmetry. On the basis of these findings, the oligomeric state of CrPRX1 is discussed and it is concluded that this PRX exists in a ring-shaped decameric form comprising a pentamer of dimers.


Subject(s)
Chlamydomonas reinhardtii/genetics , Microscopy, Atomic Force/methods , Peroxiredoxins/chemistry , Peroxiredoxins/genetics , Amino Acid Sequence , Crystallography, X-Ray/methods , Peroxiredoxins/isolation & purification
9.
Ann Hum Genet ; 82(3): 150-157, 2018 05.
Article in English | MEDLINE | ID: mdl-29282708

ABSTRACT

BACKGROUND: Mucopolysaccharidosis type I (MPS I) is a rare autosomal-recessive disorder caused by defects in alpha-L-iduronidase (IDUA), a lysosomal enzyme encoded by the IDUA gene. Herein, we characterized IDUA mutations underlying mucopolysaccharidosis type I intermediate form (Hurler-Scheie syndrome) and its molecular pathogenic mechanisms. METHODS: Clinical data, activity of the IDUA enzyme in leukocytes, and a mutation of the IDUA gene were analyzed. Pathogenesis associated with an IDUA mutation was further investigated by evaluating the mutant cDNA sequence, protein expression and activity in COS-7 cells. RESULTS: Five unrelated patients were identified to have clinical diagnosis of intermediate form of MPS I (Hurler-Scheie) and exhibited low-to-absent levels of leukocyte IDUA activity. Genetic analysis revealed homozygous c.*1T>C (p.X654R) mutation in two patients and compound heterozygosity between the c.*1T>C and another allele including c.265G>A (p.R89Q), c.935G>A (p.W312X), or c.1138 C>T (p.Q380X), each in a single patient. Sequencing the 3'RACE product of the c.*1T>C (p.X654R) allele indicated a 38-amino acids elongation of the mutant protein. COS-7 cells expressing IDUA with the mutations exhibited extremely low levels or complete absence of enzyme activity compared to wild-type IDUA. Western blot analysis detected no protein in p.W312X and p.Q380X samples, while an elevated molecular mass and a different pattern of protein bands were found in p.X654R specimen compared with the wild type IDUA. CONCLUSIONS: Mutational spectrum underlying intermediate MPS I is expanded. Our data suggest that the p.X654R is an intermediate IDUA mutant allele with residual enzyme activity. It can lead to intermediate or milder form of MPS I depending on another associated allele.


Subject(s)
Iduronidase/genetics , Mucopolysaccharidosis I/genetics , Animals , COS Cells , Child , Child, Preschool , Chlorocebus aethiops , DNA Mutational Analysis , Female , Humans , Male , Mutation , Thailand
10.
Nat Commun ; 7: 11847, 2016 06 14.
Article in English | MEDLINE | ID: mdl-27297041

ABSTRACT

Calcium (Ca(2+)) and redox signalling play important roles in acclimation processes from archaea to eukaryotic organisms. Herein we characterized a unique protein from Chlamydomonas reinhardtii that has the competence to integrate Ca(2+)- and redox-related signalling. This protein, designated as calredoxin (CRX), combines four Ca(2+)-binding EF-hands and a thioredoxin (TRX) domain. A crystal structure of CRX, at 1.6 Å resolution, revealed an unusual calmodulin-fold of the Ca(2+)-binding EF-hands, which is functionally linked via an inter-domain communication path with the enzymatically active TRX domain. CRX is chloroplast-localized and interacted with a chloroplast 2-Cys peroxiredoxin (PRX1). Ca(2+)-binding to CRX is critical for its TRX activity and for efficient binding and reduction of PRX1. Thereby, CRX represents a new class of Ca(2+)-dependent 'sensor-responder' proteins. Genetically engineered Chlamydomonas strains with strongly diminished amounts of CRX revealed altered photosynthetic electron transfer and were affected in oxidative stress response underpinning a function of CRX in stress acclimation.


Subject(s)
Calcium-Binding Proteins/chemistry , Calcium/chemistry , Chlamydomonas reinhardtii/genetics , Chloroplasts/genetics , Gene Expression Regulation, Plant , Plant Proteins/chemistry , Thioredoxins/chemistry , Binding Sites , Calcium/metabolism , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Calmodulin/chemistry , Calmodulin/genetics , Calmodulin/metabolism , Chlamydomonas reinhardtii/metabolism , Chloroplasts/metabolism , Cloning, Molecular , Crystallography, X-Ray , Electron Transport , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Models, Molecular , Oxidation-Reduction , Peroxiredoxins/chemistry , Peroxiredoxins/genetics , Peroxiredoxins/metabolism , Photosynthesis/genetics , Plant Proteins/genetics , Plant Proteins/metabolism , Plants, Genetically Modified , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Folding , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Signal Transduction , Thioredoxins/genetics , Thioredoxins/metabolism
11.
ACS Chem Biol ; 11(7): 1891-900, 2016 07 15.
Article in English | MEDLINE | ID: mdl-27115290

ABSTRACT

Human glucosylcerebrosidase 2 (GBA2) of the CAZy family GH116 is responsible for the breakdown of glycosphingolipids on the cytoplasmic face of the endoplasmic reticulum and Golgi apparatus. Genetic defects in GBA2 result in spastic paraplegia and cerebellar ataxia, while cross-talk between GBA2 and GBA1 glucosylceramidases may affect Gaucher disease. Here, we report the first three-dimensional structure for any GH116 enzyme, Thermoanaerobacterium xylanolyticum TxGH116 ß-glucosidase, alone and in complex with diverse ligands. These structures allow identification of the glucoside binding and active site residues, which are shown to be conserved with GBA2. Mutagenic analysis of TxGH116 and structural modeling of GBA2 provide a detailed structural and functional rationale for pathogenic missense mutations of GBA2.


Subject(s)
Mutation, Missense , Thermoanaerobacterium/enzymology , beta-Glucosidase/metabolism , Catalytic Domain , Crystallography, X-Ray , Glucosylceramidase , Humans , beta-Glucosidase/chemistry , beta-Glucosidase/genetics
12.
Acta Crystallogr F Struct Biol Commun ; 71(Pt 1): 41-4, 2015 Jan 01.
Article in English | MEDLINE | ID: mdl-25615966

ABSTRACT

The Thermoanaerobacterium xylanolyticum gene product TxGH116, a glycoside hydrolase family 116 protein of 806 amino-acid residues sharing 37% amino-acid sequence identity over 783 residues with human glucosylceramidase 2 (GBA2), was expressed in Escherichia coli. Purification by heating, immobilized metal-affinity and size-exclusion chromatography produced >90% pure TxGH116 protein with an apparent molecular mass of 90 kDa on SDS-PAGE. The purified TxGH116 enzyme hydrolyzed the p-nitrophenyl (pNP) glycosides pNP-ß-D-glucoside, pNP-ß-D-galactoside and pNP-N-acetyl-ß-D-glucopyranoside, as well as cellobiose and cellotriose. The TxGH116 protein was crystallized using a precipitant consisting of 0.6 M sodium citrate tribasic, 0.1 M Tris-HCl pH 7.0 by vapour diffusion with micro-seeding to form crystals with maximum dimensions of 120×25×5 µm. The TxGH116 crystals diffracted X-rays to 3.15 Šresolution and belonged to the monoclinic space group P2(1). Structure solution will allow a structural explanation of the effects of human GBA2 mutations.


Subject(s)
Bacterial Proteins/chemistry , Thermoanaerobacterium/enzymology , beta-Glucosidase/chemistry , Amino Acid Sequence , Bacterial Proteins/biosynthesis , Crystallization , Crystallography, X-Ray , Escherichia coli , Molecular Sequence Data , Protein Biosynthesis , beta-Glucosidase/biosynthesis
13.
Proteomics ; 13(14): 2088-99, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23576270

ABSTRACT

O-GlcNAcylation is a dynamic PTM of nuclear and cytoplasmic proteins, regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase, which catalyze the addition and removal of O-GlcNAc, respectively. This modification is associated with glucose metabolism, which plays important roles in many diseases including cancer. Although emerging evidence reveals that some tumor-associated proteins are O-GlcNAc modified, the total O-GlcNAcylation in cancer is still largely unexplored. Here, we demonstrate that O-GlcNAcylation was increased in primary breast malignant tumors, not in benign tumors and that this augmentation was associated with increased expression of OGT level. Using 2D O-GlcNAc immnoblotting and LC-MS/MS analysis, we successfully identified 29 proteins, with seven being uniquely O-GlcNAcylated or associated with O-GlcNAcylation in cancer. Of these identified proteins, some were related to the Warburg effect, including metabolic enzymes, proteins involved in stress responses and biosynthesis. In addition, proteins associated with RNA metabolism, gene expression, and cytoskeleton were highly O-GlcNAcylated or associated with O-GlcNAcylation. Moreover, OGT knockdown showed that decreasing O-GlcNAcylation was related to inhibition of the anchorage-independent growth in vitro. These data indicate that aberrant protein O-GlcNAcylation is associated with breast cancer. Abnormal modification of these O-GlcNAc-modified proteins might be one of the vital malignant characteristics of cancer.


Subject(s)
Breast Neoplasms/chemistry , Glycoproteins/analysis , Proteome/analysis , Acetylglucosamine/chemistry , Biomarkers, Tumor , Breast Neoplasms/metabolism , Electrophoresis, Gel, Two-Dimensional , Female , Glycoproteins/chemistry , Glycoproteins/metabolism , Heterogeneous-Nuclear Ribonucleoproteins , Humans , Immunoblotting , Lactic Acid , Models, Biological , N-Acetylglucosaminyltransferases , Proteome/chemistry , Proteome/metabolism , Reproducibility of Results
SELECTION OF CITATIONS
SEARCH DETAIL
...