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 (kcat of 0.16 μM/min) shows a Michaelis–Menten-type response to both Mg2+ (Km of 2.3 mM) and MgATP2− (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 Mg2+, in contrast with the Synechocystis magnesium chelatase. This loss of co-operativity reveals the significant regulatory role of Synechocystis ChlD.
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Research Article|
December 10 2013
Characterization of the magnesium chelatase from Thermosynechococcus elongatus
Nathan B. P. Adams;
Nathan B. P. Adams
*Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, U.K.
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Christopher J. Marklew;
Christopher J. Marklew
*Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, U.K.
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Amanda A. Brindley;
Amanda A. Brindley
*Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, U.K.
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C. Neil Hunter;
C. Neil Hunter
*Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, U.K.
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James D. Reid
James D. Reid
1
†Department of Chemistry, The University of Sheffield, Sheffield S3 7HF, U.K.
1To whom correspondence should be addressed (email j.reid@sheffield.ac.uk).
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Publisher: Portland Press Ltd
Received:
June 24 2013
Revision Received:
September 30 2013
Accepted:
October 21 2013
Accepted Manuscript online:
October 21 2013
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2014 Biochemical Society
2014
Biochem J (2014) 457 (1): 163–170.
Article history
Received:
June 24 2013
Revision Received:
September 30 2013
Accepted:
October 21 2013
Accepted Manuscript online:
October 21 2013
Citation
Nathan B. P. Adams, Christopher J. Marklew, Amanda A. Brindley, C. Neil Hunter, James D. Reid; Characterization of the magnesium chelatase from Thermosynechococcus elongatus. Biochem J 1 January 2014; 457 (1): 163–170. doi: https://doi.org/10.1042/BJ20130834
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