RESUMO
In the presence of divalent cations, inorganic pyrophosphatase is activated to hydrolyze inorganic pyrophosphate to inorganic phosphate. Here, we clone, express, purify, and characterize inorganic pyrophosphatase from the psychrophilic Shewanella sp. AS-11 (Sh-PPase). The recombinant Sh-PPase was expressed in Escherichia coli BL21 (DE3) at 20°C using pET16b as an expression vector and purified from the cell extracts by a combination of ammonium sulfate fractionation and anion-exchange chromatography. Sh-PPase was found to be a family II PPase with a subunit molecular mass of 34 kD that preferentially utilizes Mn²âº over Mg²âº ions for activity. The functional characteristics of Sh-PPase, such as activity, temperature dependency, and thermal inactivation, were greatly influenced by manganese ions. Manganese ion activation increased the enzyme's activity at low temperatures; therefore, it was required to gain the cold-adapted characteristics of Sh-PPase.
Assuntos
Pirofosfatase Inorgânica/genética , Pirofosfatase Inorgânica/isolamento & purificação , Shewanella/enzimologia , Cátions Bivalentes/metabolismo , Clonagem Molecular , Temperatura Baixa , Ativação Enzimática , Estabilidade Enzimática , Escherichia coli/genética , Infecções por Bactérias Gram-Negativas/microbiologia , Pirofosfatase Inorgânica/química , Pirofosfatase Inorgânica/metabolismo , Magnésio/metabolismo , Manganês/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Shewanella/química , Shewanella/genética , Especificidade por SubstratoRESUMO
Mn²âº ions influence the activity, temperature dependence, and thermostability of the psychrophilic Shewanella-PPase (Sh-PPase), and are required to function in cold environments. The functional characteristics of Sh-PPase on activation with Mn²âº ions are possibly related to conformational changes in the molecule. In this study, conformational changes of Sh-PPase on activation with Mn²âº ions were analyzed in solution by fluorescence spectroscopy analysis of intrinsic tryptophan residues, 1-anilino-8-naphthalene sulfonate fluorescence, and circular dichroism spectroscopy. For Sh-PPase, Mn²âº ions did not affect the flexibility of the tryptophan residues and secondary structure of the enzyme. However, the microenvironment of the tryptophan residues and surface area of Sh-PPase were more hydrophilic on activation with Mn²âº ions. These results indicate that activation with Mn²âº ions causes conformational changes around the aromatic amino acid residues and affects the hydrophobicity of the enzyme surface, which results in conformational changes. Substrate-induced conformational changes reflect that metal-free Sh-PPase in solution indicated an open structure and will be a close structure when binding substrate. In combination of our spectroscopic analyses on Sh-PPase, it can be concluded that activation with Mn²âº ions changes some conformation of Sh-PPase molecule in solution.
