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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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Detection of 8VRs in Synechocystis by Western blottingSeparated membrane (M) and soluble (S) fractions from described strains of Synechocystis, resolved by SDS/PAGE and transferred on to a membrane were probed with anti-BciA and anti-BciB antibodies. The membrane was also probed with antibodies to known membrane-associated and soluble Chl biosynthetic proteins AcsF and Gun4 respectively.
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Figure 4: Detection of 8VRs in Synechocystis by Western blottingSeparated membrane (M) and soluble (S) fractions from described strains of Synechocystis, resolved by SDS/PAGE and transferred on to a membrane were probed with anti-BciA and anti-BciB antibodies. The membrane was also probed with antibodies to known membrane-associated and soluble Chl biosynthetic proteins AcsF and Gun4 respectively.

Mentions: To determine the localization of BciB in Synechocystis, as well as the recombinant BciA, samples from cultures of WT, ΔbciB and ΔbciB::bciARs grown under moderate light intensity were disrupted by bead-beating and the soluble and membrane fractions were separated by centrifugation. These samples were resolved by SDS/PAGE and transferred on to a PVDF membrane which was probed with antibodies raised against BciB (Synechocystis) and BciA (A. thaliana), as well as those raised against known membrane-associated (AcsF, A. thaliana CHL27; Agrisera) and soluble (Gun4, Synechocystis) proteins involved in Chl biosynthesis (Figure 4). The blot indicates that the native BciB protein is found in the cytoplasm as well as being localized to the thylakoid membrane, whereas the recombinant BciA is only detected in the membrane fraction.


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

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

Detection of 8VRs in Synechocystis by Western blottingSeparated membrane (M) and soluble (S) fractions from described strains of Synechocystis, resolved by SDS/PAGE and transferred on to a membrane were probed with anti-BciA and anti-BciB antibodies. The membrane was also probed with antibodies to known membrane-associated and soluble Chl biosynthetic proteins AcsF and Gun4 respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4214422&req=5

Figure 4: Detection of 8VRs in Synechocystis by Western blottingSeparated membrane (M) and soluble (S) fractions from described strains of Synechocystis, resolved by SDS/PAGE and transferred on to a membrane were probed with anti-BciA and anti-BciB antibodies. The membrane was also probed with antibodies to known membrane-associated and soluble Chl biosynthetic proteins AcsF and Gun4 respectively.
Mentions: To determine the localization of BciB in Synechocystis, as well as the recombinant BciA, samples from cultures of WT, ΔbciB and ΔbciB::bciARs grown under moderate light intensity were disrupted by bead-beating and the soluble and membrane fractions were separated by centrifugation. These samples were resolved by SDS/PAGE and transferred on to a PVDF membrane which was probed with antibodies raised against BciB (Synechocystis) and BciA (A. thaliana), as well as those raised against known membrane-associated (AcsF, A. thaliana CHL27; Agrisera) and soluble (Gun4, Synechocystis) proteins involved in Chl biosynthesis (Figure 4). The blot indicates that the native BciB protein is found in the cytoplasm as well as being localized to the thylakoid membrane, whereas the recombinant BciA is only detected in the membrane fraction.

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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