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

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

Departments of ^*Physiology and ^‡Chemistry, Carlsberg Laboratory, Gamle Carlsberg Vej 10, 2500 Valby, Denmark

Communicated by Diter von Wettstein, Washington State University, Pullman, WA (received for review January 20, 2000)

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 B₁₂ (B₁₂)-requiring mutants of the bluE and bluB genes of Rhodobacter capsulatus, grown without B₁₂, 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 B₁₂-dependent mutants required B₁₂ 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 13¹-radical of MPE. Withdrawal of an electron gives the 13¹-cation of MPE. Hydroxyl ion attack on the cation gives 13¹-hydroxy-MPE. Withdrawal of three hydrogen atoms leads successively to 13¹-keto-MPE, its 13²-radical, and cyclization to PChlide.

Footnotes

↵ † To whom reprint requests should be sent at present address: Nyelandsvej 14, St., tv, 2000 Frederiksberg, Denmark. E-mail: gough{at}biobase.dk.
Abbreviations:

B12,

vitamin B12;

AdCob,

adenosylcobalamin;

MP,

Mg-protoporphyrin;

MDE,

Mg-protoporphyrin dimethyl ester;

MPE,

Mg-protoporphyrin monomethyl ester;

mvMPE,

3-vinyl-8-ethyl-MPE;

PChlide,

protochlorophyllide;

BChl,

bacteriochlorophyll;

LDI-TOF MS,

laser desorption/ionization time-of-flight mass spectrometry;

ANCOPROX,

anaerobic coproporphyrinogen oxidase