Figure 1.
Model for the Pdu microcompartment and the role of PduQ.
The Pdu MCP consists of a protein shell that encapsulates enzymes and cofactors for metabolizing 1,2-propanediol. The shell is thought to be made from 9 proteins (PduABB'JKMNTU). Encapsulated within are enzymes for the activation of B12(III) to coenzyme B12 (Ado-B12) (PduS-O-GH) as well as three 1,2-PD degradative enzymes: Ado-B12-dependent diol dehydratase (PduCDE), propionaldehyde dehydrogenase (PduP) and 1-propanol dehydrogenase (PduQ). The proposed function of the Pdu MCP is to sequester propionaldehyde and channel it to downstream enzymes in order to prevent toxicity and DNA damage. The PduQ enzyme recycles NADH to NAD+ internally within the MCP and this is needed for the activity of the PduP enzyme (this study). Alternatively, NADH can be recycled to NAD+ by the electron transport chain although at a slower rate compared to internal recycling by PduQ. This latter process is thought to require movement of NAD+/NADH through specific pores that span the protein shell.
Table 1.
Bacterial strains used in this study.
Table 2.
Anaerobic purification of PduQ-His6.
Table 3.
Kinetic parameters for propionaldehyde reduction and 1-propanol oxidation by anaerobically purified PduQ-His6.
Figure 2.
PduQ is a component of the Pdu MCP.
Panel A: 10–20% SDS-PAGE gel stained with Bio-Safe Coomassie. Lane 1, molecular mass markers; lane 2, 10 µg Pdu MCPs purified from wild-type Salmonella; lane 3, 10 µg Pdu MCPs purified from a pduQ deletion mutant BE903. Panel B: Western blot with antisera against PduQ peptide (51–64). Lane 1, 10 µg Pdu MCPs purified from wild-type Salmonella; lane 2, 10 µg Pdu MCPs purified from ΔpduQ mutant BE903.
Figure 3.
A pduQ deletion mutant grows slowly on 1,2-PD.
Cells were grown on minimal 1,2-PD medium with saturating (150 nM) or limiting (20 nM) CN-Cbl (vitamin B12): panels A or B, respectively. Symbols meanings are indicated on the figure.
Figure 4.
The Adh2 enzyme from Z. mobilis failed to complement a pduQ deletion mutant.
Cells were grown in 1,2-PD minimal medium with 75 nM CN-Cbl (vitamin B12). 10 µM IPTG was used for induction of pduQ and 10–500 µm IPTG was used for induction for ZmadhB from pLAC22.
Figure 5.
A ΔpduQ mutant is unimpaired for growth in a strain unable to form MCPs (ΔpduAB).
Cells were grown on 1,2-PD minimal medium supplemented with 40 or 150 nM CN-Cbl (vitamin B12).
Figure 6.
PduQ regenerates NAD+ for the PduP reaction in purified Pdu MCPs.
The PduP activity of purified MCPs was measured by monitoring the absorbance increase at 232 nm due to propionyl-CoA formation. Limiting NAD+ (40 µM) was added in the assays. The propionyl-CoA produced by wild-type MCPs and ΔpduQ MCPs, respectively, reached 62.3 and 27.6 µM in 8 min. Because the concentration of propionyl-CoA produced by the wild-type exceeded the amount of NAD+ added to the reaction (the PduP reaction produces 1 NADH per propionyl-CoA formed) recycling of NADH to NAD+ is indicated.
Figure 7.
His-tag protein affinity pull-down assays indicated binding between PduQ and PduP.
Lane 1, protein standards; lane 2, His6-PduP; lane 3, His6-PduP+PduQ; lane 4, PduP-His6; lane 5, PduP-His6+PduQ; lane 6, His6-PduQ; lane 7, His6-PduQ+PduP; lane 8, PduQ-His6; lane 9, PduQ-His6+PduP.