title: Characterization of the magnesium chelatase from Thermosynechococcus elongatus.
abstract: The first committed step in chlorophyll biosynthesis is catalysed by magnesium chelatase (E.C. 6.6.1.1), which uses the free energy of ATP hydrolysis to insert an Mg(2+) ion into the ring of protoporphyrin IX. We have characterized magnesium chelatase from the thermophilic cyanobacterium Thermosynechococcus elongatus. This chelatase is thermostable, with subunit melting temperatures between 55 and 63°C and optimal activity at 50°C. The T. elongatus chelatase (kcat of 0.16 μM/min) shows a Michaelis-Menten-type response to both Mg(2+) (Km of 2.3 mM) and MgATP(2-) (Km of 0.8 mM). The response to porphyrin is more complex; porphyrin inhibits at high concentrations of ChlH, but when the concentration of ChlH is comparable with the other two subunits the response is of a Michaelis-Menten type (at 0.4 μM ChlH, Km is 0.2 μM). Hybrid magnesium chelatases containing a mixture of subunits from the mesophilic Synechocystis and Thermosynechococcus enzymes are active. We generated all six possible hybrid magnesium chelatases; the hybrid chelatase containing Thermosynechococcus ChlD and Synechocystis ChlI and ChlH is not co-operative towards Mg(2+), in contrast with the Synechocystis magnesium chelatase. This loss of co-operativity reveals the significant regulatory role of Synechocystis ChlD.
authors.affiliation: *Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, U.K.
authors.forename: Nathan B P
authors.initials: NB
authors.lastname: Adams
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Marklew
authors.forename: Amanda A
authors.initials: AA
authors.lastname: Brindley
authors.forename: C Neil
authors.initials: CN
authors.lastname: Hunter
authors.forename: James D
authors.initials: JD
authors.lastname: Reid
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism; D000458:Cyanobacteria / Q000201:enzymology*; D004789:Enzyme Activation; D007700:Kinetics; D008190:Lyases / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology*; D008274:Magnesium / Q000494:pharmacology; D009994:Osmolar Concentration; D021122:Protein Subunits / Q000502:physiology; D046939:Synechocystis / Q000201:enzymology; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D021122:Protein Subunits; D000255:Adenosine Triphosphate; D008190:Lyases; C086788:magnesium chelatase; D008274:Magnesium