Characterisation of PduS, the pdu metabolosome corrin reductase, and evidence of substructural organisation within the bacterial microcompartment

Abstract

PduS is a corrin reductase and is required for the reactivation of the cobalamin-dependent diol dehydratase. It is one component encoded within the large propanediol utilisation (pdu) operon, which is responsible for the catabolism of 1,2-propanediol within a self-assembled proteinaceous bacterial microcompartment. The enzyme is responsible for the reactivation of the cobalamin coenzyme required by the diol dehydratase. The gene for the cobalamin reductase from Citrobacter freundii (pduS) has been cloned to allow the protein to be overproduced recombinantly in E. coli with an N-terminal His-tag. Purified recombinant PduS is shown to be a flavoprotein with a non-covalently bound FMN that also contains two coupled [4Fe-4S] centres. It is an NADH-dependent flavin reductase that is able to mediate the one-electron reductions of cob(III)alamin to cob(II)alamin and cob(II)alamin to cob(I)alamin. The [4Fe-4S] centres are labile to oxygen and their presence affects the midpoint redox potential of flavin. Evidence is presented that PduS is able to bind cobalamin, which is inconsistent with the view that PduS is merely a flavin reductase. PduS is also shown to interact with one of the shell proteins of the metabolosome, PduT, which is also thought to contain an [Fe-S] cluster. PduS is shown to act as a corrin reductase and its interaction with a shell protein could allow for electron passage out of the bacterial microcompartment.

Figure 1. Regeneration of adenosylcobalamin from aquacobalamin.

In the 1,2-propanediol utilisation metabolosome the adenosylcobalamin-dependent diol dehydratase (PduCDE) occasionally generates aquacobalamin. This inactivated form of the coenzyme is reactivated by the action of a corrin reductase, PduS, and an adenosyltransferase (PduO). This is achieved by two sequential one-electron reductions of aquacob(III)alamin to cob(I)alamin prior to the transfer of the adenosyl moiety from ATP.

Figure 2. UV-visible spectrum of purified PduS.

The UV-visible spectrum of aerobically-purified PduS in 20 mM Tris-HCl (pH 8.0) in red is compared to that of aerobically purified PduS (in blue). The blue spectrum is consistent with that of a flavoprotein whereas the red spectrum has added features around the 420 nm region consistent with the presence of additional [Fe-S] centre(s). A gel of purified PduS is shown in the inset.

Figure 3. Left hand panel: X-band EPR spectra of PduS reduced using sodium dithionite, a) WT reduced using ten equivalents of dithionite showing interaction between two [Fe-S] clusters, b) WT reduced using three equivalents of dithionite, c) C270A mutant reduced using ten equivalents of dithionite (this spectrum is the sum of 16 scans), the asterisk marks a small signal from an unassigned (non-flavin) radical species.

Right hand panel: X-band EPR spectra of PduS reduced using an excess of NADH, a) shows a wide sweep width with and is expanded vertically to reveal the features arising from the [4Fe-4S]¹⁺ centre, b) shows a spectrum of the same sample recorded under conditions that favour the accurate registration of flavosemiquinone radical spectra and over a narrower sweep width. Experimental conditions were: microwave power 0.5 mW, modulation amplitude 5 G, modulation frequency 100 KHz, temperature 12 K for all but right hand panel spectrum b) where they were: microwave power 10 µW, modulation amplitude 1 G, modulation frequency 100 KHz, temperature 70K.

Figure 4. X-band EPR spectra of hydroxocobalamin and NADH in the absence, a), and presence, b), of PduS.

Experimental conditions: microwave power 0.5 mW, modulation amplitude 5 G, modulation frequency 100 KHz, temperature 12K.

Figure 5. Determination of midpoint reduction potential of PduS.

Spectral changes associated with the reduction of PduS (70 µM), in 100 mM phosphate buffer (pH 7.5) at 25°C with dithionite during the spectroelectrochemical titration. Spectra were recorded after the sample reached equilibrium following each addition of dithionite. The arrow indicates the wavelength at which the maximal changes in spectral properties between oxidised and reduced enzyme occur. (a) Shows spectra of aerobically purified PduS housing predominantly flavin during dithionite titration. (b) Shows anaerobically purified PduS housing [4Fe-4S] and flavin cofactors during dithionite titration. Arrows indicate directions of absorption change at selected points in these spectra. (c) A fit of the spectral data from panel (a) at 450 nm to the Nernst function, giving a midpoint redox potential of the cofactors to be E^o = −150±5 mV. (d) A fit of the spectral data from panel (b) at 455 nm to the Nernst function, giving a midpoint redox potential of the flavin to be E^o = −262±5 mV. Midpoint redox potentials are shown by dashed lines.

Figure 6. SDS-PAGE of PduS/PduT copurification.

Lane 1 contains molecular mass markers as indicated. Lane 2 contains the purification eluate from a nickel column that has been applied with the crude cell extract of a strain overproducing His-tagged PduS and untagged PduT. The identities of PduS and PduT were confirmed by MALDI after trypsin digestion of the extracted protein bands.