Substrate Recognition of Nitrogenase-like Dark Operative Protochlorophyllide Oxidoreductase from Prochlorococcus marinus

Bröcker, Markus J.; Wätzlich, Denise; Uliczka, Frank; Virus, Simone; Saggu, Miguel; Lendzian, Friedhelm; Scheer, Hugo; Rüdiger, Wolfhart; Moser, Jürgen; Jahn, Dieter

doi:10.1074/jbc.m805206200

Cited by 31 publications

(59 citation statements)

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“…The activity of mutant DPOR proteins was analyzed as shown earlier (3). For the investigation of artificial substrate-reducing activities, all DPOR and nitrogenase assays were carried out as described earlier (9,13,30,31).…”

Section: Methodsmentioning

confidence: 99%

“…DPOR is a two-component metalloprotein comprising an ATP-dependent reductase (L 2 ) and a catalytic unit [(NB) 2 ], both sharing a substantial degree of structural and sequence identity with nitrogenase ( Fig. 1B) (3,4). As in nitrogenase, both components of DPOR carry redox active metallocenters (5)(6)(7)(8), which mediate the ATPdriven electron transfer from L 2 to the site of substrate reduction in (NB) 2 .…”

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confidence: 99%

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Structure of ADP-aluminium fluoride-stabilized protochlorophyllide oxidoreductase complex

Moser

Lange

Krausze

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Photosynthesis uses chlorophylls for the conversion of light into chemical energy, the driving force of life on Earth. During chlorophyll biosynthesis in photosynthetic bacteria, cyanobacteria, green algae and gymnosperms, dark-operative protochlorophyllide oxidoreductase (DPOR), a nitrogenase-like metalloenzyme, catalyzes the chemically challenging two-electron reduction of the fully conjugated ring system of protochlorophyllide a. The reduction of the C-17=C-18 double bond results in the characteristic ring architecture of all chlorophylls, thereby altering the absorption properties of the molecule and providing the basis for light-capturing and energytransduction processes of photosynthesis. We report the X-ray crystallographic structure of the substrate-bound, ADP-aluminium fluoride-stabilized (ADP·AlF 3 -stabilized) transition state complex between the DPOR components L 2 and (NB) 2 from the marine cyanobacterium Prochlorococcus marinus. Our analysis permits a thorough investigation of the dynamic interplay between L 2 and (NB) 2 . Upon complex formation, substantial ATP-dependent conformational rearrangements of L 2 trigger the protein-protein interactions with (NB) 2 as well as the electron transduction via redox-active [4Fe-4S] clusters. We also present the identification of artificial "small-molecule substrates" of DPOR in correlation with those of nitrogenase. The catalytic differences and similarities between DPOR and nitrogenase have broad implications for the energy transduction mechanism of related multiprotein complexes that are involved in the reduction of chemically stable double and/or triple bonds.dynamic switch protein | electron transfer T he biosynthesis of chlorophylls is essential for the capture of global energy. This complex, multienzymatic process generates chlorophyllide a (Chlide) through the stereospecific reduction of the C-17=C-18 double bond of ring D in protochlorophyllide a (Pchlide) (Fig. 1A). Two completely unrelated enzymes have evolved for Pchlide reduction: a monomeric, lightdependent system (1), found in angiosperms and cyanobacteria, and the dark-operative protochlorophyllide oxidoreductase (DPOR), found in anoxygenic photosynthetic bacteria, cyanobacteria, algae, and gymnosperms (2). DPOR is a two-component metalloprotein comprising an ATP-dependent reductase (L 2 ) and a catalytic unit [(NB) 2 ], both sharing a substantial degree of structural and sequence identity with nitrogenase ( Fig. 1B) (3, 4). As in nitrogenase, both components of DPOR carry redox active metallocenters (5-8), which mediate the ATPdriven electron transfer from L 2 to the site of substrate reduction in (NB) 2 . L 2 and (NB) 2 are only transiently associated with each other during catalysis (9), and ATP hydrolysis triggers their association and dissociation, permitting control of the timing of the accompanying electron transfer process between the two proteins. Previously determined structures of L 2 and (NB) 2 (5-7) provided a static picture of DPOR catalysis. However, only the structural investi...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Structure of ADP-aluminium fluoride-stabilized protochlorophyllide oxidoreductase complex

Moser

Lange

Krausze

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…Heterologous Production and Purification of P. marinus DPOR-P. marinus DPOR ChlL 2 and (ChlN/ChlB) 2 subcomplexes were recombinantly produced and purified under anaerobic conditions (oxygen partial pressure Ͻ1 ppm) in an anaerobic chamber (Coy Laboratories) as described earlier (18). The employed lysis buffer contained 100 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, and 10 mM MgCl 2 .…”

Section: Methodsmentioning

confidence: 99%

“…N-terminal GST tags fused to ChlN and ChlL were used for affinity chromatographic purification of both complexes using Protino glutathione-agarose (Macherey-Nagel). PreScission TM protease (GE Healthcare) treatment was employed to specifically elute ChlL 2 and (ChlN/ChlB) 2 from the resin, respectively (18).…”

Section: Methodsmentioning

confidence: 99%

“…In contrast, the second [4Fe-4S] cluster located on (ChlN/ChlB) 2 has no direct equivalent in nitrogenase, which carries the [8Fe-7S] P-cluster and the [1Mo-7Fe-9S-1X-1homocitrate] (Fe-Moco) metallo center instead (16,17). Substrate recognition by the DPOR subcomplex (ChlN/ ChlB) 2 tolerated minor modifications of the ring substituents on rings A, B, C, and E of Pchlide (18), whereas DPOR did not utilize substrate analogs with modifications at the catalytic target on ring D. Based on these results it was proposed that the active site of DPOR covers large parts of the macrocyclic substrate and is located in close proximity to the redox active [4Fe-4S] cluster.…”

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Biosynthesis of (Bacterio)chlorophylls

Bröcker

Wätzlich

Saggu

et al. 2010

Journal of Biological Chemistry

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Dark operative protochlorophyllide oxidoreductase (DPOR) catalyzes the light-independent two-electron reduction of protochlorophyllide a to form chlorophyllide a, the last common precursor of chlorophyll a and bacteriochlorophyll a biosynthesis. During ATP-dependent DPOR catalysis the homodimeric ChlL 2 subunit carrying a [4Fe-4S] cluster transfers electrons to the corresponding heterotetrameric catalytic subunit (ChlN/ChlB) 2 , which also possesses a redox active [4Fe-4S] cluster. To investigate the transient interaction of both subcomplexes and the resulting electron transfer reactions, the ternary DPOR enzyme holocomplex comprising subunits ChlN, ChlB, and ChlL from the cyanobacterium Prochlorococcus marinus was trapped as an octameric (ChlN/ChlB) 2 (ChlL 2 ) 2 complex after incubation with the nonhydrolyzable ATP analogs adenosine 5-(␥-thio)triphosphate, adenosine 5-(␤,␥-imido)triphosphate, or MgADP in combination with AlF 4 ؊ . Additionally, a mutant ChlL 2 protein, with a deleted Leu 153 in the switch II region also allowed for the formation of a stable octameric complex. Furthermore, efficient complex formation required the presence of protochlorophyllide. Electron paramagnetic resonance spectroscopy of ternary DPOR complexes revealed a reduced [4Fe-4S] cluster located on ChlL 2 , indicating that complete ATP hydrolysis is a prerequisite for intersubunit electron transfer. Circular dichroism spectroscopic experiments indicated nucleotide-dependent conformational changes for ChlL 2 after ATP binding. A nucleotide-dependent switch mechanism triggering ternary complex formation and electron transfer was concluded. From these results a detailed redox cycle for DPOR catalysis was deduced. Reduction of protochlorophyllide a (Pchlide)2 to chlorophyllide a (Chlide) is a central step in the biosynthesis of chlorophyll and bacteriochlorophyll (1). Two evolutionarily unrelated enzymes are capable of catalyzing the stereospecific two-electron reduction of the C17-C18 double bond of Pchlide (Fig. 1A) (2-4). Monomeric, light-dependent Pchlide oxidoreductase (POR; NADPH Pchlide oxidoreductase, EC 1.3.1.33) drives the NADPH-dependent reduction (5-7) of Pchlide bound in the active site via absorption of light energy. This light dependence of POR catalysis prevents angiosperms from synthesizing chlorophyll in the dark (8, 9). Anoxygenicphotosynthetic bacteria make use of a different ATP-dependent Pchlide reducing system, which is termed the light-independent, dark operative Pchlide oxidoreductase (DPOR), whereas gymnosperms, mosses, ferns, algae, and cyanobacteria utilize both POR and DPOR (3). In chlorophyll-synthesizing organisms DPOR is encoded by the chlN, chlB, and chlL genes (10 -12). The corresponding genes for bacteriochlorophyllsynthesizing organisms have been termed bchN, bchB, and bchL (10, 13). DPOR subunits ChlN, ChlB, and ChlL share significant amino acid sequence homologies with nitrogenase subunits NifD, NifK, and NifH, respectively (12,14).In

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Dark‐Operative Protochlorophyllide Oxidoreductase

Moser

Schubert

2012

Encyclopedia of Inorganic and Bioinorganic Chemistry

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Dark‐operative (or light‐independent) protochlorophyllide oxidoreductase is an enzyme of the chlorophyll and bacteriochlorophyll biosynthetic pathway catalyzing the reduction of protochlorophyllide to chlorophyllide. DPOR catalysis involves the ATP‐dependent, two‐electron reduction and trans‐hydrogenation of the C17–C18 double bond of the conjugated D ring system of protochlorophyllide converting the pheoporphyrin backbone of protochlorophyllide to a chlorin macrocycle characteristic of all chlorophylls. The process is mediated by the dynamic interaction of two DPOR subcomplexes, each carrying redox active 4Fe–4S clusters. We discuss all available structural and biochemical data on this enzyme and compare it to nitrogenase and other related enzyme systems.

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Substrate Recognition of Nitrogenase-like Dark Operative Protochlorophyllide Oxidoreductase from Prochlorococcus marinus

Abstract: Protochlorophyllide (Pchlide)2 a is a metabolite during chlorophyll and bacteriochlorophyll biosynthesis. Two distinct strategies have evolved for the stereo-and regioselective two electron reduction of ring D of Pchlide a to form chlorophyllide (Chlide) a (Fig.

Cited by 31 publications

References 59 publications

Structure of ADP-aluminium fluoride-stabilized protochlorophyllide oxidoreductase complex

Structure of ADP-aluminium fluoride-stabilized protochlorophyllide oxidoreductase complex

Biosynthesis of (Bacterio)chlorophylls

Dark‐Operative Protochlorophyllide Oxidoreductase

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