Radical SAM Enzymes Involved in Tetrapyrrole Biosynthesis and Insertion

Abstract

The anaerobic biosyntheses of heme, heme d ₁, and bacteriochlorophyll all require the action of radical SAM enzymes. During heme biosynthesis in some bacteria, coproporphyrinogen III dehydrogenase (CgdH) catalyzes the decarboxylation of two propionate side chains of coproporphyrinogen III to the corresponding vinyl groups of protoporphyrinogen IX. Its solved crystal structure was the first published structure for a radical SAM enzyme. In bacteria, heme is inserted into enzymes by the cytoplasmic heme chaperone HemW, a radical SAM enzyme structurally highly related to CgdH. In an alternative heme biosynthesis route found in archaea and sulfate-reducing bacteria, the two radical SAM enzymes AhbC and AhbD catalyze the removal of two acetate groups (AhbC) or the decarboxylation of two propionate side chains (AhbD). NirJ, a close homologue of AhbC, is required for propionate side chain removal during the formation of heme d ₁ in some denitrifying bacteria. Biosynthesis of the fifth ring (ring E) of all chlorophylls is based on an unusual six-electron oxidative cyclization step. The sophisticated conversion of Mg-protoporphyrin IX monomethylester to protochlorophyllide is facilitated by an oxygen-independent cyclase termed BchE, which is a cobalamin-dependent radical SAM enzyme. Most of the radical SAM enzymes involved in tetrapyrrole biosynthesis were recognized as such by Sofia et al. in 2001 (Nucleic Acids Res.2001, 29, 1097-1106) and were biochemically characterized thereafter. Although much has been achieved, the challenging tetrapyrrole substrates represent a limiting factor for enzyme/substrate cocrystallization and the ultimate elucidation of the corresponding enzyme mechanisms.

Figure 1

Overview of the branched tetrapyrrole biosynthesis pathway. Radical SAM reaction steps are highlighted by the respective enzyme names in violet. Note that the CgdH step is also carried out by CgdC in many organisms, and the AhbD-like step is also catalyzed by ChdC, both under aerobic conditions (see also main text). Arrows with solid lines represent one reaction step, and arrows with dashed lines indicate more than one reaction.

Figure 2

Currently known routes to heme and heme d₁. Radical SAM reactions are shown with chemical structures and are highlighted in pink. Note that the CgdH step is also carried out by CgdC in many organisms, and the AhbD-like step is also catalyzed by ChdC, both under aerobic conditions (see also main text).

Figure 3

Crystal structure of CgdH from E. coli and catalytic mechanism. The partial TIM-barrel is shown in pale cyan, and the additional structural elements are depicted in green. On the right, the cofactor arrangement within the CgdH structure is shown in detail, with labels for both SAM molecules and the cysteine ligands coordinating the [4Fe–4S] cluster. The catalytic mechanism shown at the bottom is described in the main text.

Figure 4

Model for the activity of the heme chaperone HemW. Heme b from heme biosynthesis (final enzyme protoporphyrin IX ferrochelatase, PpfC) is most likely transferred via bacterioferritin (BfrB, green) to the heme chaperone HemW (blue). Subsequently, the [4Fe–4S] cluster-containing HemW dimerizes and localizes to the membrane, where it interacts with its target protein NarI (yellow), the heme-containing subunit of the respiratory nitrate reductase NarGHI. After heme incorporation into apo-NarI, the holo-NarGHI is able to catalyze the reduction of nitrate to nitrite. PgoX = protoporphyrinogen IX oxidase.

Figure 5

Proposed reaction intermediates of BchE catalysis. The synthesis of the fifth ring (ring E) of bacteriochlorophyll is formally described as oxidative six-electron (e^–) cyclization. R stands for ethyl or vinyl.

Figure 6

Two potential mechanisms for the cobalamin-dependent catalysis of BchE (left and right trace). The left-hand reaction sequence involves the attack of the hydroxyl group of hydroxocobalamin and proceeds via the geminal diol 2. The right-hand mechanism has no direct cobalamin involvement and includes the formation of the enol 5.