THE D1 C-TERMINAL PROCESSING PROTEASE OF PHOTOSYSTEM-II FROM SCENEDESMUS-OBLIQUUS - PROTEIN-PURIFICATION AND GENE CHARACTERIZATION IN WILD-TYPE AND PROCESSING MUTANTS
Jt. Trost et al., THE D1 C-TERMINAL PROCESSING PROTEASE OF PHOTOSYSTEM-II FROM SCENEDESMUS-OBLIQUUS - PROTEIN-PURIFICATION AND GENE CHARACTERIZATION IN WILD-TYPE AND PROCESSING MUTANTS, The Journal of biological chemistry, 272(33), 1997, pp. 20348-20356
Polypeptide D1 of the photosystem II reaction center of oxygenic photo
synthesis is expressed in precursor form (pre-D1), and it must be prot
eolytically processed at its C terminus to enable assembly of the mang
anese cluster responsible for photosynthetic water oxidation. A rapid
and highly sensitive enzyme-linked immunosorbent assay-based microtite
r plate method is described for assaying this D1 C-terminal processing
protease. A protocol is described for the isolation and purification
to homogeneity of the enzyme from the green alga, Scenedesmus obliquus
. Amino acid sequence information on the purified protease was used to
clone the corresponding gene, the translated sequence of which is pre
sented. A comparison of the gene product with homologous proteases poi
nts to a region of conserved residues that likely corresponds to the a
ctive site of a new class of serine protease, The LF-1 mutant strain o
f Scenedesmus (isolated by Dr, Norman Bishop) is incapable of processi
ng pre-D1. We show here that the C-terminal processing protease gene i
n this strain contains a single base deletion that causes a frame shif
t and a premature stop of translation within the likely active site of
the enzyme. A suppressor strain, LF-1-RVT-1, which is photoautotrophi
c and capable of processing pre-D1 has a nearby single base insertion
that restores the expression of active enzyme. These observations prov
ide the first definitive proof that the enzyme isolated is responsible
for in vivo proteolytic processing of pre-D1 and that no other protea
se can compensate for its loss.