The Radical SAM enzyme NirJ catalyzes the removal of two propionate side chains during heme d₁ biosynthesis

Abstract

Heme d is a modified tetrapyrrole playing an important role in denitrification by acting as the catalytically essential cofactor in the cytochrome cd nitrite reductase of many denitrifying bacteria. In the course of heme d biosynthesis, the two propionate side chains on pyrrole rings A and B of the intermediate 12,18-didecarboxysiroheme are removed from the tetrapyrrole macrocycle. In the final heme d molecule, the propionate groups are replaced by two keto functions. Although it was speculated that the Radical S-adenosyl-l-methionine (SAM) enzyme NirJ might be responsible for the removal of the propionate groups and introduction of the keto functions, this has not been shown experimentally, so far. Here, we demonstrate that NirJ is a Radical SAM enzyme carrying two iron-sulfur clusters. While the N-terminal [4Fe-4S] cluster is essential for the initial SAM cleavage reaction, it is not required for substrate binding. NirJ tightly binds its substrate 12,18-didecarboxysiroheme and, thus, can be purified in complex with the substrate. By using the purified NirJ/substrate complex in an in vitro enzyme activity assay, we show that NirJ indeed catalyzes the removal of the two propionate side chains under simultaneous SAM cleavage. However, under the reaction conditions employed, no keto group formation is observed indicating that an additional cofactor or enzyme is needed for this reaction.

Crystal structure of NirF: Insights into its role in hemed1biosynthesis

“…Alternatively, a role of NirF as a biosynthetic enzyme could be envisaged, potentially involved in the introduction of the carbonyl functions at C3 and C8 of rings A and B. While it has been shown that the cytoplasmic Radical SAM enzyme NirJ removes the two propionate moieties at C3 and C8 of 12,18-didecarboxysiroheme (DDSH) [17] and that the periplasmic NirN converts the propionate at C17 to an acrylate group in the final step of the pathway [18,17], the formation of the carbonyl functions at C3 and C8 is still enigmatic. Although it was speculated that NirJ might also be responsible for the oxidation of these position, the installation of the carbonyls by NirJ has not been observed experimentally so far [17,31].…”

“…The first steps of heme d1 biosynthesis are the conversion of uroporphyrinogen III to 12,18didecarboxysiroheme (DDSH) via percorrin-2, sirohydrochlorin and siroheme, successively catalyzed by NirE, CysG (in some organisms) and NirDLGH (Figure 1) [13][14][15][16]. The intermediate DDSH is further converted by the Radical SAM enzyme NirJ, which sequentially removes the propionate groups from pyrrole rings A and B [17]. Based on mass spectrometry, a tetrapyrrole with two γ-lactones involving the acetate groups at C2 and C7 has been observed as the final product of this reaction, but these lactones may be artifacts of the extraction procedure and alternatives such as hydroxyl groups or hydrogen replacing the propionate chains were proposed [17].…”

“…The intermediate DDSH is further converted by the Radical SAM enzyme NirJ, which sequentially removes the propionate groups from pyrrole rings A and B [17]. Based on mass spectrometry, a tetrapyrrole with two γ-lactones involving the acetate groups at C2 and C7 has been observed as the final product of this reaction, but these lactones may be artifacts of the extraction procedure and alternatives such as hydroxyl groups or hydrogen replacing the propionate chains were proposed [17]. Importantly, the generation of dihydro-heme d1 (dd1), the last intermediate of heme d1 biosynthesis and substrate of the oxidoreductase NirN, was not observed in experiments with NirJ, suggesting that dd1 may be produced by NirF, the only remaining less well characterized protein encoded in the nir-operon [18,17].…”

Carbon isotopic heterogeneity of coenzyme F430 and membrane lipids in methane‐oxidizing archaea

“…Regardless of how glutamate is formed, tetrapyrrole biosynthesis proceeds by combining eight glutamate molecules into the 40‐carbon uroporphyrinogen III. Two additional methyl carbons are added to uroporphyrinogen III from methionine, via a S ‐adenosyl methionine (SAM) reaction to form precorrin‐2 (Boss, Oehme, Billig, Birkemeyer, & Layer, ; Singh et al, ; Storbeck et al, , ; Zajicek et al, ). These methyl groups likely come from methyl‐H 4 MPT which delivers the methyl group to homocysteine to form methionine.…”

“…The oxidative pathway in class II methanogens produces 2-oxoglutarate with this ratio at 2:3. Birkemeyer, & Layer, 2017;Singh et al, 2016;Storbeck et al, 2011Storbeck et al, , 2009Zajicek et al, 2009). These methyl groups likely come from methyl-H 4 MPT which delivers the methyl group to homocysteine to form methionine.…”