RFFBR Second Annual Paper Award Summary

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Most of chlorophylls (Chls) and bacteriochlorophylls (BChls) found in nature have an ethyl side chain at the C-8 position. In most cases, the tetrapyrrole precursor to all these molecules is divinyl-protochlorophyllide (DV-PChlide), which must be reduced during the biosynthesis of these molecules (Gomez Maqueo Chew and Bryant (2007) Ann. Rev.. Microbiol. 61: 113-129). In 2005, Nagata et al. reported that a protein encoded by AT5G18660 was responsible for the reduction of the C-8 vinyl moiety during the biosynthesis of Chl a and Chl b in Arabidopsis thaliana (Nagata et al. (2005) Plant Cell 17: 233-240). Surprisingly, a Chlorobium tepidum protein encoded by CT1063 exhibited a very high degree of sequence similarity to AT5G18660. All green sulfur bacteria synthesize three types of (B)Chls, and in the case of C. tepidum, these Chls are Chl aPD, BChl cF, and BChl aP. The C-8 vinyl group of each of these (B)Chls must be reduced to an ethyl moiety, and in the case of the major antenna pigment, BChl c, the C-8 ethyl side chain is further methylated to different degrees to form propionyl, isobutyl, and rarely neo-pentyl side chains. In 1995 Suzuki and Bauer had reported that a Rhodobacter capsulatus bchJ mutant accumulated large amounts of DV-Pchlide and some monovinyl Mg-protoporphyrin monoester in the growth medium, and concluded that bchJ, a gene found in the photosynthetic gene cluster of purple bacteria, encoded the enzyme responsible for the reduction of the C-8 vinyl group of DV-PChlide (Suzuki and Bauer, (1995), J. Biol. Chem. 270: 3732-3740). An ortholog of bchJ (CT2014) was identified in the genome of the green sulfur bacterium C. tepidum and was initially presumed to encode the C-8 vinyl reductase.

In order to determine which of these two gene products actually encoded the vinyl reductase activity, both the bchJ (CT2014) and CT1063 genes were insertionally inactivated in C. tepidum. Our results showed that all three Chls found in C. tepidum had a C-8 vinyl group in the CT1063 mutant. This result clearly suggested that CT1063, renamed bciA, encoded the divinyl reductase in C. tepidum (or a component thereof). On the other hand, the bchJ (CT2014) mutant produced low but detectable amounts of Chl aPD, BChl aP and BChl cF that had reduced C-8 ethyl sidechains. This indicated that the C-8 reductase activity was still present in the bchJ mutant. However, consistent with the results of Suzuki and Bauer in R. capsulatus, the C. tepidum bchJ mutant excreted large amounts of DV-PChlide a into the growth medium and had a greatly reduced (B)Chl content. The presence of a highly conserved bciA gene homolog in R. capsulatus suggests that BciA is responsible for the reduction of the DV-PChlide in this organism as well. Together, these results suggested that BchJ might play a role in substrate channeling and/or regulation of Chl biosynthesis.

When the C. tepidum BciA was heterologously expressed in E. coli and using DV-PChlide purified from the medium of the bchJ mutant as substrate, NADPH was shown to be the reductant for this reaction and there was no requirement for additional proteins to reduce DV-PChlide in vitro. This result conclusively established that BciA was the only gene product required for the DVR activity. In follow-up experiments the steady-state kinetics of this reaction have been studied, and the DVR has been shown to react by an ordered sequential mechanism (F. Shen and D. A. Bryant, unpublished results). The absence of a bciA homolog in two green sulfur bacterial genomes, some purple bacterial genomes, and most cyanobacterial genomes implies that there must be at least two structural classes of divinyl-reductases. Tanaka and coworkers (Ito et al., (2008), J. Biol. Chem. 283: 9002-9011) recently showed that slr1923 in Synechocystis sp. PCC 6803 plays a role in reduction of C-8 vinyl groups in this cyanobacterium. However, some organisms (e.g., Roseiflexus castenholzii) synthesize BChl a with C-8 ethyl side chains but lack homologs of both bciA and slr1923, implying that at least three types of vinyl reductases occur among chlorophototrophs.

Photos of Don Bryant and Aline Gomez Maqueo Chew are displayed

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Top photo: Donald Bryant in his office

Bottom photo: Aline Gomez Maqueo Chew holding her RFFBR Annual Reward certificate

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