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

  • Jürgen Moser
  • Jan Jasper
  • José Vazquez Ramos
  • Sven T. Sowa
  • Gunhild Layer
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1876)

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.

Key words

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

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Institut für MikrobiologieTechnische Universität BraunschweigBraunschweigGermany
  2. 2.Institut für BiochemieUniversität LeipzigLeipzigGermany
  3. 3.Institut für Pharmazeutische WissenschaftenAlbert-Ludwigs-Universität FreiburgFreiburgGermany

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