Functional Diversity of HemN-like Proteins

Abstract

HemN is a radical S-adenosylmethionine (SAM) enzyme that catalyzes the anaerobic oxidative decarboxylation of coproporphyrinogen III to produce protoporphyrinogen IX, a key intermediate in heme biosynthesis. Proteins homologous to HemN (HemN-like proteins) are widespread in both prokaryotes and eukaryotes. Although these proteins are in most cases annotated as anaerobic coproporphyrinogen III oxidases (CPOs) in the public database, many of them are actually not CPOs but have diverse functions such as methyltransferases, cyclopropanases, heme chaperones, to name a few. This Perspective discusses the recent advances in the understanding of HemN-like proteins, and particular focus is placed on the diverse chemistries and functions of this growing protein family.

Figure 1

HemN binds two SAM molecules (PDB 1OLT). (A) Active site of HemN that binds (S)-SAM (∼60% occupancy). (B) Active site of HemN that binds (R)-SAM (∼40% occupancy). The distances from the SAM1 C5′ atom to the sulfur and methyl carbon of SAM2 are shown in brown and blue, respectively.

Figure 2

Catalytic mechanism of HemN. (A) Common pathway of HemN-like enzymes in the generation of a SAM-based methylene radical intermediate 1. (B) Mechanism of HemN-catalyzed oxidative decarboxylation reaction. (C) Working hypothesis for the detailed HemN catalysis. The [4Fe-4S] cluster is represented as a cube, and the porphyrinogen ring is represented as a purple diamond. P and V represent propionate and vinyl moieties, respectively. 2-I, 2-II, 2-III, and 2-IV are coproporphyrinogen III (substrate), mono-decarboxylated product (harderoporphyrinogen), protoporphyrinogen IX (di-decarboxylated product), and the SAM adduct of the mono-decarboxylated product, respectively.

Figure 3

HemN-catalyzed homolytic substitution (S_H) reactions. The reaction occurs on SAM and the sulfoxide analogueue of SAM (SAHO) but not on the sulfone analogueue of SAM (SAHO₂).

Figure 4

Common paradigm in the catalysis of the class C RSMs. The sp²-hydridized carbons are highlighted by a light blue ellipse.

Figure 5

Methylation reactions catalyzed by the class C RSMs. (A) Consecutive methylation of menaquinone. (B) Thiazole methylation by TbtI in the biosynthesis of thiomuracin A1.

Figure 6

NosN-mediated biosynthesis of the nosiheptide indolyl ring.

Figure 7

Cyclopropanation reactions catalyzed by the class C RSMs. (A) The structure of spirocyclopropylcyclohexadienone family compounds. 7-I is a metabolite produced by the ytkT-knockout mutant, and 7-II is produced in vitro in the YtkT assay. (B) Cyclopropanation in CC-1065 biosynthesis. (C) Cyclopropanation in jawsamycin biosynthesis.

Figure 8

Anaerobic degradation of heme by ChuW or HutW. The solvent-derived hydrogen atom is shown in blue.

Figure 9

Bayesian MCMC phylogeny of Hem-like proteins. The major clades are shown in different colors, and the functionally characterized proteins are shown along the branches. Bayesian inferences of posterior probabilities (PPs) are indicated by the filled circles with different colors. It is worth noting that in some literature HemZ is annotated as a HemN-homologous protein,^, while it is sometimes also annotated as HemH and CpfC, and the latter enzymes are not radical SAM enzymes and not HemN-homologous. Caution should be taken for these different nomenclature systems. See the Supporting Information for the detailed information on the sequences discussed in this Perspective.