Characterization of the magnesium chelatase from Thermosynechococcus elongatus

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 Mg²⁺ 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 (k_cat of 0.16 μM/min) shows a Michaelis–Menten-type response to both Mg²⁺ (K_m of 2.3 mM) and MgATP²⁻ (K_m 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, K_m 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 Mg²⁺, in contrast with the Synechocystis magnesium chelatase. This loss of co-operativity reveals the significant regulatory role of Synechocystis ChlD.