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Methods Mol Biol. 2019;1876:125-140. doi: 10.1007/978-1-4939-8864-8_8.

Expression, Purification, and Activity Analysis of Chlorophyllide Oxidoreductase and Ni2+-Sirohydrochlorin a,c-Diamide Reductase.

Author information

1
Institut für Mikrobiologie, Technische Universität Braunschweig, Braunschweig, Germany. j.moser@tu-bs.de.
2
Institut für Mikrobiologie, Technische Universität Braunschweig, Braunschweig, Germany.
3
Institut für Biochemie, Universität Leipzig, Leipzig, Germany.
4
Institut für Pharmazeutische Wissenschaften, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany. gunhild.layer@pharmazie.uni-freiburg.de.

Abstract

Nitrogenase-like enzymes play a vital role in the reduction of the conjugated ring systems of diverse tetrapyrrole molecules. The biosynthesis of all bacteriochlorophylls involves the two-electron reduction of the C7-C8 double bond of the green pigment chlorophyllide, which is catalyzed by the nitrogenase-like two-component metalloenzyme chlorophyllide oxidoreductase (COR); whereas in all methanogenic microbes, another nitrogenase-like system, CfbC/D, is responsible for the sophisticated six-electron reduction of Ni2+-sirohydrochlorin a,c-diamide in the course of coenzyme F430 biosynthesis. The first part of this chapter describes the production and purification of the COR components (BchY/BchZ)2 and BchX2, the measurement of COR activity, and the trapping of the ternary COR complex; and the second part describes the strategy for obtaining homogenous and catalytically active preparations of CfbC2 and CfbD2 and a suitable method for extracting the reaction product Ni2+-hexahydrosirohydrochlorin a,c-diamide.

KEYWORDS:

Chlorophyll biosynthesis; Chlorophyllide oxidoreductase (COR); Coenzyme F430 biosynthesis; Dynamic switch protein; Ni2+-sirohydrochlorin a,c-diamide reductase (CfbC/D); Nitrogenase-like enzymes

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