The chondroitin sulfate/dermatan sulfate 4-O-endosulfatase from marine bacterium Vibrio sp FC509 is a dimeric species: Biophysical characterization of an endosulfatase.
Sulfatases catalyze hydrolysis of sulfate groups. They have a key role in regulating the sulfation states that determine the function of several scaffold molecules. Currently, there are no studies of the conformational stability of endosulfatases. In this work, we describe the structural features and conformational stability of a 4-O-endosulfatase (EndoV) from a marine bacterium, which removes specifically the 4-O-sulfate from chondroitin sulfate/dermatan sulfate. For that purpose, we have used several biophysical techniques, namely, fluorescence, circular dichroism (CD), FTIR spectroscopy, analytical ultracentrifugation (AUC), differential scanning calorimetry (DSC), mass spectrometry (MS), dynamic light scattering (DLS) and size exclusion chromatography (SEC). The protein was a dimer with an elongated shape. EndoV acquired a native-like structure in a narrow pH range (7.0-9.0); it is within this range where the protein shows the maximum of enzymatic activity. The dimerization did not involve the presence of disulphide-bridges as suggested by AUC, SEC and DLS experiments in the presence of β-mercaptoethanol (β-ME). EndoV secondary structure is formed by a mixture of α and β-sheet topology, as judged by deconvolution of CD and FTIR spectra. Thermal and chemical denaturations showed irreversibility and the former indicates that protein did not unfold completely during heating.
Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Elche, Alicante, Spain; Biocomputation and Complex Systems Physics Institute, Zaragoza, Spain. Electronic address: jlneira@umh.es.
José L
JL
Neira
Department of Physical Chemistry, University of Granada, Granada, Spain.
Encarnación
E
Medina-Carmona
Department of Physical Chemistry, University of Murcia, Murcia, Spain.
José G
JG
Hernández-Cifre
Departament de Bioquímica i Biologia Molecular, Unitat de Biociències, Universitat Autònoma de Barcelona, Bellaterra, Cerdanyola del Vallés, Barcelona, Spain.
Laia
L
Montoliu-Gaya
Department of Physical Chemistry, Biochemistry and Inorganic Chemistry, University of Almería, Agrifood Campus of International Excellence (ceiA3), Almería, Spain.
Ana
A
Cámara-Artigás
CNRS UMR 8587, Laboratoire Analyse et Modélisation pour la Biologie et l'Environnement, Evry, France; Université Evry-Val-d'Essonne, Laboratoire Analyse et Modélisation pour la Biologie et l'Environnement, Evry, France.
Ilham
I
Seffouh
CNRS UMR 8587, Laboratoire Analyse et Modélisation pour la Biologie et l'Environnement, Evry, France; Université Evry-Val-d'Essonne, Laboratoire Analyse et Modélisation pour la Biologie et l'Environnement, Evry, France.
Florence
F
Gonnet
CNRS UMR 8587, Laboratoire Analyse et Modélisation pour la Biologie et l'Environnement, Evry, France; Université Evry-Val-d'Essonne, Laboratoire Analyse et Modélisation pour la Biologie et l'Environnement, Evry, France.
Régis
R
Daniel
Departament de Bioquímica i Biologia Molecular, Unitat de Biociències, Universitat Autònoma de Barcelona, Bellaterra, Cerdanyola del Vallés, Barcelona, Spain.
Sandra
S
Villegas
Department of Physical Chemistry, University of Murcia, Murcia, Spain.
José García
JG
de la Torre
Department of Physical Chemistry, University of Granada, Granada, Spain.
Angel L
AL
Pey
National Glyco-engineering Research Center and State Key Laboratory of Microbial Technology, Shandong University, Jinan, China.
Fuchuan
F
Li
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D055162:Biocatalysis; D055592:Biophysical Phenomena; D002152:Calorimetry, Differential Scanning; D002809:Chondroitin Sulfates / Q000378:metabolism*; D002810:Chondroitinases and Chondroitin Lyases / Q000737:chemistry / Q000378:metabolism*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003871:Dermatan Sulfate / Q000031:analogs & derivatives* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D011489:Protein Denaturation; D055503:Protein Multimerization; D058767:Protein Unfolding; D017550:Spectroscopy, Fourier Transform Infrared; D013431:Sulfates / Q000378:metabolism; D013696:Temperature; D014733:Vibrio / Q000201:enzymology*
D001426:Bacterial Proteins; D013431:Sulfates; C069524:dermatan sulfate chondroitin sulfate; D003871:Dermatan Sulfate; D002809:Chondroitin Sulfates; D002810:Chondroitinases and Chondroitin Lyases
Circular dichroism; Differential scanning calorimetry; Fluorescence; Irreversibility; Mass spectrometry; Protein stability