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.
