Characterization of the PduS Cobalamin Reductase of Salmonella enterica and Its Role in the Pdu Microcompartment

Abstract

ABSTRACT Salmonella enterica degrades 1,2-propanediol (1,2-PD) in a coenzyme B 12 (adenosylcobalamin, AdoCbl)-dependent fashion. Salmonella obtains AdoCbl by assimilation of complex precursors, such as vitamin B 12 and hydroxocobalamin. Assimilation of these compounds requires reduction of their central cobalt atom from Co 3+ to Co 2+ to Co + , followed by adenosylation to AdoCbl. In this work, the His 6 -tagged PduS cobalamin reductase from S. enterica was produced at high levels in Escherichia coli , purified, and characterized. The anaerobically purified enzyme reduced cob(III)alamin to cob(II)alamin at a rate of 42.3 ± 3.2 μmol min −1 mg −1 , and it reduced cob(II)alamin to cob(I)alamin at a rate of 54.5 ± 4.2 nmol min −1 mg −1 protein. The apparent K m values of PduS-His 6 were 10.1 ± 0.7 μM for NADH and 67.5 ± 8.2 μM for hydroxocobalamin in cob(III)alamin reduction. The apparent K m values for cob(II)alamin reduction were 27.5 ± 2.4 μM with NADH as the substrate and 72.4 ± 9.5 μM with cob(II)alamin as the substrate. High-performance liquid chromatography (HPLC) and mass spectrometry (MS) indicated that each monomer of PduS contained one molecule of noncovalently bound flavin mononucleotide (FMN). Genetic studies showed that a pduS deletion decreased the growth rate of Salmonella on 1,2-PD, supporting a role in cobalamin reduction in vivo . Further studies demonstrated that the PduS protein is a component of the Pdu microcompartments (MCPs) used for 1,2-PD degradation and that it interacts with the PduO adenosyltransferase, which catalyzes the terminal step of AdoCbl synthesis. These studies further characterize PduS, an unusual MCP-associated cobalamin reductase, and, in conjunction with prior results, indicate that the Pdu MCP encapsulates a complete cobalamin assimilation system.