Molecular dissection of the Cereibacter sphaeroides spheroidene monooxygenase.

In some purple phototrophic bacteria, the enzyme spheroidene monooxygenase (CrtA) catalyses the final step of carotenoid biosynthesis, introducing a keto group at the C2 position of spheroidene to produce spheroidenone. CrtA from the model purple bacterium Cereibacter (previously Rhodobacter) sphaeroides has a C-terminal extension consisting of a disordered, proline-rich sequence followed by a short region containing a significant proportion of glycine residues; the role of this extension is not understood. In addition to the accumulation of spheroidene, a C. sphaeroides ΔcrtA mutant generated in a previous study had a slower growth rate and made fewer photosynthetic complexes than the wild-type strain. We show here that these phenotypes are largely due to a polar effect of the crtA deletion on the downstream bchID bacteriochlorophyll biosynthesis genes. We generated a crtA mutant where bchID expression was not interrupted and used this background to test a series of CrtA C-terminal truncations to identify the minimal enzyme that retains spheroidene monooxygenase activity. Using structural modelling, we identify a histidine residue that acts as the axial ligand to a heme group required for spheroidene monooxygenase activity, and two threonine residues found proximal to the bound carotenoid. We show that the subtle photoheterotrophic growth phenotype of this new ΔcrtA mutant is due to the loss of the C-terminus of the enzyme rather than the altered carotenoid content of this strain. We further demonstrate that the C-terminal extension appears to mediate the association of CrtA with other membrane proteins, forming a range of high-molecular-mass complexes.