The D1 C-terminal processing protease of photosystem II from Scenedesmus obliquus. Protein purification and gene characterization in wild type and processing mutants.
Polypeptide D1 of the photosystem II reaction center of oxygenic photosynthesis is expressed in precursor form (pre-D1), and it must be proteolytically processed at its C terminus to enable assembly of the manganese cluster responsible for photosynthetic water oxidation. A rapid and highly sensitive enzyme-linked immunosorbent assay-based microtiter 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 presented. A comparison of the gene product with homologous proteases points to a region of conserved residues that likely corresponds to the active site of a new class of serine protease. The LF-1 mutant strain of Scenedesmus (isolated by Dr. Norman Bishop) is incapable of processing pre-D1. We show here that the C-terminal processing protease gene in this strain contains a single base deletion that causes a frame shift and a premature stop of translation within the likely active site of the enzyme. A suppressor strain, LF-1-RVT-1, which is photoautotrophic and capable of processing pre-D1 has a nearby single base insertion that restores the expression of active enzyme. These observations provide the first definitive proof that the enzyme isolated is responsible for in vivo proteolytic processing of pre-D1 and that no other protease can compensate for its loss.
Trost
J T
JT
Central Research and Development Department, Experimental Station, E. I. du Pont de Nemours & Co., Wilmington, Delaware 19880-0173, USA.
Chisholm
D A
DA
Jordan
D B
DB
Diner
B A
BA
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000235:genetics / Q000302:isolation & purification*; D056890:Eukaryota / Q000201:enzymology*; D008969:Molecular Sequence Data; D009154:Mutation; D045322:Photosynthetic Reaction Center Complex Proteins / Q000378:metabolism*; D045332:Photosystem II Protein Complex; D011480:Protease Inhibitors / Q000494:pharmacology
D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D011480:Protease Inhibitors; D010450:Endopeptidases