Expression of the chlI, chlD, and chlH genes from the Cyanobacterium synechocystis PCC6803 in Escherichia coli and demonstration that the three cognate proteins are required for magnesium-protoporphyrin chelatase activity.
Magnesium-protoporphyrin chelatase catalyzes the first step unique to chlorophyll synthesis: the insertion of Mg2+ into protoporphyrin IX. Genes from Synechocystis sp. PCC6803 with homology to the bchI and bchD genes of Rhodobacter sp. were cloned using degenerate oligonucleotides. The function of these genes, putatively encoding subunits of magnesium chelatase, was established by overexpression in Escherichia coli, including the overexpression of Synechocystis chlH, previously cloned as a homolog of the Rhodobacter bchH gene. The combined cell-free extracts were able to catalyze the insertion of Mg2+ into protoporphyrin IX in an ATP-dependent manner and only when the products of all three genes were present. The ChlH, ChlI, and ChlD gene products are therefore assigned to the magnesium chelatase step in chlorophyll a biosynthesis in Synechocystis PCC6803. The primary structure of the Synechocystis ChlD protein reveals some interesting features; the N-terminal half of the protein shows 40-41% identity to Rhodobacter BchI and Synechocystis ChlI, whereas the C-terminal half displays 33% identity to Rhodobacter BchD. This suggests a functional as well as an evolutionary relationship between the "I" and "D" genes.
Jensen
P E
PE
Krebs Institute for Biomolecular Research, University of Sheffield, Sheffield S10 2TN, United Kingdom.
Gibson
L C
LC
Henningsen
K W
KW
Hunter
C N
CN
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000201:enzymology / Q000235:genetics*; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D008190:Lyases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; C100608:ChlI protein, Synechocystis sp. PCC 6803; D017931:DNA Primers; D008190:Lyases; C086788:magnesium chelatase; C100607:ChlD protein, Synechocystis sp. PCC 6803; C100609:ChlH protein, Synechocystis