Three separate proteins constitute the magnesium chelatase of Rhodobacter sphaeroides.
The insertion of magnesium into protoporphyrin IX is the first step unique to chlorophyll production and is catalyzed by magnesium chelatase. The Rhodobacter sphaeroides genes, bchI and bchD together, and bchH alone, were cloned and expressed with the pET3a vector in Escherichia coli strain BL21 (DE3). The 40-kDa BchI protein was synthesized in greater abundance compared to the 70-kDa BchD protein when both were expressed together from the same plasmid. The production of large amounts of the 140-kDa BchH protein in E. coli was accompanied by an accumulation of protoporphyrin IX. The accumulated protoporphyrin IX was bound specifically to BchH in an approximate molar ratio of 1:1. All three recombinant proteins were soluble; BchH was monomeric, Bchl was dimeric, while BchD appeared to be polymeric with a molecular mass of approximately 550 kDa. The BchH and BchI proteins were purified to apparent homogeneity while BchD was separated from BchI and partially purified. Magnesium was inserted into protoporphyrin IX and deuteroporphyrin by combining these three proteins in the presence of ATP. One monomer of BchH to one dimer of BchI gave the optimal magnesium chelatase activity and the activity was dependent on the amount of partially purified BchD added to the assay at the optimum BchH:BchI ratio. The reaction was dissected into two parts with an activation step requiring BchI, BchD, and Mg2+-ATP, and a metal-insertion step which in addition requires Mg2+, protoporphyrin IX, and BchH. The stoichiometric binding of protoporphyrin IX to BchH in vitro is direct evidence for BchH carrying out such a role in vivo whereas the other two proteins are involved in ATP activation and magnesium insertion.
Willows
R D
RD
Carlsberg Laboratory, Department of Physiology, Copenhagen Valby, Denmark.
Gibson
L C
LC
Kanangara
C G
CG
Hunter
C N
CN
von Wettstein
D
D
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D007700:Kinetics; D008190:Lyases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D008190:Lyases; C086788:magnesium chelatase; D008274:Magnesium