Characterization of the PduS cobalamin reductase of Salmonella enterica and its role in the Pdu microcompartment.
Salmonella enterica degrades 1,2-propanediol (1,2-PD) in a coenzyme B12 (adenosylcobalamin, AdoCbl)-dependent fashion. Salmonella obtains AdoCbl by assimilation of complex precursors, such as vitamin B12 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 His6-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 Km values of PduS-His6 were 10.1±0.7 μM for NADH and 67.5±8.2 μM for hydroxocobalamin in cob(III)alamin reduction. The apparent Km 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.
Cheng
Shouqiang
S
Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA.
Bobik
Thomas A
TA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002413:Cations, Divalent / Q000378:metabolism; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005816:Genetic Complementation Test; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D010641:Phenotype; D011485:Protein Binding; D019779:Salmonella enterica / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D013696:Temperature; D020798:Two-Hybrid System Techniques
D001426:Bacterial Proteins; D002413:Cations, Divalent