Radical S-adenosylmethionine enzyme coproporphyrinogen III oxidase HemN: functional features of the [4Fe-4S] cluster and the two bound S-adenosyl-L-methionines (original) (raw)
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Crystal structure of coproporphyrinogen III oxidase reveals cofactor geometry of Radical SAM enzymes
The EMBO Journal, 2003
Radical SAM' enzymes generate catalytic radicals by combining a 4Fe±4S cluster and S-adenosylmethionine (SAM) in close proximity. We present the ®rst crystal structure of a Radical SAM enzyme, that of HemN, the Escherichia coli oxygen-independent coproporphyrinogen III oxidase, at 2.07 A Ê resolution. HemN catalyzes the essential conversion of coproporphyrinogen III to protoporphyrinogen IX during heme biosynthesis. HemN binds a 4Fe±4S cluster through three cysteine residues conserved in all Radical SAM enzymes. A juxtaposed SAM coordinates the fourth Fe ion through its amide nitrogen and carboxylate oxygen. The SAM sulfonium sulfur is near both the Fe (3.5 A Ê) and a neighboring sulfur of the cluster (3.6 A Ê), allowing single electron transfer from the 4Fe±4S cluster to the SAM sulfonium. SAM is cleaved yielding a highly oxidizing 5¢-deoxyadenosyl radical. HemN, strikingly, binds a second SAM immediately adjacent to the ®rst. It may thus successively catalyze two propionate decarboxylations. The structure of HemN reveals the cofactor geometry required for Radical SAM catalysis and sets the stage for the development of inhibitors with antibacterial function due to the uniquely bacterial occurrence of the enzyme.
Biological Chemistry, 2000
During heme biosynthesis the oxygen-independent coproporphyrinogen III oxidase HemN catalyzes the oxidative decarboxylation of the two propionate side chains on rings A and B of coproporphyrinogen III to the corresponding vinyl groups to yield protoporphyrinogen IX. Here, the sequence of the two decarboxylation steps during HemN catalysis was investigated. A reaction intermediate of HemN activity was isolated by HPLC analysis and identified as monovinyltripropionic acid porphyrin by mass spectrometry. This monovinylic reaction intermediate exhibited identical chromatographic behavior during HPLC analysis as harderoporphyrin (3-vinyl-8,13,17-tripropionic acid-2,7,12,18-tetramethylporphyrin). Furthermore, HemN was able to utilize chemically synthesized harderoporphyrinogen as substrate and converted it to protoporphyrinogen IX. These results suggest that during HemN catalysis the propionate side chain of ring A of coproporphyrinogen III is decarboxylated prior to that of ring B.
Fems Microbiology Letters, 2003
During heme biosynthesis in Escherichia coli two structurally unrelated enzymes, one oxygen-dependent (HemF) and one oxygen-independent (HemN), are able to catalyze the oxidative decarboxylation of coproporphyrinogen III to form protoporphyrinogen IX. Oxygendependent coproporphyrinogen III oxidase was produced by overexpression of the E. coli hemF in E. coli and purified to apparent homogeneity. The dimeric enzyme showed a K m value of 2.6 M for coproporphyrinogen III with a k cat value of 0.17 min ؊1 at its optimal pH of 6. HemF does not utilize protoporphyrinogen IX or coproporphyrin III as substrates and is inhibited by protoporphyrin IX. Molecular oxygen is essential for the enzymatic reaction. Single turnover experiments with oxygen-loaded HemF under anaerobic conditions demonstrated electron acceptor function for oxygen during the oxidative decarboxylation reaction with the concomitant formation of H 2 O 2 . Metal chelator treatment inactivated E. coli HemF. Only the addition of manganese fully restored coproporphyrinogen III oxidase activity. Evidence for the involvement of four highly conserved histidine residues (His-96, His-106, His-145, and His-175) in manganese coordination was obtained. One catalytically important tryptophan residue was localized in position 274. None of the tested highly conserved cysteine (Cys-167), tyrosine (Tyr-135, Tyr-160, Tyr-170, Tyr-213, Tyr-240, and Tyr-276), and tryptophan residues (Trp-36, Trp-123, Trp-166, and Trp-298) were found important for HemF activity. Moreover, mutation of a potential nucleotide binding motif (GGGXXTP) did not affect HemF activity. Two alternative routes for HemFmediated catalysis, one metal-dependent, the other metal-independent, are proposed.
Structural diversity in the AdoMet radical enzyme superfamily
AdoMet radical enzymes are involved in processes such as cofactor biosynthesis, anaerobic metabolism, and natural product biosynthesis. These enzymes utilize the reductive cleavage of S-adenosylmethionine (AdoMet) to afford l-methionine and a transient 5′-deoxyadenosyl radical, which subsequently generates a substrate radical species. By harnessing radical reactivity, the AdoMet radical enzyme superfamily is responsible for an incredible diversity of chemical transformations. Structural analysis reveals that family members adopt a full or partial Triose-phosphate Isomerase Mutase (TIM) barrel protein fold, containing core motifs responsible for binding a catalytic [4Fe–4S] cluster and AdoMet. Here we evaluate over twenty structures of AdoMet radical enzymes and classify them into two categories: ‘traditional’ and ‘ThiC-like’ (named for the structure of 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase (ThiC)). In light of new structural data, we reexamine the ‘traditional’ structural motifs responsible for binding the [4Fe–4S] cluster and AdoMet, and compare and contrast these motifs with the ThiC case. We also review how structural data combine with biochemical, spectroscopic, and computational data to help us understand key features of this enzyme superfamily, such as the energetics, the triggering, and the molecular mechanisms of AdoMet reductive cleavage. This article is part of a Special Issue entitled: Radical SAM Enzymes and Radical Enzymology.
Proceedings of the National Academy of Sciences, 2013
Significance AdoMet radical enzymes harness the power of radical-based chemistry to carry out complex chemical transformations. The structure of butirosin biosynthethic enzyme BtrN reveals both unforeseen differences and surprising similarities compared with other members of this rapidly expanding enzyme superfamily. In particular, variations in how BtrN binds S -adenosyl- L -methionine (AdoMet) warrant redefinition of the core fold responsible for adenosyl-radical generation whereas similarities in how BtrN binds an auxiliary iron–sulfur cluster provide the basis for assignment of a previously undescribed structural motif.
Archives of biochemistry and biophysics, 2015
Genes for chlorite dismutase-like proteins are found widely among heme-synthesizing bacteria and some Archaea. It is now known that among the Firmicutes and Actinobacteria these proteins do not possess chlorite dismutase activity but instead are essential for heme synthesis. These proteins, named HemQ, are iron-coproporphyrin (coproheme) decarboxylases that catalyze the oxidative decarboxylation of coproheme III into protoheme IX. As purified, HemQs do not contain bound heme, but readily bind exogeneously supplied heme with low micromolar affinity. The heme-bound form of HemQ has low peroxidase activity and in the presence of peroxide the bound heme may be destroyed. Thus, it is possible that HemQ may serve a dual role as a decarboxylase in heme biosynthesis and a regulatory protein in heme homeostasis.
Heme Iron Catalysis: Contrast to Non-Heme Iron Enzymes
2017
Metalloenzymes catalyze a wide range of chemical transformations. Their remarkable versatility is imparted to them by their metal centers that are redox active or function as Lewis acids. They utilize transition metals such as iron that has accessibility to a variety of redox states, allowing them to efficiently activate and insert molecular oxygen into a wide range of unactivated organic substrates. The work described in this dissertation is the structural and mechanistic characterization of both heme and non-heme iron-dependent metalloenzymes. Precisely, the focus is placed on three enzymes, a cytochrome P450, CYP121, a tyrosine hydroxylase, LmbB2, and an extradiol dioxygenase, 3-hydroxyanthranilate 3,4-dioxygenase (HAO). The first two enzymes expand the repertoire of activities performed by hemoproteins. CYP121 catalyzes an unusual C-C crosslinking reaction that is distinct from traditional oxygenase chemistry performed by this family. LmbB2 is one of the first enzymes that media...
Journal of Biological Chemistry, 1999
Hmu O, a heme degradation enzyme in Corynebacterium diphtheriae, forms a stoichiometric complex with iron protoporphyrin IX and catalyzes the oxygen-dependent conversion of hemin to biliverdin, carbon monoxide, and free iron. Using a multitude of spectroscopic techniques, we have determined the axial ligand coordination of the heme-Hmu O complex. The ferric complex shows a pH-dependent reversible transition between a water-bound hexacoordinate high spin neutral pH form and an alkaline form, having high spin and low spin states, with a pK a of 9. 1 H NMR, EPR, and resonance Raman of the heme-Hmu O complex establish that a neutral imidazole of a histidine residue is the proximal ligand of the complex, similar to mammalian heme oxygenase. EPR of the deoxy cobalt porphyrin IX-Hmu O complex confirms this proximal histidine coordination. Oxy cobalt-Hmu O EPR reveals a hydrogen-bonding interaction between the O 2 and an exchangeable proton in the Hmu O distal pocket and two distinct orientations for the bound O 2. Mammalian heme oxygenase has only one O 2 orientation. This difference and the mixed spin states at alkaline pH indicate structural differences in the distal environment between Hmu O and its mammalian counterpart.