Journal
JOURNAL OF BACTERIOLOGY
Volume 192, Issue 19, Pages 5071-5080Publisher
AMER SOC MICROBIOLOGY
DOI: 10.1128/JB.00575-10
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Funding
- NSF [MCB0956451]
- Div Of Molecular and Cellular Bioscience
- Direct For Biological Sciences [0956451] Funding Source: National Science Foundation
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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 Co3+ to Co2+ 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 mu mol 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 mu M for NADH and 67.5 +/- 8.2 mu M for hydroxocobalamin in cob(III) alamin reduction. The apparent K-m values for cob(II) alamin reduction were 27.5 +/- 2.4 mu M with NADH as the substrate and 72.4 +/- 9.5 mu M 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.
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