Heterologous expression and purification of the manganese(II) oxidizing peroxidase of alpha-proteobacterium Erythrobacter sp. SD21.

摘要

Manganese (II)-oxidation by bacteria and fungi is an important biogeochemical process in terrestrial and marine environments. Manganese(II) oxidation (Mn(II) → Mn(III)) in the alpha-proteobacterium Erythrobacter sp. SD21 is catalyzed by a manganese oxidizing peroxidase (Mop), a novel enzyme with an unusual combination of an animal heme peroxidase domain and multiple calcium binding motifs. Because the native expression in Erythrobacter sp. SD21 produces insufficient amounts of the enzyme to study on a molecular level, Mop was heterologously expressed in Escherichia coli. The SD21 Mop gene (238 kDa) was cloned into three different vectors (pBAD/Thio, pSpeedET, and pSpeedET-gIII) and expressed in E. coli to determine the optimal conditions for high-level expression of soluble and active purified Mop. Heterologous expression of Mop was indicated by bands at ∼250 and ∼150 kDa on Coomassie stained SDS-PAGE gels. The recombinant enzyme activity was stimulated by both pyrroloquinoline quinone (PQQ) and hydrogen peroxide. However, Mop purified by nickel affinity chromatography resulted in a loss of activity. A series of experiments were performed to regain Mop activity, which resulted in discovering that a small molecule associated with an E. coli protein acts as a stimulating factor required for Mop activity. Mop is a novel protein that represents a new class of Mn(II) oxidizing proteins. Understanding the molecular mechanism of Mop and similar Mn(II) oxidizing proteins will provide insight into bacterial Mn(II) oxidation that may contribute to the application of these biological processes to bioremediation.