Aerobic Enzymes and Their Radical SAM Enzyme Counterparts in

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Aerobic enzymes and their radical SAM enzyme counterparts in tetrapyrrole pathways Bin Li, and Jennifer Bridwell-Rabb Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00906 • Publication Date (Web): 15 Oct 2018 Downloaded from http://pubs.acs.org on October 16, 2018

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Biochemistry

Title: Aerobic enzymes and their radical SAM enzyme counterparts in tetrapyrrole pathways Authors: Bin Li and Jennifer Bridwell-Rabb* *Contact Information: email: [email protected] Department of Chemistry, University of Michigan, Ann Arbor, MI, 48109, USA Abstract: Microorganisms have lifestyles and metabolism adapted to environmental niches, which can be very broad or highly restricted. Molecular oxygen (O2) is currently variably present in microenvironments and has driven adaptation and microbial differentiation over the course of evolution on Earth. Obligate anaerobes use enzymes and cofactors susceptible to low levels of O2 and are restricted to O2-free environments, whereas aerobes typically take advantage of O2 as a reactant in many biochemical pathways and may require O2 for essential biochemical reactions. In this perspective, we focus on analogous enzymes found in tetrapyrrole biosynthesis, modification, and degradation that are catalyzed by O2-sensitive radical Sadenosylmethionine (SAM) enzymes and by O2-dependent metalloenzymes. We will showcase four transformations, for which aerobic organisms use O2 as a co-substrate, but anaerobic organisms do not. These reactions include oxidative decarboxylation, methyl and methylene oxidation, ring formation, and ring cleavage. Further, we will highlight biochemically uncharacterized enzymes implicated in reactions that resemble those catalyzed by the parallel aerobic and anaerobic enzymes. Intriguingly, several of these reactions require insertion of an oxygen atom into substrate, which in aerobic enzymes is facilitated by activation of O2, but in anaerobic organisms requires an alternative mechanism. Table of Contents Graphic:

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Introduction: The tetrapyrrole biosynthetic pathway is responsible for construction of the so-called “pigments of life” chlorophyll, heme, siroheme, heme d1, cobalamin, and coenzyme F430 (Figure 1A). This pathway begins with conversion of 5-aminolevulinic acid into uroporphyrinogen III and then uses a series of chemical steps that include changes to the oxidation state of the scaffold, the addition of chemical moieties, and/or coordination of metal ions to confer different functionalities to the macrocyclic products1. Each of these additional biosynthetic steps tailors the end products to the role(s) they play in catalysis, signaling, and/or pigmentation. This seemingly ancient pathway is found in all domains of life, but shows variations in the mechanisms of different enzymatic steps (Figure 1A). For example, several reactions, including those catalyzed by coproporphyrinogen III oxidase and Mg-protoporphyrin IX monomethylester cyclase, which are involved in oxidative decarboxylation and cyclization reactions, respectively, are catalyzed by parallel enzymes, or those that catalyze the same reaction, but work in oxic and/or anoxic conditions2, 3. The enzymes involved in these reactions are found amidst those that are universally conserved and have presumably developed during evolution guided by the availability of molecular oxygen (O2)3. O2 is an essential resource for aerobic organisms. O2 serves as the terminal electron acceptor for respiration, and is often exploited as a co-substrate for biosynthetic transformations. In catabolic and anabolic pathways, the kinetic stability of O2 is overcome through activation by transition metals (iron, manganese, and copper) or cofactors (flavins) in an enzyme active site. In particular, reduced transition metal ions bind O2 and promote its cleavage through formation of metal-oxygen intermediates: superoxo (O-O•), peroxo (O-O-), hydroperoxo (O-OH), and oxo (=O) during their catalytic cycles4. The majority of these enzymes contain iron and can be classified as heme-based or non-heme-based enzymes (mono- and di-nuclear), and further differentiated based on whether they incorporate one (monooxygenases), two (dioxygenases), or no oxygen atoms (oxidases) from O2 into the product(s). Most heme-based enzymes employ a high-valent Fe(IV)-oxo compound I intermediate in their catalytic cycle, but heme oxygenases instead use a Fe(III)-OOH species5,6 (Figure 1B). Non-heme-based enzymes employ a protein ligated Fe(II) center and depending on the system, have been proposed to proceed through Fe(IV) or Fe(V) intermediates4, 7-9 (Figure 1B). In either case, the resultant high-valent iron-oxo species can typically abstract a H-atom from substrate to initiate subsequent steps7-9. In contrast, anaerobic organisms are restricted to environments devoid of O2 as they lack O2detoxification mechanisms and use enzymes and cofactors susceptible to low O2-levels. In these organisms, the problem of generating a strong oxidant is often solved through the use of a O2independent organic radical, such as the 5'-deoxyadenosyl radical (5'-dAdo•). This species is a hallmark of the radical S-adenosylmethionine (SAM) superfamily, which is currently estimated to include more than 113,000 protein sequences collectively implicated in catalyzing over 80 different reactions10. These enzymes contain a trademark Cys-X3-Cys-X2-Cys motif for ligating an O2-sensitive [4Fe-4S] cluster11. In many radical SAM enzymes, this cluster facilitates cleavage of SAM and formation of 5'-dAdo•, which can abstract a H-atom from substrate11 (Figure 1C). Distinct subclasses within the superfamily bind auxiliary cofactors to extend their accessible range of chemistry11-13. A large proportion of radical SAM enzymes are predicted to have Nterminal cobalamin (Cbl)-binding domains or C-terminal domains for binding auxiliary [4Fe-4S] clusters12-14. Cbl-dependent radical SAM enzymes use a combination of methyl-Cbl and the


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Biochemistry

radical SAM machinery to catalyze methylation of inert C- and P-centers15, 16, or non-methylated Cbl to facilitate a radical-mediated ring contraction17. The enzymes that bind auxiliary C-terminal Fe-S clusters are members of the SPASM/TWITCH subclass and have been implicated in an array of reactions where the auxiliary cluster(s) bind substrate18, 19 or transfer electrons20, 21. The use of O2-dependent and O2-independent enzymes to catalyze the same reaction is not the only strategy that organisms use to grow irrespective of aerobic or anaerobic environmental conditions. Indeed, some biosynthetic pathways, like that seen for cobalamin, have alternate aerobic and anaerobic routes that differ in reaction sequence22, 23. The use of parallel enzymes to catalyze the same reaction is also not exclusive to tetrapyrrole biosynthesis, but instead seems to be a common feature of several metabolic pathways24. In many instances, the mechanisms of the enzymes that catalyze these parallel transformations are unknown and may involve unprecedented chemistry. Furthermore, in cases where the aerobic enzyme activates O2 for insertion into substrate, the source of the oxygen atom and mechanism of its insertion by the anaerobic counterpart remains mysterious. In this perspective, we will feature four parallel reactions found in the tetrapyrrole pathways to highlight current knowledge about the exceptional number of ways that chemistry is facilitated using an activated oxygen species in aerobic organisms and using 5'-dAdo• in anaerobic organisms.

Figure 1. Aerobic and anaerobic enzymes catalyze parallel reactions in tetrapyrrole biosynthesis and degradation. (A) O2-dependent (red) and O2-independent (blue) tetrapyrrole biosynthetic and degradative enzymes discussed in this perspective. End products of tetrapyrrole biosynthesis are boxed in tan. Uroporphyrinogen III, the branchpoint for each end product is boxed in green. Black arrows are reactions highlighted in this perspective and gray arrow indicate intermediate reactions. Cobalamin biosynthesis proceeds through either an aerobic route from precorrin-2 or an anaerobic route from sirohydrochlorin22,23. Abbreviations used here, include ALA, 5-aminolevulinic acid; Bchlide, bacteriochlorophyllide; HMB, hydroxymethylbilane; HO, heme oxygenase; MPE, Mg-protoporphyrin IX monomethylester; PBG, porphobilinogen; Pchilide, 3,8-divinyl protochlorophyllide. (B) Dioxygen derived high-valent metal-oxo and hydroperoxo species are potent oxidants. Top left, heme-based Compound I used by cytochrome P450 enzymes and peroxidases, which are characterized by a proximal cysteine and histidine ligand, respectively5. Top right, a heme-based ferric-hydroperoxo species is considered the hydroxylating reagent in heme oxygenases6. Bottom left, a proposed Fe(V) species used by Rieske non-heme iron oxygenases58. Bottom middle, a common species used by mononuclear non-heme iron oxygenases, which have a 2-His-1-carboxylate motif, including -ketoglutarate (KG)-dependent oxygenases7. Bottom right, Compound Q from the diiron enzyme methane monooxygenase9. (C) Radical SAM enzymes use a 5'deoxyadenosyl radical derived from the reductive of S-adenosylmethionine (SAM) as the oxidant11. ACS homolysis Paragon Plus Environment

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Oxidative sidechain decarboxylation proceeds through common radical intermediates The iron-containing protoporphyrin, protoheme or heme, is one of the most ubiquitous compounds in nature and is involved in key biological processes, including electron transfer, signaling, catalysis and the transport and storage of O225. Starting with uroporphyrinogen III, there are three known routes for assembling protoheme2 (Figure 1A). Each of these pathways requires an enzyme to catalyze decarboxylation of two propionate sidechains appended to the core macrocycle. In the traditional, or protoporphyrin-dependent pathway, either the O2dependent coproporphyrinogen III oxidase (HemF or CgdC), or the O2-independent counterpart (HemN or CgdH), iteratively oxidizes the C3 and C8 propionate groups of coproporphyrinogen III to form the vinyl groups of protoporphyrinogen IX2, 26, 27 (Figure 2A). A subsequent oxidation forms protoporphyrin IX and addition of Fe2+ completes synthesis of protoheme2. HemF is found in eukaryotes, proteobacteria, and cyanobacteria, whereas HemN is found in prokaryotes2. Some organisms carry both genes and differentially regulate their expression in response to O2 levels3. For the O2-dependent enzyme HemF, two main mechanisms have been proposed. Based on stimulation of E.coli HemF activity through addition of manganese and inactivation by metal chelators, a metal-dependent mechanistic proposal that resonates with that of the manganesedependent enzyme oxalate oxidase has been suggested26, 28. Here, formation of a Mn(III)superoxo species facilitates removal of a H-atom from substrate and begins the catalytic cycle26 (Figure 2B). Although chemically satisfying, this proposal is complicated by conflicting reports about the need for a metal ion in HemF homologs and crystal structures of human and yeast HemF that show the conserved residues implicated in metal binding are located too far apart to serve as ligands without significant structural rearrangements29, 30. An alternative metalindependent proposal places HemF in a class of oxidases and oxygenases that proceed in a cofactor-independent manner31. Here, a pyrrole peroxide anion formed through base-mediated addition of O2 to the coproporphyrinogen III scaffold could abstract a proton from the β-position of the propionate group. Subsequent elimination of CO2 and H2O2, both of which have been detected in vitro, would complete the first of two decarboxylation reactions required to form protoporphyrinogen IX26, 31, 32 (Figure 2C). Unlike HemF, the O2-independent enzyme HemN is a member of the radical SAM superfamily. HemN initiates catalysis using 5'-dAdo• formed via reductive cleavage of SAM to abstract a Hatom from the β-position of the coproporphyrinogen III substrate propionate sidechain33 (Figure 2B). Once the substrate radical is formed, the remaining catalytic steps are shared with those proposed in the metal-dependent HemF mechanism: decarboxylation, loss of an electron, and a second catalytic cycle forms protoporphyrinogen IX33. Structural studies of HemN revealed two molecules of SAM within the HemN active site34. SAM1 binds in the canonical orientation and coordinates the [4Fe-4S] cluster. SAM2 binds adjacent to SAM1 and is stabilized by a few conserved residues. Mutation of these residues abolishes HemN activity and suggest SAM2 is important for catalysis34. SAM2 has been proposed to serve as the elusive electron acceptor for the first decarboxylation reaction, which would permit formation of 5'-dAdo• and initiation of the second round33 (Figure 2B). This type of reaction is similar to a proposal for the radical SAM enzyme DesII that suggests a product radical could donate an electron back to the oxidized [4Fe-4S] cluster35. In both cases, a second round of catalysis could occur without an external


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Biochemistry

supply of electrons35. Alternatively, SAM2 has been proposed to facilitate the first decarboxylation reaction by accepting the initial electron transferred from the [4Fe-4S] cluster33. However, for this transfer to happen, the electron would need to be tunneled through SAM1. Although these proposals are intriguing, both lack precedent, and the exact role of SAM2 remains to be elucidated.


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Figure 2. Oxidative decarboxylation of propionate groups on the tetrapyrrole macrocycle. (A) Different enzymes are involved in decarboxylating the C3 and C8 (positions numbered in blue) propionate sidechains in the three protoheme biosynthetic pathways (see Figure 1). These sidechains are converted to vinyl groups (red circles). In the traditional protoporphyrindependent pathway, the decarboxylation reaction is catalyzed by the O2-dependent coproporphyrinogen III oxidase HemF or O2-independent coproporphyrinogen III oxidase HemN (top panel). In the siroheme or coproporphyrin-dependent pathways, coproheme decarboxylase HemQ or an O2-independent alternative heme biosynthetic protein AhbD catalyzes the reaction. Additional enzymes highlighted in this panel are: HemY (or CgoX/PgoX), copro/protoporphyrinogen oxidase; HemH (or CpfC/PpfC), copro/protoporphyrin ferrochelatase; HemG/J (or PgdH1/H2), protoporphyrinogen dehydrogenase2. (B) Proposed reaction mechanisms for HemF (Mn(II)-dependent, top arrows), and HemN/AhbD (SAM-dependent, bottom arrows). Only the first of two-propionate sidechain decarboxylation reactions is shown. A Mn(III)-superoxo species or 5’-Ado• is used to abstract a H-atom from the β-position of the propionate group to generate a substrate radical. Subsequent one-electron oxidation and elimination of CO2 leads to the formation of the C3-vinyl group intermediate. For the HemF reaction, the destination of the electron is proposed to be the Mn(III)-OOH species. Subsequent release of H2O2 would regenerate the Mn(II) center for a second round of catalysis (purple boxes)26. Identity of the electron acceptor for one-electron oxidation of the radical intermediate is unknown for HemN and AhbD (gold box). However, for the HemN reaction, it is proposed the electron could be used for the reductive cleavage of a second molecule of SAM to initiate the second decarboxylation reaction (blue box)27. In AhbD, the electron is proposed to be shuttled to an external acceptor via the coproheme iron center and an auxiliary [4Fe4S] cluster (green box)42. (C) A metal-independent reaction mechanism for HemF. A conserved Asp residue found in the HemF active site is proposed to serve as the general base for this reaction31. (D) A proposed reaction mechanism for HemQ that uses an active site Tyrosyl radical to abstract a H-atom from the substrate to facilitate catalysis. Only the first decarboxylation reaction is shown, the second is thought to involve an active site lysine residue41.

Alternative routes of decarboxylation exploit the substrate macrocycle metal ion for catalysis In the protoporphyrin-dependent pathway described above, coproporphyrinogen III is twice decarboxylated by HemN or HemF (Figure 1A and 2A). The two remaining protoheme forming pathways, also require decarboxylation of the C3- and C8-propionate sidechains, but act on an Fe-bound coproporphyrin (coproheme) that originates from coproporphyrin and siroheme precursors36, 37 (Figure 1A and 2A). The corproporphyrin-dependent pathway is found in Grampositive bacteria and uses the aerobic coproheme decarboxylase (HemQ or ChdC)36, 38, whereas the siroheme-dependent pathway is found in anaerobic sulfate-reducing bacteria and archaea and requires AhbD, an O2-independent enzyme37, 39. Similar to organisms that contain


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Biochemistry

copies of HemN and HemF, some Gram-positive bacteria have genes that encode both HemQ and AhbD, suggesting protoheme biosynthesis can be maintained regardless of O2 availability2. In the corproporphyrin-dependent pathway, uroporphyrinogen III is converted into coproporphyrinogen III and then oxidized to coproporphyrin and H2O2 by the coproporphyrinogen oxidase (HemY or CgoX)40. Coproporphyrin is loaded with Fe2+ to form coproheme and a final two decarboxylation reactions catalyzed by HemQ, convert the C3- and C8-propionate sidechains of coproheme into the vinyl groups of protoheme (Figure 1A and 2A). Unlike the HemN and HemF-catalyzed decarboxylation reactions, HemQ uses coproheme as both a substrate and cofactor in this reaction40, 41. HemQ is predicted to use the H2O2 produced in the upstream HemY reaction to facilitate formation of a coproheme compound I-like intermediate2, 41 (Figure 2D). Rather than employing this species for direct oxidation of the propionate sidechain by removal of a H-atom, structural studies with a substrate analog revealed that the propionate groups are located on the opposite side of the porphyrin plane from where an activated oxygen species would form41. Thus, one current mechanistic proposal for HemQ requires participation of multiple active site residues to facilitate the H-atom abstraction steps41 (Figure 2D). However, the heterogeneity of observed coproheme orientations in HemQ crystal structures and lack of sequence conservation among HemQ homologs complicate proposals that rely on active site residues to facilitate catalysis and leaves many open questions as to how the propionyl substrate radical is generated40, 41. As mentioned above, the third pathway for synthesizing protoheme proceeds through a siroheme intermediate (Figure 1A). This pathway requires a set of alternative heme biosynthetic enzymes to first convert siroheme into coproheme (AhbA-C) and a radical SAM enzyme (AhbD) to convert coproheme into protoheme. AhbD is thought to facilitate this transformation using similar chemical steps to those proposed for HemN (Figure 2B)42. However, unlike HemN, AhbD is a member of the SPASM/TWITCH subclass of the radical SAM superfamily and binds at least one auxiliary [4Fe-4S] cluster42. Loss of this cluster corresponds to loss of AhbD decarboxylase activity, but AhbD retains the ability to cleave SAM, bind substrate, and generate a substrate radical42. These results and measured reduction potentials are consistent with the auxiliary cluster acting as an electron acceptor for the decarboxylation reaction42. Further, as AhbD displays lower activity when non-metal bound coproporphyrin or Cu/Zn-coproporphyin III are used as substrates, it is thought that the central iron of coproheme could serve as a conduit for electron transfer from substrate to the auxiliary cluster42 (Figure 2B). As there are no structures of AhbD available, the feasibility of this path for electron transfer is unclear. Putative enzymes introduce keto groups onto the tetrapyrrole scaffold Besides AhbD, two additional radical SAM enzymes AhbC and NirJ are found in sirohemedependent pathways (Figure 1A). These enzymes, similar to AhbD, are members of the SPASM/TWITCH subclass of the radical SAM superfamily37. AhbC catalyzes removal of the C2and C7-acetate groups of 12,18-didecarboxysiroheme37, whereas NirJ catalyzes removal of the C3- and C8-propionate groups43. NirJ has also been putatively assigned as the enzyme responsible for introducing two keto groups onto the tetrapyrrole scaffold2, 43 (Figure 3A). Evidence is currently lacking for keto group incorporation, which may require participation of an additional protein43. Two keto groups are also found on the scaffold of tolyporphins44. These molecules are thought to originate from uroporphyrinogen III via the removal of four propionate


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groups, introduction of two keto groups, and several other chemical steps44 (Figure 3B). Currently, there is no biochemical information available about the enzymes encoded by the proposed Nostocales cyanobacterium HT-58-2 tolyporphin gene cluster (Figure 3B). However, this cluster contains several genes, including two homologs of HemF and two cytochrome P450 enzymes44, which are attractive candidates for O2-dependent decarboxylation and keto group incorporation, respectively.

Figure 3. Putative enzymes involved in removal of a propionate group and oxygenation of the tetrapyrrole macrocycle. (A) The conversion of 12,18-didecarboxysiroheme to pre-heme d1 involves removal and replacement of two propionate groups at the C3- and C8- scaffold positions (labeled with blue numbers) with two keto groups (blue circles). This reaction has been proposed to be catalyzed by a radical SAM enzyme NirJ, but introduction of the keto group may require an additional protein43. (B) A similar keto group to that shown in panel A is found on the scaffold of tolyporphin A44. Here, a possible candidate from the gene cluster44 for introduction of keto groups (blue circles) is a cytochrome P450 enzyme. Genes for the core tetrapyrrole synthesis pathway are red, P450 enzymes are light blue and additional genes are purple.

Dual function enzymes catalyze oxygenation and extension of the porphyrin macrocycle in chlorophyll biosynthesis Following the HemF/HemN catalyzed formation of protoporphyrinogen IX, a reaction catalyzed by protoporphyrinogen oxidase generates protoporphyrin IX, which can be loaded with Fe2+, or reconstituted with Mg2+ and diverted towards making chlorophyll (Figure 1A). Formation of a fifth ring and several additional modifications chemically distinguish this molecule from other products of tetrapyrrole biosynthesis, and produce an optimally tuned compound for absorbing and transferring light energy45. Formation of the fifth ring is an intricate chemical process: it involves introduction of a keto group at C131 of Mg-protopophyrin IX monomethylester (MPE) and formation of a bond between C132 and C15 to form 3,8-divinyl-protochlorophyllide (Pchlide). This reaction is catalyzed by an O2-dependent enzyme ChlA, whose catalytic subunit is often termed AcsF (aerobic cyclization system Fe-containing subunit), and an O2-independent


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Biochemistry

enzyme BchE, which proceed through common C131-hydroxo and C131-keto intermediates46-48 (Figure 4A). ChlA is found in plants, algae, cyanobacteria and some other phototrophic bacteria, whereas BchE is predominantly found in anoxygenic phototrophic bacteria49. Some organisms carry genes that encode both enzymes, and their co-occurrence is thought to offer advantages for surviving fluctuations of environmental light and O2-levels3,50. ChlA is a diiron oxygenase that based on isotopic labeling experiments is known to require O246. Diiron oxygenases typically initiate catalysis using a high-valent iron(IV)-iron(IV) species to abstract a H-atom from an inert substrate9 (Figure 1B). A subsequent radical rebound is then used to complete functionalization of the C-H bond with an oxygen atom9. Unfortunately, biochemical characterization of ChlA has been thwarted by previous studies that suggest multiple soluble and insoluble components are required for ChlA activity46. More recently, however, Chen et al., have identified the elusive subunits (BciE and Ycf54), outlined the protein requirements for catalysis from different organisms49, and laid the foundation for future in vitro mechanistic studies. Unlike ChlA, BchE is predicted based on sequence to be a Cbl-dependent radical SAM enzyme. Consistent with this annotation, disruption of cobalamin biosynthetic genes in Rhodobacter capsulatus47 and Rhodospirillum rubrum51 leads to accumulation of MPE. However, as noted for other members of this class52, difficulties in overexpressing soluble forms of BchE, have resulted in essentially no mechanistic details being available about how BchE functions. Nevertheless, several mechanistic proposals exist with regards to how BchE could use radical SAM and Cbl chemistry to convert MPE into Pchlide. First is a mechanism that is reminiscent of Cbl-dependent methyltransferases (Figure 4B)15, 16, 53. Here, 5'-dAdo• generated via reductive cleavage of SAM abstracts a H-atom from C131 of MPE53. This substrate radical reacts with hydroxo-cobalamin to form the C131-hydroxo intermediate. A subsequent round of hydroxylation using the same mechanism leads to formation of a geminal-diol that collapses into a keto group. An alternate path that we can envision from the C131-hydroxo intermediate to the C131-keto species resonates with the mechanisms of the radical SAM dehydrogenases BtrN and anSME21, 54. Here, the hydroxyalkyl radical (•C-OH) species is oxidized by the auxiliary cofactor (Figure 4B). In the dehydrogenases, the auxiliary cofactor is a [4Fe-4S] cluster, whereas in BchE, the auxiliary Cbl cofactor could accept the electron. Through either route, this mechanism is attractive as it uses 5'-dAdo• to abstract a H-atom from a position analogous to that used by HemN33 and agrees with data that show the C131 carbonyl oxygen is derived from water47, 48. A recent proposal instead suggests that BchE catalyzes sequential dehydrogenation and hydration reactions55 (Figure 4B). 5'-dAdo• abstracts a H-atom from C132 of MPE. A one-electron oxidation of the substrate radical forms an unsaturated bond that facilitates nucleophilic attack of water and formation of the C131-hydroxo intermediate. A second dehydrogenation forms C131-keto MPE. In both proposals, cyclization occurs following keto-group formation and requires a third molecule of SAM to generate a radical at the C132 position53, 55. Interestingly, this position is the same as that proposed in the most recent model for the initial H-atom abstraction step55. The resultant radical attacks C15 to complete cyclization. In this proposal, Cbl could serve as an electron acceptor in the final step, similar to that proposed above and for the Cbl-dependent radical SAM enzyme OxsB17, 55. As noted for OxsB, such a role of Cbl seems frivolous based on its biosynthetic cost and requires further investigation. Regardless, the


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fundamental differences between the two proposals concerns how H2O is activated for incorporation into substrate and the position of the first H-atom abstraction.

Figure 4. Formation of the fifth ring in chlorophyll and bacteriocholorophyll biosynthesis (green circles). (A) The conversion of MPE to Pchlide is catalyzed by O2-depedent and O2-independent cyclases, ChlA and BchE, respectively. The reaction cycles of these enzymes share the intermediates MPE-131-hydroxo and MPE-131-keto46,47. The keto group oxygen atom originates from O2 and H2O in the ChlA and BchE reactions, respectively48. The C13- and C15-atoms on the scaffold are numbered in blue. (B) Proposed mechanisms for BchE53,55. The introduction of the keto group at the 131 position could proceed in a fashion similar to cobalamin-dependent methyltransferases, with hydroxo-cobalamin as a donor of the incorporated oxygen atom (top panel, black arrows) or through an iterative dehydrogenase-hydratase-dehydrogenase mechanism, where an enol tautomerizes into a keto group (bottom panel, gray arrows). Ring formation proceeds via a substrate radical at the 132 position. Blue arrows suggest a dehydrogenation step as a possible alternative route for formation of the keto group. In this case, we suggest Cbl could serve as an electron acceptor. The boxed portion in panel A is the only part of the molecule represented in panel B.

Chlorophyll modifications require sequential oxygenations with diverse oxygen donors In the chlorophyll biosynthetic branch, following isocyclic ring formation, the 8-vinyl group and macrocycle of Pchlide are reduced to form chlorophyllide a, a precursor to the primary light-


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harvesting pigment of photosynthesis, chlorophyll a (Figure 1A). In general, derivatives of chlorophyll a that are tuned to absorb available light, arise from chlorophyllide a and are differentiated by addition of a functional group and/or modification of an existing substituent on the precursor scaffold. For example, both chlorophyll f synthase and chlorophyllide a oxygenase (CAO) are enzymes involved in converting the methyl groups at the C2- and C7-positions of chlorophyllide a into formyl groups56, 57 (Figure 5A). Chlorophyll f synthase is a recently discovered light-dependent photo-oxidoreductase related to photosystem II57, whereas CAO is a Rieske non-heme iron oxygenase56. Rieske oxygenases are a subclass of non-heme iron oxygenases that couple a 2-His/2-Cys ligated [2Fe-2S] cluster, or Rieske cluster, with a catalytic non-heme iron site58. The Rieske cluster accepts external electrons and shuttles them to the non-heme iron site for O2 activation. CAO is proposed to catalyze two oxygenation reactions, producing 7-hydroxymethyl and 7-dihydroxymethyl intermediates56, 59. Consistent with this proposal, the formyl-group oxygen is known to originate from O260. Further, chlorophyll bdeficient mutants belong to a single complementation group, the 7-hydroxymethyl intermediate accumulates when CAO is incubated with chlorophyllide a in cell lysate, and CAO converts 7hydroxymethyl zinc-chlorophyllide a into zinc-chlorophyllide b59 . This sequential oxygenation proposed for CAO is unusual for the Rieske oxygenase class as all currently characterized members accept a single substrate and function as monooxygenases or dioxygenases58, 61. Figure 5. A methyl- to formyl-group modification is found in the biosynthetic pathways of chlorophyll and bacteriochlorophyll derivatives. (A) Oxidation of the C7-methyl group of chlorophyllide a is catalyzed by an O2-dependent Rieske non-heme iron oxygenase CAO56. The C7-methyl group of bacteriochlorophyllide c is transformed into a formylgroup (yellow circle) of bacteriochlorophyllide d by an O2-independent radical SAM enzyme BciD63. The origin of the oxygen atom here is presumably water. A C2-methyl group oxidation is catalyzed by a lightdependent photo-oxidoreductase and required for synthesis of chlorophyll f. This reaction may or may not require O2 to occur57. (B) Proposed mechanisms for CAO and BciD suggest the C7-methyl group oxidation proceeds through sequential hydroxylation steps59,63. The dihydroxymethyl intermediate is thought to spontaneously lose water to form the product formyl group (yellow circle). The boxed portion in panel A is the only part of the molecule represented in panel B.

A C7-methyl group oxidation that resembles the CAO catalyzed reaction is also important for conversion of bacteriochlorophyllide c into bacteriochlorophyllide e (Figure 5A). However, this reaction is catalyzed by BciD, an enzyme identified through gene inactivation studies in Chlorobaculum limnaeum and later confirmed in vitro to be sufficient for converting the C7methyl of bacteriochlorophyllide c into a formyl group62, 63. Despite containing a non-canonical cysteine motif (Cys-X8-Cys-X2-Cys), BciD binds a [4Fe-4S] cluster and produces 5'-dAdo• when incubated with SAM, substrate, and reductant55. The current mechanistic proposal for how BciD functions is reminiscent of that proposed for CAO as it is thought to catalyze sequential


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hydroxylation reactions56 (Figure 5B). However, unlike CAO, which uses O2 to facilitate its reaction, BciD is thought to use water as a source of the oxygen atom62, 63. BchE, described above, also introduces an oxygen atom into its substrate, but BchE binds an auxiliary Cbl cofactor, which has been suggested to activate water for insertion53. BciD is not known to use any auxiliary cofactors. The chemical feasibility of this reaction using only the radical SAM machinery, similar to the mechanism of CAO, awaits further exploration. Degradation of protoheme requires functional group incorporation Protoheme degradation is essential for iron homeostasis and is also important for the virulence of pathogens, which use this pathway to acquire iron from their host64. In most organisms, protoheme is degraded using an O2-dependent enzyme (Figure 6A). The most common and well-studied is heme oxygenase, which catalyzes the breakdown of protoheme in three steps using three molecules of O26. First, an Fe(III)-OOH species is used to hydroxylate the α-meso position of protoheme to form α-meso-hydroxyheme. This compound is oxidized to α-verdoheme and CO. Finally, α-verdoheme is cleaved to an Fe(III)-biliverdin IXα complex and Fe2+ is liberated following reduction6 (Figure 6B). Alternatively, IsdG/IsdI (Iron-regulated surface determinant system) converts proteoheme into Fe2+, formaldehyde, and staphylobilin65 and MhuD (Mycobacterium heme utilization) breaks down protoheme into Fe2+ and mycobilin66 (Figure 6A). Despite differences in products and protein architecture67, all three O2-dependent enzymes use protoheme as a substrate and cofactor of the reaction. Figure 6. There are multiple routes for degradation of protoheme. (A) Canonical heme oxygenases break down protoheme into biliverdin and release Fe2+ and CO6. Two other heme oxygenases, IsdG/I and MhuD, also use O2 to cleave the protoheme macrocycle, but form different products. IsdG/I convert protoheme into staphylobilin and releases formaldehyde65. MhuD produces mycobilin, which retains the meso-carbon as a formyl group66. The radical SAM enzyme ChuW degrades protoheme in the absence of O2 and produces a linear tetrapyrrole product anaerobilin68. ChuW presumably functions as a methyltransferase, but the mechanistic details of this reaction are currently unknown. Different derivatives of mycobilin, staphylobilin, and anaerobilin exist, but only one representative structure is shown here for simplicity. (B) The canonical heme oxygenase enzyme catalyzes three steps that each require the participation of O2 to occur6. Sidechains are not shown for simplicity. Purple circles highlight where functional groups have been incorporated during degradation.

Recently, an O2-independent protoheme degradation enzyme ChuW, which uses a radical oxidant to break down protoheme into a linear product anaerobilin, was identified in Escherichia coli O157:H768 (Figure 6A). This enzyme is thought to give pathogens an adaptive advantage in anaerobic niches. ChuW is predicted to be a HemN-like radical SAM enzyme that uses two molecules of SAM to catalyze methylation at an sp2-hybridized carbon center15,68. Like other enzymes in this HemN-like subclass, which are arguably some of the least-well characterized


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Biochemistry

radical SAM enzymes, the mechanism of ChuW has not been established. However, in vitro assays using [d3-methyl]-SAM showed incorporation of two deuterium atoms into product68. This data is consistent with the mechanistic proposals for other members of this class and suggests that 5'-dAdo• abstracts a H-atom from the methyl group of SAM2 to form a methylene radical15 (Figure 6A). Although molecular details for the subsequent chemical steps are lacking, it has been proposed that addition of the methylene radical to protoheme and a rearrangement would result in cleavage of the macrocycle. Final thoughts: Assembly, modification, and degradation of tetrapyrroles is catalyzed by structurally distinct enzymes adapted for environments with different O2 levels. In aerobic organisms, these reactions are catalyzed by enzymes that show variations in architecture, mechanism, and enzyme superfamily, and speak to the diverse ways that nature has of activating O2. In contrast, there are only a few ways to make oxidizing radicals anaerobically, and the mechanisms by which enzymes both oxidize and oxygenate substrates in the absence of O2 remains elusive. In the featured parallel reactions described here, we see that the anaerobic answer uniformly includes 5'-dAdo• produced by a radical SAM enzyme. For these enzymes, the repertoire of chemistry achieved seems to require the addition of a domain and/or cofactor, such as an extra molecule of SAM, auxiliary Fe-S cluster, or Cbl. There are also examples of facultative aerobic/anaerobic organisms for which the O2-dependent and O2-independent enzymes of the tetrapyrrole pathways complement each other. Expression of the required catalyst is controlled at the level of transcription based on environmental signals. In either case, regardless of the enzyme architecture chosen for catalysis, the main challenge is to guide the chemistry following the initial oxidation, whether that includes directing sequential decarboxylation reactions, steering a radical rearrangement reaction for ring opening and formation, or functionalizing an organic moiety with an oxygen atom. Acknowledgments: We thank Dr. Michael Funk and Percival Yang-Ting Chen for their careful reading of this perspective. References: [1] Warren, M. J., Smith, A. G., and SpringerLink. (2009) Tetrapyrroles Birth, Life and Death, In Molecular Biology Intelligence Unit, Springer New York,, New York, NY. [2] Dailey, H. A., Dailey, T. A., Gerdes, S., Jahn, D., Jahn, M., O'Brian, M. R., and Warren, M. J. (2017) Prokaryotic Heme Biosynthesis: Multiple Pathways to a Common Essential Product, Microbiol Mol Biol Rev 81. [3] Fujita, Y., Tsujimoto, R., and Aoki, R. (2015) Evolutionary Aspects and Regulation of Tetrapyrrole Biosynthesis in Cyanobacteria under Aerobic and Anaerobic Environments, Life (Basel) 5, 1172-1203. [4] Ray, K., Pfaff, F. F., Wang, B., and Nam, W. (2014) Status of reactive non-heme metaloxygen intermediates in chemical and enzymatic reactions, J Am Chem Soc 136, 13942-13958. [5] Poulos, T. L. (2014) Heme enzyme structure and function, Chem Rev 114, 3919-3962. [6] Matsui, T., Unno, M., and Ikeda-Saito, M. (2010) Heme oxygenase reveals its strategy for catalyzing three successive oxygenation reactions, Acc Chem Res 43, 240-247.


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[7] Krebs, C., Galonic Fujimori, D., Walsh, C. T., and Bollinger, J. M., Jr. (2007) Non-heme Fe(IV)-oxo intermediates, Acc Chem Res 40, 484-492. [8] Solomon, E. I., Brunold, T. C., Davis, M. I., Kemsley, J. N., Lee, S. K., Lehnert, N., Neese, F., Skulan, A. J., Yang, Y. S., and Zhou, J. (2000) Geometric and electronic structure/function correlations in non-heme iron enzymes, Chem Rev 100, 235-350. [9] Wallar, B. J., and Lipscomb, J. D. (1996) Dioxygen Activation by Enzymes Containing Binuclear Non-Heme Iron Clusters, Chem Rev 96, 2625-2658. [10] Akiva, E., Brown, S., Almonacid, D. E., Barber, A. E., 2nd, Custer, A. F., Hicks, M. A., Huang, C. C., Lauck, F., Mashiyama, S. T., Meng, E. C., Mischel, D., Morris, J. H., Ojha, S., Schnoes, A. M., Stryke, D., Yunes, J. M., Ferrin, T. E., Holliday, G. L., and Babbitt, P. C. (2014) The Structure-Function Linkage Database, Nucleic Acids Res 42, D521-530. [11] Broderick, J. B., Duffus, B. R., Duschene, K. S., and Shepard, E. M. (2014) Radical Sadenosylmethionine enzymes, Chem Rev 114, 4229-4317. [12] Grell, T. A., Goldman, P. J., and Drennan, C. L. (2015) SPASM and twitch domains in Sadenosylmethionine (SAM) radical enzymes, J Biol Chem 290, 3964-3971. [13] Sofia, H. J., Chen, G., Hetzler, B. G., Reyes-Spindola, J. F., and Miller, N. E. (2001) Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods, Nucleic Acids Res 29, 1097-1106. [14] Bridwell-Rabb, J., Grell, T. A. J., and Drennan, C. L. (2018) A Rich Man, Poor Man Story of S-Adenosylmethionine and Cobalamin Revisited, Annu Rev Biochem 87, 555-584. [15] Bauerle, M. R., Schwalm, E. L., and Booker, S. J. (2015) Mechanistic diversity of radical S-adenosylmethionine (SAM)-dependent methylation, J Biol Chem 290, 3995-4002. [16] Zhang, Q., van der Donk, W. A., and Liu, W. (2012) Radical-mediated enzymatic methylation: a tale of two SAMS, Acc Chem Res 45, 555-564. [17] Bridwell-Rabb, J., Zhong, A., Sun, H. G., Drennan, C. L., and Liu, H. W. (2017) A B12dependent radical SAM enzyme involved in oxetanocin A biosynthesis, Nature 544, 322-326. [18] Hanzelmann, P., and Schindelin, H. (2006) Binding of 5'-GTP to the C-terminal FeS cluster of the radical S-adenosylmethionine enzyme MoaA provides insights into its mechanism, Proc Natl Acad Sci U S A 103, 6829-6834. [19] Grove, T. L., Himes, P. M., Hwang, S., Yumerefendi, H., Bonanno, J. B., Kuhlman, B., Almo, S. C., and Bowers, A. A. (2017) Structural Insights into Thioether Bond Formation in the Biosynthesis of Sactipeptides, J Am Chem Soc 139, 11734-11744. [20] Maiocco, S. J., Grove, T. L., Booker, S. J., and Elliott, S. J. (2015) Electrochemical Resolution of the [4Fe-4S] Centers of the AdoMet Radical Enzyme BtrN: Evidence of Proton Coupling and an Unusual, Low-Potential Auxiliary Cluster, J Am Chem Soc 137, 8664-8667. [21] Goldman, P. J., Grove, T. L., Sites, L. A., McLaughlin, M. I., Booker, S. J., and Drennan, C. L. (2013) X-ray structure of an AdoMet radical activase reveals an anaerobic solution for formylglycine posttranslational modification, Proc Natl Acad Sci U S A 110, 85198524. [22] Moore, S. J., Lawrence, A. D., Biedendieck, R., Deery, E., Frank, S., Howard, M. J., Rigby, S. E., and Warren, M. J. (2013) Elucidation of the anaerobic pathway for the


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Biochemistry

corrin component of cobalamin (vitamin B12), Proc Natl Acad Sci U S A 110, 1490614911. [23] Debussche, L., Thibaut, D., Cameron, B., Crouzet, J., and Blanche, F. (1993) Biosynthesis of the corrin macrocycle of coenzyme B12 in Pseudomonas denitrificans, J Bacteriol 175, 7430-7440. [24] Galperin, M. Y., Walker, D. R., and Koonin, E. V. (1998) Analogous enzymes: independent inventions in enzyme evolution, Genome Res 8, 779-790. [25] Zhang, L. (2011) Heme biology : the secret life of heme in regulating diverse biological processes, World Scientific, Singapore ; Hackensack, NJ. [26] Breckau, D., Mahlitz, E., Sauerwald, A., Layer, G., and Jahn, D. (2003) Oxygendependent coproporphyrinogen III oxidase (HemF) from Escherichia coli is stimulated by manganese, J Biol Chem 278, 46625-46631. [27] Layer, G., Verfurth, K., Mahlitz, E., and Jahn, D. (2002) Oxygen-independent coproporphyrinogen-III oxidase HemN from Escherichia coli, J Biol Chem 277, 3413634142. [28] Tanner, A., Bowater, L., Fairhurst, S. A., and Bornemann, S. (2001) Oxalate decarboxylase requires manganese and dioxygen for activity. Overexpression and characterization of Bacillus subtilis YvrK and YoaN, J Biol Chem 276, 43627-43634. [29] Lee, D. S., Flachsova, E., Bodnarova, M., Demeler, B., Martasek, P., and Raman, C. S. (2005) Structural basis of hereditary coproporphyria, Proc Natl Acad Sci U S A 102, 14232-14237. [30] Phillips, J. D., Whitby, F. G., Warby, C. A., Labbe, P., Yang, C., Pflugrath, J. W., Ferrara, J. D., Robinson, H., Kushner, J. P., and Hill, C. P. (2004) Crystal structure of the oxygen-dependant coproporphyrinogen oxidase (Hem13p) of Saccharomyces cerevisiae, J Biol Chem 279, 38960-38968. [31] Lash, T. D. (2005) The enigma of coproporphyrinogen oxidase: how does this unusual enzyme carry out oxidative decarboxylations to afford vinyl groups?, Bioorg Med Chem Lett 15, 4506-4509. [32] Elder, G. H., Evans, J. O., Jackson, J. R., and Jackson, A. H. (1978) Factors determining the sequence of oxidative decarboxylation of the 2- and 4-propionate substituents of coproporphyrinogen III by coproporphyrinogen oxidase in rat liver., Biochem J 169, 215223. [33] Layer, G., Pierik, A. J., Trost, M., Rigby, S. E., Leech, H. K., Grage, K., Breckau, D., Astner, I., Jansch, L., Heathcote, P., Warren, M. J., Heinz, D. W., and Jahn, D. (2006) The substrate radical of Escherichia coli oxygen-independent coproporphyrinogen III oxidase HemN, J Biol Chem 281, 15727-15734. [34] Layer, G., Moser, J., Heinz, D. W., Jahn, D., and Schubert, W. D. (2003) Crystal structure of coproporphyrinogen III oxidase reveals cofactor geometry of Radical SAM enzymes, EMBO J 22, 6214-6224. [35] Ruszczycky, M. W., Choi, S. H., and Liu, H. W. (2010) Stoichiometry of the redox neutral deamination and oxidative dehydrogenation reactions catalyzed by the radical SAM enzyme DesII, J Am Chem Soc 132, 2359-2369. [36] Dailey, H. A., Gerdes, S., Dailey, T. A., Burch, J. S., and Phillips, J. D. (2015) Noncanonical coproporphyrin-dependent bacterial heme biosynthesis pathway that does not use protoporphyrin, Proc Natl Acad Sci U S A 112, 2210-2215.


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[37] Bali, S., Lawrence, A. D., Lobo, S. A., Saraiva, L. M., Golding, B. T., Palmer, D. J., Howard, M. J., Ferguson, S. J., and Warren, M. J. (2011) Molecular hijacking of siroheme for the synthesis of heme and d1 heme, Proc Natl Acad Sci U S A 108, 18260-18265. [38] Dailey, T. A., Boynton, T. O., Albetel, A. N., Gerdes, S., Johnson, M. K., and Dailey, H. A. (2010) Discovery and Characterization of HemQ: an essential heme biosynthetic pathway component, J Biol Chem 285, 25978-25986. [39] Storbeck, S., Rolfes, S., Raux-Deery, E., Warren, M. J., Jahn, D., and Layer, G. (2010) A novel pathway for the biosynthesis of heme in Archaea: genome-based bioinformatic predictions and experimental evidence, Archaea 2010, 175050. [40] Hofbauer, S., Mlynek, G., Milazzo, L., Puhringer, D., Maresch, D., Schaffner, I., Furtmuller, P. G., Smulevich, G., Djinovic-Carugo, K., and Obinger, C. (2016) Hydrogen peroxidemediated conversion of coproheme to heme b by HemQ-lessons from the first crystal structure and kinetic studies, FEBS J 283, 4386-4401. [41] Celis, A. I., Gauss, G. H., Streit, B. R., Shisler, K., Moraski, G. C., Rodgers, K. R., LukatRodgers, G. S., Peters, J. W., and DuBois, J. L. (2017) Structure-Based Mechanism for Oxidative Decarboxylation Reactions Mediated by Amino Acids and Heme Propionates in Coproheme Decarboxylase (HemQ), J Am Chem Soc 139, 1900-1911. [42] Kuhner, M., Schweyen, P., Hoffmann, M., Ramos, J. V., Reijerse, E. J., Lubitz, W., Broring, M., and Layer, G. (2016) The auxiliary [4Fe-4S] cluster of the Radical SAM heme synthase from Methanosarcina barkeri is involved in electron transfer, Chem Sci 7, 4633-4643. [43] Boss, L., Oehme, R., Billig, S., Birkemeyer, C., and Layer, G. (2017) The Radical SAM enzyme NirJ catalyzes the removal of two propionate side chains during heme d1 biosynthesis, FEBS J 284, 4314-4327. [44] Hughes, R. A., Zhang, Y. L., Zhang, R., Williams, P. G., Lindsey, J. S., and Miller, E. S. (2017) Genome Sequence and Composition of a Tolyporphin-Producing Cyanobacterium-Microbial Community, Appl Environ Microb 83. [45] Mauzerall, D. (1976) Chlorophyll and Photosynthesis, Philos T Roy Soc B 273, 287-294. [46] Bollivar, D. W., and Beale, S. I. (1996) The Chlorophyll Biosynthetic Enzyme MgProtoporphyrin IX Monomethyl Ester (Oxidative) Cyclase (Characterization and Partial Purification from Chlamydomonas reinhardtii and Synechocystis sp. PCC 6803), Plant Physiol 112, 105-114. [47] Gough, S. P., Petersen, B. O., and Duus, J. O. (2000) Anaerobic chlorophyll isocyclic ring formation in Rhodobacter capsulatus requires a cobalamin cofactor, Proc Natl Acad Sci U S A 97, 6908-6913. [48] Porra, R. J., Schafer, W., Gad'on, N., Katheder, I., Drews, G., and Scheer, H. (1996) Origin of the two carbonyl oxygens of bacteriochlorophyll a. Demonstration of two different pathways for the formation of ring E in Rhodobacter sphaeroides and Roseobacter denitrificans, and a common hydratase mechanism for 3-acetyl group formation, Eur J Biochem 239, 85-92. [49] Chen, G. E., Canniffe, D. P., and Hunter, C. N. (2017) Three classes of oxygen-dependent cyclase involved in chlorophyll and bacteriochlorophyll biosynthesis, Proc Natl Acad Sci U S A 114, 6280-6285.


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[50] Ouchane, S., Steunou, A. S., Picaud, M., and Astier, C. (2004) Aerobic and anaerobic Mgprotoporphyrin monomethyl ester cyclases in purple bacteria: a strategy adopted to bypass the repressive oxygen control system, J Biol Chem 279, 6385-6394. [51] Gray, M. J., and Escalante-Semerena, J. C. (2007) Single-enzyme conversion of FMNH2 to 5,6-dimethylbenzimidazole, the lower ligand of B12, Proc Natl Acad Sci U S A 104, 2921-2926. [52] Lanz, N. D., Blaszczyk, A. J., McCarthy, E. L., Wang, B., Wang, R. X., Jones, B. S., and Booker, S. J. (2018) Enhanced Solubilization of Class B Radical S-Adenosylmethionine Methylases by Improved Cobalamin Uptake in Escherichia coli, Biochemistry 57, 14751490. [53] Booker, S. J. (2009) Anaerobic functionalization of unactivated C-H bonds, Curr Opin Chem Biol 13, 58-73. [54] Goldman, P. J., Grove, T. L., Booker, S. J., and Drennan, C. L. (2013) X-ray analysis of butirosin biosynthetic enzyme BtrN redefines structural motifs for AdoMet radical chemistry, Proc Natl Acad Sci U S A 110, 15949-15954. [55] Yokoyama, K., and Lilla, E. A. (2018) C-C bond forming radical SAM enzymes involved in the construction of carbon skeletons of cofactors and natural products, Nat Prod Rep 35, 660-694. [56] Tanaka, A., Ito, H., Tanaka, R., Tanaka, N. K., Yoshida, K., and Okada, K. (1998) Chlorophyll a oxygenase (CAO) is involved in chlorophyll b formation from chlorophyll a, Proc Natl Acad Sci U S A 95, 12719-12723. [57] Ho, M. Y., Shen, G., Canniffe, D. P., Zhao, C., and Bryant, D. A. (2016) Light-dependent chlorophyll f synthase is a highly divergent paralog of PsbA of photosystem II, Science 353. [58] Barry, S. M., and Challis, G. L. (2013) Mechanism and Catalytic Diversity of Rieske NonHeme Iron-Dependent Oxygenases, ACS Catal 3. [59] Oster, U., Tanaka, R., Tanaka, A., and Rudiger, W. (2000) Cloning and functional expression of the gene encoding the key enzyme for chlorophyll b biosynthesis (CAO) from Arabidopsis thaliana, Plant J 21, 305-310. [60] Schneegurt, M. A., and Beale, S. I. (1992) Origin of the chlorophyll b formyl oxygen in Chlorella vulgaris, Biochemistry 31, 11677-11683. [61] Kal, S., and Que, L. (2017) Dioxygen activation by nonheme iron enzymes with the 2-His1-carboxylate facial triad that generate high-valent oxoiron oxidants, J Biol Inorg Chem 22, 339-365. [62] Harada, J., Mizoguchi, T., Satoh, S., Tsukatani, Y., Yokono, M., Noguchi, M., Tanaka, A., and Tamiaki, H. (2013) Specific gene bciD for C7-methyl oxidation in bacteriochlorophyll e biosynthesis of brown-colored green sulfur bacteria, PLoS One 8, e60026. [63] Thweatt, J. L., Ferlez, B. H., Golbeck, J. H., and Bryant, D. A. (2017) BciD Is a Radical SAdenosyl-l-methionine (SAM) Enzyme That Completes Bacteriochlorophyllide e Biosynthesis by Oxidizing a Methyl Group into a Formyl Group at C-7, J Biol Chem 292, 1361-1373. [64] Wilks, A., and Burkhard, K. A. (2007) Heme and virulence: how bacterial pathogens regulate, transport and utilize heme, Nat Prod Rep 24, 511-522. [65] Matsui, T., Nambu, S., Ono, Y., Goulding, C. W., Tsumoto, K., and Ikeda-Saito, M. (2013) Heme degradation by Staphylococcus aureus IsdG and IsdI liberates formaldehyde rather than carbon monoxide, Biochemistry 52, 3025-3027.


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[66] Nambu, S., Matsui, T., Goulding, C. W., Takahashi, S., and Ikeda-Saito, M. (2013) A new way to degrade heme: the Mycobacterium tuberculosis enzyme MhuD catalyzes heme degradation without generating CO, J Biol Chem 288, 10101-10109. [67] Wilks, A., and Ikeda-Saito, M. (2014) Heme utilization by pathogenic bacteria: not all pathways lead to biliverdin, Acc Chem Res 47, 2291-2298. [68] LaMattina, J. W., Nix, D. B., and Lanzilotta, W. N. (2016) Radical new paradigm for heme degradation in Escherichia coli O157:H7, Proc Natl Acad Sci U S A 113, 12138-12143.


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Aerobic Enzymes and Their Radical SAM Enzyme Counterparts in

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