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.
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References
Kiesel S, Watzlich D, Lange C et al (2015) Iron–sulfur cluster-dependent catalysis of chlorophyllide a oxidoreductase from Roseobacter denitrificans. J Biol Chem 290:1141–1154
Watzlich D, Brocker MJ, Uliczka F et al (2009) Chimeric nitrogenase-like enzymes of (bacterio)chlorophyll biosynthesis. J Biol Chem 284:15530–15540
Takahashi Y, Nakamura M (1999) Functional assignment of the ORF2-iscS-iscU-iscA-hscB-hscA-fdx-ORF3 gene cluster involved in the assembly of Fe-S clusters in Escherichia coli. J Biochem 126:917–926
Brocker MJ, Virus S, Ganskow S et al (2008) ATP-driven reduction by dark-operative protochlorophyllide oxidoreductase from Chlorobium tepidum mechanistically resembles nitrogenase catalysis. J Biol Chem 283:10559–10567
Muller AH, Gough SP, Bollivar DW et al (2011) Methods for the preparation of chlorophyllide a: an intermediate of the chlorophyll biosynthetic pathway. Anal Biochem 419:271–276
Bollivar DW, Suzuki JY, Beatty JT et al (1994) Directed mutational analysis of bacteriochlorophyll a biosynthesis in Rhodobacter capsulatus. J Mol Biol 237:622–640
Moore SJ, Sowa ST, Schuchardt C et al (2017) Elucidation of the biosynthesis of the methane catalyst coenzyme F430. Nature 543:78–82
Pfaltz A, Livingston DA, Jaun B et al (1985) Zur Kenntnis des Faktors F430 aus methanogenen Bakterien: Über die Natur der Isolierungsartefakte von F430, ein Beitrag zur Chemie von F430 und zur konformationellen Stereochemie der Ligandperipherie von hydroporphinoiden Nickel(II)-Komplexen. Helv Chim Acta 68:1338–1358
Walther J, Bröcker MJ, Wätzlich D et al (2009) Protochlorophyllide: a new photosensitizer for the photodynamic inactivation of gram-positive and gram-negative bacteria. FEMS Microbiol Lett 290:156–163
Nomata J, Mizoguchi T, Tamiaki H, Fujita Y (2006) A second nitrogenase-like enzyme for bacteriochlorophyll biosynthesis: reconstitution of chlorophyllide a reductase with purified X-protein (BchX) and YZ-protein (BchY-BchZ) from Rhodobacter capsulatus. J Biol Chem 281:15021–15028
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Moser, J., Jasper, J., Ramos, J.V., Sowa, S.T., Layer, G. (2019). Expression, Purification, and Activity Analysis of Chlorophyllide Oxidoreductase and Ni2+-Sirohydrochlorin a,c-Diamide Reductase. In: Hu, Y. (eds) Metalloproteins. Methods in Molecular Biology, vol 1876. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8864-8_8
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DOI: https://doi.org/10.1007/978-1-4939-8864-8_8
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