title: Chimeric nitrogenase-like enzymes of (bacterio)chlorophyll biosynthesis.
abstract: Nitrogenase-like light-independent protochlorophyllide oxidoreductase (DPOR) is involved in chlorophyll biosynthesis. Bacteriochlorophyll formation additionally requires the structurally related chlorophyllide oxidoreductase (COR). During catalysis, homodimeric subunit BchL(2) or ChlL(2) of DPOR transfers electrons to the corresponding heterotetrameric catalytic subunit, (BchNB)(2) or (ChlNB)(2). Analogously, subunit BchX(2) of the COR enzymes delivers electrons to subunit (BchYZ)(2). Various chimeric DPOR enzymes formed between recombinant subunits (BchNB)(2) and BchL(2) from Chlorobaculum tepidum or (ChlNB)(2) and ChlL(2) from Prochlorococcus marinus and Thermosynechococcus elongatus were found to be enzymatically active, indicating a conserved docking surface for the interaction of both DPOR protein subunits. Biotin label transfer experiments revealed the interaction of P. marinus ChlL(2) with both subunits, ChlN and ChlB, of the (ChlNB)(2) tetramer. Based on these findings and on structural information from the homologous nitrogenase system, a site-directed mutagenesis approach yielded 10 DPOR mutants for the characterization of amino acid residues involved in protein-protein interaction. Surface-exposed residues Tyr(127) of subunit ChlL, Leu(70) and Val(107) of subunit ChlN, and Gly(66) of subunit ChlB were found essential for P. marinus DPOR activity. Next, the BchL(2) or ChlL(2) part of DPOR was exchanged with electron-transferring BchX(2) subunits of COR and NifH(2) of nitrogenase. Active chimeric DPOR was generated via a combination of BchX(2) from C. tepidum or Roseobacter denitrificans with (BchNB)(2) from C. tepidum. No DPOR activity was observed for the chimeric enzyme consisting of NifH(2) from Azotobacter vinelandii in combination with (BchNB)(2) from C. tepidum or (ChlNB)(2) from P. marinus and T. elongatus, respectively.
authors.affiliation: Institute of Microbiology, Technische Universität Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany.
authors.forename: Denise
authors.initials: D
authors.lastname: Wätzlich
authors.forename: Markus J
authors.initials: MJ
authors.lastname: Bröcker
authors.forename: Frank
authors.initials: F
authors.lastname: Uliczka
authors.forename: Markus
authors.initials: M
authors.lastname: Ribbe
authors.forename: Simone
authors.initials: S
authors.lastname: Virus
authors.forename: Dieter
authors.initials: D
authors.lastname: Jahn
authors.forename: Jürgen
authors.initials: J
authors.lastname: Moser