The ATPase activity of the ChlI subunit of magnesium chelatase and formation of a heptameric AAA+ ring.
The AAA(+) ATPase component of magnesium chelatase (ChlI) drives the insertion of Mg(2+) into protoporphyrin IX; this is the first step in chlorophyll biosynthesis. We describe the ATPase activity, nucleotide binding kinetics, and structural organization of the ChlI protein. A consistent reaction scheme arises from our detailed steady state description of the ATPase activity of the ChlI subunit and from transient kinetic analysis of nucleotide binding. We provide the first demonstration of metal ion binding to a specific subunit of any of the multimeric chelatases and characterize binding of Mg(2+) to the free and MgATP(2)(-) bound forms of ChlI. Transient kinetic studies with the fluorescent substrate analogue TNP-ATP show that there are two forms of monomeric enzyme, which have distinct magnesium binding properties. Additionally, we describe the self-association properties of the subunit and provide a structural analysis of the multimeric ring formed by this enzyme in the presence of nucleotide. This single particle analysis demonstrates that this species has a 7-fold rotational symmetry, which is in marked contrast to most members of the AAA(+) family that tend to form hexamers.
Reid
James D
JD
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, United Kingdom.
Siebert
C Alistair
CA
Bullough
Per A
PA
Hunter
C Neil
CN
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D002850:Chromatography, Gel; D020285:Cryoelectron Microscopy / Q000379:methods; D006868:Hydrolysis; D007091:Image Processing, Computer-Assisted; D007536:Isomerism; D007700:Kinetics; D008190:Lyases / Q000737:chemistry* / Q000378:metabolism*; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D021122:Protein Subunits; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D008190:Lyases; C086788:magnesium chelatase; D008274:Magnesium