Rhodobacter sphaeroides mutants overexpressing chlorophyllide a oxidoreductase of Blastochloris viridis elucidate functions of enzymes in late bacteriochlorophyll biosynthetic pathways.

Yusuke Tsukatani,Jiro Harada,Tadashi Mizoguchi,Jiro Nomata,Yuichi Fujita,Haruki Yamamoto,Hitoshi Tamiaki

doi:10.1038/srep09741

Abstract

In previous studies we have demonstrated that chlorophyllide a oxidoreductases (CORs) from bacteriochlorophyll (BChl) a-producing Rhodobacter species and BChl b-producing Blastochloris viridis show distinct substrate recognition and different catalytic hydrogenation reactions, and that these two types of CORs therefore cause committed steps for BChls a and b biosynthesis. In this study, COR genes from B. viridis were incorporated and overexpressed in a series of Rhodobacter sphaeroides mutants. We found that the following two factors are essential in making R. sphaeroides produce BChl b: the loss of functions of both intrinsic COR and 8-vinyl reductase (BciA) in the host R. sphaeroides strain; and expression of the BchYZ catalytic components of COR from B. viridis, not the complete set of COR (BchXYZ), in the host strain. In addition, we incorporated bchYZ of B. viridis into the R. sphaeroides mutant lacking BchJ and BciA, resulting in the strain accumulating both BChl a and BChl b. This is the first example of an anoxygenic photosynthetic bacterium producing BChls a and b together. The results suggest that BchJ enhances activity of the intrinsic COR. The physiological significance of BchJ in pigment biosynthetic pathways will be discussed.

Highlights

Rhodobacter sphaeroides mutants overexpressing chlorophyllide a oxidoreductase of Blastochloris viridis elucidate functions of enzymes in late bacteriochlorophyll biosynthetic pathways
In previous studies we have demonstrated that chlorophyllide a oxidoreductases (CORs) from bacteriochlorophyll (BChl) a-producing Rhodobacter species and BChl b-producing Blastochloris viridis show distinct substrate recognition and different catalytic hydrogenation reactions, and that these two types of CORs cause committed steps for BChls a and b biosynthesis
We found that the following two factors are essential in making R. sphaeroides produce BChl b: the loss of functions of both intrinsic COR and 8-vinyl reductase (BciA) in the host R. sphaeroides strain; and expression of the BchYZ catalytic components of COR from B. viridis, not the complete set of COR (BchXYZ), in the host strain

Summary

Results and discussion

Construction and pigment analysis of the platform R. sphaeroides mutant strains. The wild-type strain of R. sphaeroides was used as a host strain to construct the single mutants, DbchZ and DbchJ (Fig. S1). Our previous study[9] has clearly indicated that the suitable substrate for the B. viridis COR proteins is 8V-Chlide a, not Chlide a, and the present study supports the idea that BchYZ small amount of 3-(1-hydroxyethyl)-Chlide a; in-line absorption and mass spectra of a minor elution peak from the DbchZ mutant (Supplementary Fig. S2) are almost identical to those of 3-(1-hydroxyethyl)-Chlide a from the DbchZ1BvYZ strain (Fig. 4CD, traces 1). Because the amounts of BChls a and b produced in the DbchJ/bciA1BvYZ mutant were almost the same (Fig. 6A, trace iii), both intrinsic a-COR and exogenous b-COR catalytic components were likely to be almost functional This implies that BchJ may form a tertiary complex with pigment substrates and pigment biosynthesis enzymes. It is highly likely that BchJ are involved in most of the BChl a biosynthetic pathways to facilitate enzymes working in the pigment biosynthesis

Methods

Author contributions

Additional information