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. 2000 Jun 6;97(12):6908-13.
doi: 10.1073/pnas.97.12.6908.

Anaerobic chlorophyll isocyclic ring formation in Rhodobacter capsulatus requires a cobalamin cofactor

S P Gough  1 , B O Petersen, J O Duus
Affiliations

Anaerobic chlorophyll isocyclic ring formation in Rhodobacter capsulatus requires a cobalamin cofactor

S P Gough et al. Proc Natl Acad Sci U S A. .

Abstract

The isocyclic ring of bacteriochlorophyll (BChl) is formed by the conversion of Mg-protoporphyrin monomethyl ester (MPE) to protochlorophyllide (PChlide). Similarities revealed by blast searches with the putative anaerobic MPE-cyclase BchE suggested to us that this protein also uses a cobalamin cofactor. We found that vitamin B(12) (B(12))-requiring mutants of the bluE and bluB genes of Rhodobacter capsulatus, grown without B(12), accumulated Mg-porphyrins. Laser desorption/ionization time-of-flight (LDI-TOF) MS and NMR spectroscopy identified them as MPE and its 3-vinyl-8-ethyl (mvMPE) derivative. An in vivo assay was devised for the cyclase converting MPE to PChlide. Cyclase activity in the B(12)-dependent mutants required B(12) but not protein synthesis. The following reaction mechanism is proposed for this MPE-cyclase reaction. Adenosylcobalamin forms the adenosyl radical, which leads to withdrawal of a hydrogen atom and formation of the benzylic-type 13(1)-radical of MPE. Withdrawal of an electron gives the 13(1)-cation of MPE. Hydroxyl ion attack on the cation gives 13(1)-hydroxy-MPE. Withdrawal of three hydrogen atoms leads successively to 13(1)-keto-MPE, its 13(2)-radical, and cyclization to PChlide.

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Figures

Figure 1
Figure 1
Some intermediates of the BChl and cobalamin biosynthetic pathway. The role of the MPE-cyclase is shown.
Figure 2
Figure 2
Pileup of BchE homologs and P-methylase. Organism, accession no., Swiss Prot or other database name, and Ψ-blast E values (exponential notation) with C. tepidum BchE as search profile (7 iterations) are shown at lower right. ANCOPROX, anaerobic coproporphyrinogen oxidase.
Figure 3
Figure 3
The down-field region of the 1H NMR spectra of the MPE (major component) and mvMPE (minor component) from the bluE mutant and of Mg-protoporphyrin and its diester (MDE). The meso protons are indicated for the peaks.
Figure 4
Figure 4
Percent growth, BChl formation, and MPE content as a function of B12 concentration in cultures of B12-deficient mutants bluE and bluB. Growth (shown as percent of maximal growth observed, OD700 = 2.4) was for 26 h in the dark from inocula sizes predetermined to cause MPE accumulation.
Figure 5
Figure 5
(A) PChlide formation induced by B12 in B12-deficient bluE cells. Absorption spectra of extracts of bluE cells (inoculum size 10 μl), grown to OD700 = 1.7, in 50 ml. Nicotinamide (10 mM) and 34 μg⋅ml−1 chloramphenicol (CAP) were added, followed by incubation for 6 h, with or without 5 μM B12. (B) Fluorescence emission spectra of extracts of cells from in vivo cyclase assays of wild-type R. capsulatus. FU, fluorescence units. The assays were performed with and without 10 mM nicotinamide with 8 μM MPE in 2.8 μl of DMSO. (C) Fluorescence emission spectra of cells from in vivo cyclase assays of bluE mutant. Chloramphenicol, 34 μg⋅ml−1; adenosylcobalamin (AdCob), 15 μM; B12, 15 μM; nicotinamide, 10 mM.
Figure 6
Figure 6
Proposed radical reaction mechanism for an AdCob-dependent anaerobic MPE-cyclase.
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