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Elucidation of the preferred routes of C8-vinyl reduction in chlorophyll and bacteriochlorophyll biosynthesis.

Canniffe DP, Chidgey JW, Hunter CN - Biochem. J. (2014)

Bottom Line: Two unrelated classes of C8-vinyl reductase are known to exist, BciA and BciB, found in the purple phototroph Rhodobacter sphaeroides and the cyanobacterium Synechocystis sp.PCC6803 respectively.In vivo data indicate that the preferred substrates for both classes of the enzyme is C8-vinyl chlorophyllide, with C8-vinyl protochlorophyllide reduced only under conditions in which this pigment accumulates as a result of perturbed formation of chlorophyllide.

View Article: PubMed Central - PubMed

Affiliation: *Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, U.K.

ABSTRACT
Most of the chlorophylls and bacteriochlorophylls utilized for light harvesting by phototrophic organisms carry an ethyl group at the C8 position of the molecule, the product of a C8-vinyl reductase acting on a chlorophyll/bacteriochlorophyll biosynthetic precursor. Two unrelated classes of C8-vinyl reductase are known to exist, BciA and BciB, found in the purple phototroph Rhodobacter sphaeroides and the cyanobacterium Synechocystis sp. PCC6803 respectively. We constructed strains of each bacterium with the native C8-vinyl reductase swapped for the other class of the enzyme, and combined these replacements with a series of deletions of the native bch and chl genes. In vivo data indicate that the preferred substrates for both classes of the enzyme is C8-vinyl chlorophyllide, with C8-vinyl protochlorophyllide reduced only under conditions in which this pigment accumulates as a result of perturbed formation of chlorophyllide.

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HPLC elution profiles of pigments extracted from R. sphaeroides strainsHPLC elution profiles of extracts from pellets of (A) WT, (B) ΔbciA, (C) ΔbchCXF, (D) ΔbchCXF/ΔbciA, (E) V3 and (F) V3/ΔbciA strains. Retention times and Soret maxima of peaks are used to identify pigment species (inset). Traces are normalized to major peak height for clarity.
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Figure 2: HPLC elution profiles of pigments extracted from R. sphaeroides strainsHPLC elution profiles of extracts from pellets of (A) WT, (B) ΔbciA, (C) ΔbchCXF, (D) ΔbchCXF/ΔbciA, (E) V3 and (F) V3/ΔbciA strains. Retention times and Soret maxima of peaks are used to identify pigment species (inset). Traces are normalized to major peak height for clarity.

Mentions: Our previous study on BciA in R. sphaeroides indicated that a second unrelated 8VR was active in reducing a BChl precursor, compensating for the loss of BciA [15]. Subsequently, Tsukatani et al. [19] discovered that COR, which catalyses the reduction of the C7=C8 double bond, was also able to reduce the 8V group. This dual function of COR, encoded by the bchX, bchY and bchZ genes, accounts for the lack of a mutant phenotype in our previously described ΔbciA strain. Therefore, in order to determine the preferred substrate of the conventional 8VR in R. sphaeroides, mutants in several genes essential for (B)Chl biosynthesis were constructed in both WT and ΔbciA backgrounds. The pigments accumulated in these strains were extracted from pellets of semi-aerobically grown cultures and analysed by HPLC (Figure 2). Neither the WT (Figure 2, A) nor ΔbciA (Figure 2, B) accumulates BChl precursors in measurable amounts. A mutant in which the steps exclusive to BChl biosynthesis are blocked, ΔbchCXF, accumulated 8E Chlide and 8V Pchlide (Figure 2, C), whereas the 8V forms of both Chlide and Pchlide accumulated following additional deletion of bciA (Figure 2, D). These data indicate that before any 8V reduction by COR, BciA will reduce only 8V Chlide, as 8E Pchlide is not detected. Interestingly, the V3 mutant of R. sphaeroides, an unmapped mutant in a subunit of DPOR (dark-operative POR) [34], accumulates Pchlide reduced at the C8 position (Figure 2E), whereas this strain lacking bciA accumulates 8V Pchlide (Figure 2F). These data indicate that BciA will reduce the 8V group of Pchlide, but only in the absence of Chlide.


Elucidation of the preferred routes of C8-vinyl reduction in chlorophyll and bacteriochlorophyll biosynthesis.

Canniffe DP, Chidgey JW, Hunter CN - Biochem. J. (2014)

HPLC elution profiles of pigments extracted from R. sphaeroides strainsHPLC elution profiles of extracts from pellets of (A) WT, (B) ΔbciA, (C) ΔbchCXF, (D) ΔbchCXF/ΔbciA, (E) V3 and (F) V3/ΔbciA strains. Retention times and Soret maxima of peaks are used to identify pigment species (inset). Traces are normalized to major peak height for clarity.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 2: HPLC elution profiles of pigments extracted from R. sphaeroides strainsHPLC elution profiles of extracts from pellets of (A) WT, (B) ΔbciA, (C) ΔbchCXF, (D) ΔbchCXF/ΔbciA, (E) V3 and (F) V3/ΔbciA strains. Retention times and Soret maxima of peaks are used to identify pigment species (inset). Traces are normalized to major peak height for clarity.
Mentions: Our previous study on BciA in R. sphaeroides indicated that a second unrelated 8VR was active in reducing a BChl precursor, compensating for the loss of BciA [15]. Subsequently, Tsukatani et al. [19] discovered that COR, which catalyses the reduction of the C7=C8 double bond, was also able to reduce the 8V group. This dual function of COR, encoded by the bchX, bchY and bchZ genes, accounts for the lack of a mutant phenotype in our previously described ΔbciA strain. Therefore, in order to determine the preferred substrate of the conventional 8VR in R. sphaeroides, mutants in several genes essential for (B)Chl biosynthesis were constructed in both WT and ΔbciA backgrounds. The pigments accumulated in these strains were extracted from pellets of semi-aerobically grown cultures and analysed by HPLC (Figure 2). Neither the WT (Figure 2, A) nor ΔbciA (Figure 2, B) accumulates BChl precursors in measurable amounts. A mutant in which the steps exclusive to BChl biosynthesis are blocked, ΔbchCXF, accumulated 8E Chlide and 8V Pchlide (Figure 2, C), whereas the 8V forms of both Chlide and Pchlide accumulated following additional deletion of bciA (Figure 2, D). These data indicate that before any 8V reduction by COR, BciA will reduce only 8V Chlide, as 8E Pchlide is not detected. Interestingly, the V3 mutant of R. sphaeroides, an unmapped mutant in a subunit of DPOR (dark-operative POR) [34], accumulates Pchlide reduced at the C8 position (Figure 2E), whereas this strain lacking bciA accumulates 8V Pchlide (Figure 2F). These data indicate that BciA will reduce the 8V group of Pchlide, but only in the absence of Chlide.

Bottom Line: Two unrelated classes of C8-vinyl reductase are known to exist, BciA and BciB, found in the purple phototroph Rhodobacter sphaeroides and the cyanobacterium Synechocystis sp.PCC6803 respectively.In vivo data indicate that the preferred substrates for both classes of the enzyme is C8-vinyl chlorophyllide, with C8-vinyl protochlorophyllide reduced only under conditions in which this pigment accumulates as a result of perturbed formation of chlorophyllide.

View Article: PubMed Central - PubMed

Affiliation: *Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, U.K.

ABSTRACT
Most of the chlorophylls and bacteriochlorophylls utilized for light harvesting by phototrophic organisms carry an ethyl group at the C8 position of the molecule, the product of a C8-vinyl reductase acting on a chlorophyll/bacteriochlorophyll biosynthetic precursor. Two unrelated classes of C8-vinyl reductase are known to exist, BciA and BciB, found in the purple phototroph Rhodobacter sphaeroides and the cyanobacterium Synechocystis sp. PCC6803 respectively. We constructed strains of each bacterium with the native C8-vinyl reductase swapped for the other class of the enzyme, and combined these replacements with a series of deletions of the native bch and chl genes. In vivo data indicate that the preferred substrates for both classes of the enzyme is C8-vinyl chlorophyllide, with C8-vinyl protochlorophyllide reduced only under conditions in which this pigment accumulates as a result of perturbed formation of chlorophyllide.

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