Abstract
The sensory rhodopsin from
Anabaena
(
Nostoc
) sp. PCC7120 is the first cyanobacterial retinylidene protein identified. Here, we report on NosACO (
Nostoc
apo-carotenoid oxygenase), encoded by the ORF (open reading frame)
all4284
, as the candidate responsible for the formation of the required chromophore, retinal. In contrast with the enzymes from animals, NosACO converts β-apo-carotenals instead of β-carotene into retinal
in vitro
. The identity of the enzymatic products was proven by HPLC and gas chromatography–MS. NosACO exhibits a wide substrate specificity with respect to chain lengths and functional end-groups, converting β-apo-carotenals, (3
R
)-3-hydroxy-β-apo-carotenals and the corresponding alcohols into retinal and (3
R
)-3-hydroxyretinal respectively. However, kinetic analyses revealed very divergent
K
m
and
V
max
values. On the basis of the crystal structure of SynACO (
Synechocystis
sp. PCC6803 apo-carotenoid oxygenase), a related enzyme showing similar enzymatic activity, we designed a homology model of the native NosACO. The deduced structure explains the absence of β-carotene-cleavage activity and indicates that NosACO is a monotopic membrane protein. Accordingly, NosACO could be readily reconstituted into liposomes. To localize SynACO
in vivo
, a
Synechocystis
knock-out strain was generated expressing SynACO as the sole carotenoid oxygenase. Western-blot analyses showed that the main portion of SynACO occurred in a membrane-bound form.