Refolding and characterization of methionine adenosyltransferase from Euglena gracilis
Methionine adenosyltransferase from Euglena gracilis (MATX) is a recently discovered member of the MAT family of proteins that synthesize S-adenosylmethionine. Heterologous overexpression of MATX in Escherichia coli rendered the protein mostly in inclusion bodies under all conditions tested. Therefo...
| Autores: | , , , , , |
|---|---|
| Formato: | artículo |
| Fecha de publicación: | 2012 |
| País: | España |
| Recursos: | Consejo Superior de Investigaciones Científicas (CSIC) |
| Repositorio: | DIGITAL.CSIC. Repositorio Institucional del CSIC |
| OAI Identifier: | oai:digital.csic.es:10261/55441 |
| Acesso em linha: | http://hdl.handle.net/10261/55441 |
| Access Level: | acceso abierto |
| Palavra-chave: | Methionine adenosyltransferase Refolding Protein characterization Inclusion bodies |
| Resumo: | Methionine adenosyltransferase from Euglena gracilis (MATX) is a recently discovered member of the MAT family of proteins that synthesize S-adenosylmethionine. Heterologous overexpression of MATX in Escherichia coli rendered the protein mostly in inclusion bodies under all conditions tested. Therefore, a refolding and purification procedure from these aggregates was developed to characterize the enzyme. Maximal recovery was obtained using inclusion bodies devoid of extraneous proteins by washing under mild urea (2 M) and detergent (5%) concentrations. Refolding was achieved in two steps following solubilization in the presence of Mg2+; chaotrope dilution to <1 M and dialysis under reducing conditions. Purified MATX is a homodimer that exhibits Michaelis kinetics with a Vmax of 1.46 μmol/min/mg and Km values of approximately 85 and 260 μM for methionine and ATP, respectively. The activity is dependent on Mg2+ and K+ ions, but is not stimulated by dimethylsulfoxide. MATX exhibits tripolyphosphatase activity that is stimulated in the presence of S-adenosylmethionine. Far-UV circular dichroism revealed β-sheet and random coil as the main secondary structure elements of the protein. The high level of sequence conservation allowed construction of a structural model that preserved the main features of the MAT family, the major changes involving the N-terminal domain. |
|---|