Heme biosynthesis in prokaryotes Layer, Gunhild
Biochimica et biophysica acta. Molecular cell research,
January 2021, 2021-01-00, 20210101, Letnik:
1868, Številka:
1
Journal Article
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The cyclic tetrapyrrole heme is used as a prosthetic group in a broad variety of different proteins in almost all organisms. Often, it is essential for vital biochemical processes such as aerobic and ...anaerobic respiration as well as photosynthesis. In Nature, heme is made from the common tetrapyrrole precursor 5-aminolevulinic acid, and for a long time it was assumed that heme is biosynthesized by a single, common pathway in all organisms. However, although this is indeed the case in eukaryotes, heme biosynthesis is more diverse in the prokaryotic world, where two additional pathways exist. The final elucidation of the two ‘alternative’ heme biosynthesis routes operating in some bacteria and archaea was achieved within the last decade. This review summarizes the three different heme biosynthesis pathways with a special emphasis on the two ‘new’ prokaryotic routes.
•Three distinct pathways for heme biosynthesis in prokaryotes•Novel insights into structure and mechanisms of heme biosynthesis enzymes•Relationship of biosynthesis routes to heme, siroheme and heme d1
Tetrapyrroles like hemes, chlorophylls, and cobalamin are complex macrocycles which play essential roles in almost all living organisms. Heme serves as prosthetic group of many proteins involved in ...fundamental biological processes like respiration, photosynthesis, and the metabolism and transport of oxygen. Further, enzymes such as catalases, peroxidases, or cytochromes P450 rely on heme as essential cofactors. Heme is synthesized in most organisms via a highly conserved biosynthetic route. In humans, defects in heme biosynthesis lead to severe metabolic disorders called porphyrias. The elucidation of the 3D structures for all heme biosynthetic enzymes over the last decade provided new insights into their function and elucidated the structural basis of many known diseases. In terms of structure and function several rather unique proteins were revealed such as the V‐shaped glutamyl‐tRNA reductase, the dipyrromethane cofactor containing porphobilinogen deaminase, or the “Radical SAM enzyme” coproporphyrinogen III dehydrogenase. This review summarizes the current understanding of the structure–function relationship for all heme biosynthetic enzymes and their potential interactions in the cell.
Abstract
The biological formation of methane (methanogenesis) is a globally important process, which is exploited in biogas technology, but also contributes to global warming through the release of a ...potent greenhouse gas into the atmosphere. The last and methane-releasing step of methanogenesis is catalysed by the enzyme methyl-coenzyme M reductase (MCR), which carries several exceptional posttranslational amino acid modifications. Among these, a 5-C-(
S
)-methylarginine is located close to the active site of the enzyme. Here, we show that a unique Radical
S
-adenosyl-L-methionine (SAM) methyltransferase is required for the methylation of the arginine residue. The gene encoding the methyltransferase is currently annotated as “methanogenesis marker 10” whose function was unknown until now. The deletion of the methyltransferase gene
ma4551
in
Methanosarcina acetivorans
WWM1 leads to the production of an active MCR lacking the C-5-methylation of the respective arginine residue. The growth behaviour of the corresponding
M
.
acetivorans
mutant strain and the biophysical characterization of the isolated MCR indicate that the methylated arginine is important for MCR stability under stress conditions.
The heme synthase AhbD catalyzes the last step of the siroheme-dependent heme biosynthesis pathway, which is operative in archaea and sulfate-reducing bacteria. The AhbD-catalyzed reaction consists ...of the oxidative decarboxylation of two propionate side chains of iron-coproporphyrin III to the corresponding vinyl groups of heme b . AhbD is a Radical SAM enzyme employing radical chemistry to achieve the decarboxylation reaction. Previously, it was proposed that the central iron ion of the substrate iron-coproporphyrin III participates in the reaction by enabling electron transfer from the initially formed substrate radical to an iron-sulfur cluster in AhbD. In this study, we investigated the substrate radical that is formed during AhbD catalysis. While the iron-coproporphyrinyl radical was not detected by electron paramagnetic resonance (EPR) spectroscopy, trapping and visualization of the substrate radical was successful by employing substrate analogs such as coproporphyrin III and zinc-coproporphyrin III. The radical signals detected by EPR were analyzed by simulations based on density functional theory (DFT) calculations. The observed radical species on the substrate analogs indicate that hydrogen atom abstraction takes place at the β-position of the propionate side chain and that an electron donating ligand is located in proximity to the central metal ion of the porphyrin.
Methionine synthases are essential enzymes for amino acid and methyl group metabolism in all domains of life. Here, we describe a putatively anciently derived type of methionine synthase yet unknown ...in bacteria, here referred to as core-MetE. The enzyme appears to represent a minimal MetE form and transfers methyl groups from methylcobalamin instead of methyl-tetrahydrofolate to homocysteine. Accordingly, it does not possess the tetrahydrofolate binding domain described for canonical bacterial MetE proteins. In Dehalococcoides mccartyi strain CBDB1, an obligate anaerobic, mesophilic, slowly growing organohalide-respiring bacterium, it is encoded by the locus cbdbA481. In line with the observation to not accept methyl groups from methyl-tetrahydrofolate, all known genomes of bacteria of the class Dehalococcoidia lack metF encoding for methylene-tetrahydrofolate reductase synthesizing methyl-tetrahydrofolate, but all contain a core-metE gene. We heterologously expressed core-MetE
in E. coli and purified the 38 kDa protein. Core-MetE
exhibited Michaelis-Menten kinetics with respect to methylcob(III)alamin (K
≈ 240 µM) and L-homocysteine (K
≈ 50 µM). Only methylcob(III)alamin was found to be active as methyl donor with a k
≈ 60 s
. Core-MetE
did not functionally complement metE-deficient E. coli strain DH5α (ΔmetE::kan) suggesting that core-MetE
and the canonical MetE enzyme from E. coli have different enzymatic specificities also in vivo. Core-MetE appears to be similar to a MetE-ancestor evolved before LUCA (last universal common ancestor) using methylated cobalamins as methyl donor whereas the canonical MetE consists of a tandem repeat and might have evolved by duplication of the core-MetE and diversification of the N-terminal part to a tetrahydrofolate-binding domain.
Methane biogenesis in methanogens is mediated by methyl-coenzyme M reductase, an enzyme that is also responsible for the utilization of methane through anaerobic methane oxidation. The enzyme uses an ...ancillary factor called coenzyme Fsub.430, a nickel-containing modified tetrapyrrole that promotes catalysis through a methyl radical/Ni(ii)-thiolate intermediate. However, it is unclear how coenzyme Fsub.430 is synthesized from the common primogenitor uroporphyrinogen iii, incorporating 11 steric centres into the macrocycle, although the pathway must involve chelation, amidation, macrocyclic ring reduction, lactamization and carbocyclic ring formation. Here we identify the proteins that catalyse the biosynthesis of coenzyme Fsub.430 from sirohydrochlorin, termed CfbACfbE, and demonstrate their activity. The research completes our understanding of how the repertoire of tetrapyrrole-based pigments are constructed, permitting the development of recombinant systems to use these metalloprosthetic groups more widely.
In archaea and sulfate-reducing bacteria, heme is synthesized via the siroheme-dependent pathway. The last step of this route is catalyzed by the Radical SAM enzyme AhbD and consists of the ...conversion of iron-coproporphyrin III into heme. AhbD belongs to the subfamily of Radical SAM enzymes containing a SPASM/Twitch domain carrying either one or two auxiliary iron–sulfur clusters in addition to the characteristic Radical SAM cluster. In previous studies, AhbD was reported to contain one auxiliary 4Fe-4S cluster. In this study, the amino acid sequence motifs containing conserved cysteine residues in AhbD proteins from different archaea and sulfate-reducing bacteria were reanalyzed. Amino acid sequence alignments and computational structural models of AhbD suggested that a subset of AhbD proteins possesses the full SPASM motif and might contain two auxiliary iron–sulfur clusters (AuxI and AuxII). Therefore, the cluster content of AhbD from Methanosarcina barkeri was studied using enzyme variants lacking individual clusters. The purified enzymes were analyzed using UV/Visible absorption and EPR spectroscopy as well as iron/sulfide determinations showing that AhbD from M. barkeri contains two auxiliary 4Fe-4S clusters. Heme synthase activity assays suggested that the AuxI cluster might be involved in binding the reaction intermediate and both clusters potentially participate in electron transfer.
Heme d1 is a modified tetrapyrrole playing an important role in denitrification by acting as the catalytically essential cofactor in the cytochrome cd1 nitrite reductase of many denitrifying ...bacteria. In the course of heme d1 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 d1 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.
The heme d1 biosynthesis enzyme NirJ belongs to the Radical SAM protein family and binds two iron–sulfur clusters. One of the clusters is required for the typical SAM cleavage reaction. Overall NirJ catalyzes the removal of two propionate side chains from the substrate 12,18‐didecarboxysiroheme.
Iron-sulfur (Fe-S) clusters are key metal cofactors of metabolic, regulatory, and stress response proteins in most organisms. The unique properties of these clusters make them susceptible to ...disruption by iron starvation or oxidative stress. Both iron and sulfur can be perturbed under stress conditions, leading to Fe-S cluster defects. Bacteria and higher plants contain a specialized system for Fe-S cluster biosynthesis under stress, namely the Suf pathway. In Escherichia coli the Suf pathway consists of six proteins with functions that are only partially characterized. Here we describe how the SufS and SufE proteins interact with the SufBCD protein complex to facilitate sulfur liberation from cysteine and donation for Fe-S cluster assembly. It was previously shown that the cysteine desulfurase SufS donates sulfur to the sulfur transfer protein SufE. We have found here that SufE in turn interacts with the SufB protein for sulfur transfer to that protein. The interaction occurs only if SufC is present. Furthermore, SufB can act as a site for Fe-S cluster assembly in the Suf system. This provides the first evidence of a novel site for Fe-S cluster assembly in the SufBCD complex.
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