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.

Structure of ADP-aluminium fluoride-stabilized protochlorophyllide oxidoreductase complex

“…S1-S3). Interestingly, the amino acid residues themselves are not conserved between the two enzymes, which may be nature's design to prevent deleterious "cross-talks" between different biological systems (7,13). The spatial distributions of the protein surfaces of L 2 and NifH 2 that are involved in the binding of (NB) 2 and (NifDK) 2 , respectively, are largely conserved between DPOR and nitrogenase ( Fig.…”

“…by nitrogenase contained 1.5 mg NifH 2 and either 0.15 mg (NifDK) 2 or 0.15 mg apo (NifDK) 2 variant in a total volume of 1 mL, and assays of CO reduction by nitrogenase contained 20 mg NifH and either 3 mg (NifDK) 2 or 3 mg apo (NifDK) 2 variant in a total volume of 1 mL. The products of these assays were analyzed as described previously (13,15,30,31). Apo (NifDK) 2 variant was generated by deletion of nifH, which results in a (NifDK) 2 2 , whereas apo nitrogenase variant consists of NifH 2 and apo (NifDK) 2 variant.…”

“…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). The substrate concentrations are detailed in Table 1.…”

Biosynthesis of (Bacterio)chlorophylls

“…DPOR Enzyme Assay-The standard DPOR activity assay containing 13 M Pchlide, 2 mM dithionite, as an artificial electron donor, 2 mM ATP, and an ATP regenerating system was performed and analyzed as described earlier in the presence of 100 pmol of purified (ChlN/ChlB) 2 and 200 pmol of ChlL 2 (125 l final volume) (15,18).…”

“…Hydrolysis of two ATP molecules by dimeric subunit ChlL 2 facilitates electron transfer to a second [4Fe-4S] cluster located on the heterotetrameric complex (ChlN/ChlB) 2 . Transient interaction of ChlL 2 and (ChlN/ ChlB) 2 of ChlL was proposed (15). This initial step of DPOR catalysis was shown to resemble the corresponding electron transfer reactions in nitrogenase catalysis.…”