A new regime of heme-dependent aromatic oxygenase superfamily

Edited by William F. DeGrado, University of California, San Francisco, CA, and approved August 31, 2021 (received for review May 19, 2021)
October 19, 2021
118 (43) e2106561118


Two histidine-ligated heme-dependent monooxygenase proteins, TyrH and SfmD, have recently been found to resemble enzymes from the dioxygenase superfamily currently named after tryptophan 2,3-dioxygenase (TDO), that is, the TDO superfamily. These latest findings prompted us to revisit the structure and function of the superfamily. The enzymes in this superfamily share a similar core architecture and a histidine-ligated heme. Their primary functions are to promote O-atom transfer to an aromatic metabolite. TDO and indoleamine 2,3-dioxygenase (IDO), the founding members, promote dioxygenation through a two-step monooxygenation pathway. However, the new members of the superfamily, including PrnB, SfmD, TyrH, and MarE, expand its boundaries and mediate monooxygenation on a broader set of aromatic substrates. We found that the enlarged superfamily contains eight clades of proteins. Overall, this protein group is a more sizeable, structure-based, histidine-ligated heme-dependent, and functionally diverse superfamily for aromatics oxidation. The concept of TDO superfamily or heme-dependent dioxygenase superfamily is no longer appropriate for defining this growing superfamily. Hence, there is a pressing need to redefine it as a heme-dependent aromatic oxygenase (HDAO) superfamily. The revised concept puts HDAO in the context of thiol-ligated heme-based enzymes alongside cytochrome P450 and peroxygenase. It will update what we understand about the choice of heme axial ligand. Hemoproteins may not be as stringent about the type of axial ligand for oxygenation, although thiolate-ligated hemes (P450s and peroxygenases) more frequently catalyze oxygenation reactions. Histidine-ligated hemes found in HDAO enzymes can likewise mediate oxygenation when confronted with a proper substrate.
Heme-based enzymes mediate a wide variety of essential chemistry reactions (13). It has been generally regarded that thiolate-ligated heme and histidine-ligated heme differ in the preference of chemical reaction. Indeed, the type of the proximal axial ligand of the heme is paramount to the heme-based chemistry outcomes (47). The thiolate-ligated ferryl intermediates typically carry more oxidizing power than the histidine-ligated counterparts. The thiolate-ligated ferryl hemes are capable of mediating hydrogen atom abstraction and O-atom transfer on unactivated and completely saturated hydrocarbons, whereas the histidine-ligated ferryl intermediates tend to prefer electron transfer reactions. The middle point reduction potentials determined for thiolate- and histidine-ligated heme-based ferryl intermediates are distinct (Scheme 1A) (810), explaining the thermodynamics ground for the tendency of O-atom transfer vs. sequential one-electron transfer in these ferryl-based heme systems.
Scheme 1.
Heme-based oxidation cycles. (A) Redox potential of thiolate or histidine-ligated compounds I and II reported in the literature (810). (B) Cysteine-ligated cytochromes P450, histidine-ligated peroxidases, and cysteine-ligated peroxygenases share a peroxo and a compound I intermediate; L = Cys in cytochromes P450 and peroxygenases, His in peroxidases. (C) The newly defined heme-dependent aromatic oxygenase (HDAO) superfamily performs two major types of reactions on aromatics, dioxygenation, and monooxygenation. The proposed mechanism is illustrated in a pathway primarily based on the current understanding of the IDO/TDO dioxygenation mechanism.
The most well-described heme-based oxidizing enzymes are cytochromes P450, peroxygenases, and peroxidases (Scheme 1B). Cytochromes P450 have a thiolate-ligated heme capable of utilizing both molecular dioxygen and hydrogen peroxide. The O2-dependent reactions in cytochrome P450s require a reductase system, while hydrogen peroxide-mediated reactions take a short circuit of the classic catalytic cycle called catalytic shunt (11). Peroxygenases also employ a cysteine residue as an axial ligand but utilize only hydrogen peroxide as the natural oxidant, but not dioxygen. Peroxidases are histidine-ligated heme enzymes and use hydrogen peroxide to catalyze sequential one-electron oxidation reactions, typically generating organic substrate-based radicals. A high-valent iron(IV) oxo species with porphyrin cation radical, compound I (cpd I), and the one-electron reduced iron(IV) oxo (or ferryl) intermediate, compound II (cpd II), are frequently observed in the heme-dependent chemistry. The strong electron-donating nature of the thiolate ligand enhances the reactivity of cpd I, which is critical for C−H bond activation and protecting the enzyme from self-oxidation (12). The enhancement comes from increased basicity (pKa) of the ferryl unit FeIV=O contributed by the axial thiolate donor. In contrast, histidine-ligated cpd II is more electrophilic due to its less electron-donating nature (13).
Intriguingly, tryptophan 2,3-dioxygenase (TDO) and its sibling enzyme, indoleamine 2,3-dioxygenase (IDO), are among the few exceptions that employ a histidine-ligated heme center to promote oxygen atom transfer from dioxygen to ʟ-tryptophan (1417). A heme-dependent dioxygenase superfamily, therefore, has been named after TDO, that is, tryptophan dioxygenase superfamily (TDO superfamily), which occupies a unique position in heme-based chemistry because of its choice of histidyl-ligated heme as the catalytic cofactor for oxygenation reactions. Recently, a 3-methyl-ʟ-tyrosine hydroxylase, SfmD (18), and a heme-dependent tyrosine hydroxylase (TyrH) (19) have been characterized. The de novo crystal structures and oxygen-dependent reactivity of SfmD and TyrH reveal that these enzymes belong to the TDO superfamily. These latest findings inspired us to revisit this protein group and propose an updated name, that is, a heme-dependent aromatic oxygenase (HDAO) superfamily.

The Structures and Functions of the Members in the Previous Tryptophan Dioxygenase Superfamily

The current TDO superfamily is a small, structurally related group of proteins. Up to 2020, this superfamily had only four founding members, that is, two IDO proteins IDO1 and IDO2, TDO, and PrnB (20). A putative member, MarE, is proposed based on the protein primary structure similarity to the above four members (21). IDO and TDO proteins catalyze the first and committed step of tryptophan degradation in the kynurenine pathway, generating N-formylkynurenine. TDO shows a more strict specificity for ʟ-tryptophan and is a tetrameric enzyme, while IDO is a monomeric enzyme with a relaxed substrate specificity (22, 23).
Since its discovery, when it was named tryptophan pyrrolase in 1936 by Kotake and Masayama (24), TDO has served as one of the historically critical exemplary enzymes demonstrating the direct enzymatic incorporation of molecular dioxygen into an organic compound without the need of additional cofactor or cosubstrate (25). IDO and TDO have demonstrated biological and medicinal significance because the kynurenine pathway metabolites serve as neurotransmission and immune regulators. Malignant tumor cells overexpress IDO/TDO to evade immune surveillance because they are among the immune response checkpoint proteins (2629). The mechanism of IDO and TDO has been described as two-step O-atom incorporations into tryptophan via epoxyindole and ferryl intermediates (Scheme 1C) (3033).
In 2005, the first structure of TDO from Xanthomonas campestris was determined by a group within the Northeast Structural Genomics Consortium, and its coordinates became available as Protein Data Bank (PDB) entry 1YW0. This structure is an apo form lacking the heme prosthetic group. Later in the same year, the first IDO structure became available. The heme incorporated holoenzyme structure of human IDO1 in complex with a weak noncompetitive inhibitor, 4-phenylimidazole, was determined by Shiro and colleagues (34) at the RIKEN Spring-8 Center and deposited to the PDB with an accession number 2D0T. The human IDO1 structure is described by two α-helical domains with a b-type heme bound in the larger domain (34). Later, the structures of heme-bound TDO were also solved from two bacterial sources, X. campestris (35) and Cupriavidus metallidurans (36), and a fruit fly (Drosophila melanogaster) (37). The human TDO structure was initially solved as an apo form (38), and the structure of the holoprotein with the heme bound and in complex with substrate ʟ-tryptophan was determined through the collaboration of the Yeh and Tong laboratories in 2016 (39). Until 2017, the human IDO1 structure was limited to the inhibitor-bound form. Yeh and Poulos solved the human IDO1 structure bound with ʟ-tryptophan and a cyanide-locked heme, and also a complex structure with an IDO1 inhibitor, epacadostat (40). Tong and coworkers (41) achieved higher-resolution IDO1 structures with various inhibitors and a complete substrate-free form using a surface entropy reduction variant of human IDO1. In contrast to IDO1, the TDO structure has only one domain that largely resembles the heme-binding domain of IDO, with a root-mean-square deviation (rmsd) value of 2.21 Å over 182 Cα atoms (Fig. 1A). The substrate-binding pocket and the second coordination sphere residues are highly conserved between these two enzymes (Fig. 1B). Overall, these advances established that IDO and TDO are closely related siblings, and they catalyze a dioxygenase reaction with a histidine-ligated heme and other unique structural features absent in other proteins.
Fig. 1.
Structural comparison of previously established members in histidine-ligated HDAO superfamily. Superpositions were conducted with A chains in PDB entries 2NW8, 6E46, and 2X68 for TDO, IDO, and PrnB. (A) Overall protein structures of TDO (blue) and IDO (white cartoon with red heme). (B) Active site views of TDO (left) and IDO (right). (C) Overall protein structures of PrnB (orange) and IDO (white cartoon with red heme). (D) Active site view of PrnB. Cartoons are colored from light to deep, representing the transition from N to C terminus.
The structurally validated member, PrnB, governs the second enzymatic step of pyrrolnitrin biosynthesis (42, 43). As a natural metabolite isolated from rhizospheric pseudomonads, pyrrolnitrin is widely used to defeat fungal infections (44). PrnB was believed to catalyze a remarkable indole rearrangement and decarboxylation of 7-chloro-ʟ-tryptophan (Scheme 2) and other tryptophan-based secondary metabolites (45, 46). However, the enzyme only exhibited in vitro activity when supplemented with fresh crude cellular extract (20, 46). A more accurate delineation of the catalytic process is required to describe PrnB's chemistry reaction. PrnB is a monomeric enzyme and is structurally more similar to IDO (rmsd of 2.46 Å over 288 Cα) rather than TDO (rmsd of 3.18 Å over 175 Cα), due to the two-domain assembly feature (Fig. 1C) (20). The ternary structure of the PrnB complex bound with substrate and cyanide exhibits an indole orientation that is distinct from the case of IDO/TDO (Fig. 1D) (20), which may explain the specific reaction outcome. Given the high degree of structural similarity, PrnB was considered to share some common reaction intermediates and catalytic features with IDO/TDO, as shown in Scheme 1C (20). PrnB had been previously thought to be a functional outlier of the TDO superfamily (20).
Scheme 2.
The chemical reactions catalyzed by IDO, TDO, MarE, SfmD, TyrH, and PrnB. All the enzymes utilize a mononuclear, histidine-ligated heme for the O-atom transfer reaction(s). The newly identified members of the superfamily, MarE, SfmD, and TryH, perform a monooxygenation type of reaction.
MarE is a functionally characterized monooxygenase in the biosynthetic pathway of maremycins, catalyzing the formation of 2-oxindole product from its native substrate β-methyl-ʟ-tryptophan using molecular dioxygen and ascorbate as cosubstrates (21). Natural products with a 2-oxindole moiety, such as maremycins, exhibit potential anticancer and other biological activities (47). MarE's catalytic cycle was proposed to highly mimic the ones of IDO/TDO and PrnB, with the formation of a superoxo intermediate, followed by the homolytic cleavage of the O−O bond to yield a cpd II−like species and an epoxide intermediate (21). MarE shows 23.83% of sequence identity with a bacterial TDO from C. metallidurans (CmTDO) (SI Appendix, Table S1), containing most of the essential residues conserved in well-known TDO homologs (21). The characterization of MarE enriches the functional diversities of the superfamily by adding a monooxygenation catalytic activity (21). The proposal of MarE as part of the TDO superfamily casts the first doubt about the definition of a heme-dependent dioxygenase superfamily, as it reinforces PrnB for a monooxygenase type of function. However, a three-dimensional structure of MarE is not available yet for unambiguously establishing its proteins superfamily assignment, although it is anticipated to have a similar fold with TDO and constitutes a separate new subgroup in the phylogenetic tree.

Functionally and Structurally Validated New Members of the Dioxygenase Superfamily with a Monooxygenase Activity on Tyrosine-Based Metabolites

Two recent studies on tyrosine-oxidizing enzymes, SfmD and TyrH, with histidine-ligated heme from the largest genus of actinobacteria, Streptomyces, have attracted attention due to their oxygen-utilizing capability and sequence/structure homology to the proteins of the TDO superfamily. These two enzymes do not react with tryptophan metabolites. As shown in Scheme 2, SfmD is a 3-methyl-ʟ-tyrosine hydroxylase in the biosynthetic pathway of saframycin A (48). SfmD can utilize hydrogen peroxide, or molecular dioxygen with ascorbate as a cosubstrate, to produce 3-hydroxy-5-methyl-ʟ-tyrosine (18). The crystal structure of SfmD has been characterized as a homolog of TDO superfamily containing a novel c-type heme cofactor with a single thioether covalent linkage and a bis-histidine ligand set in the unique HxnHxxxC (n ≈ 38) heme-binding motif (18). The overall structure of SfmD resembles those other members of the superfamily (Fig. 2A). In particular, the C terminus of SfmD aligns with CmTDO with an rmsd of 2.26 Å for 102 Cα atoms (18). Although SfmD shares less than 20% of sequence identity with the founding members of the superfamily, the crystal structure establishes SfmD as a structural homolog of the superfamily.
Fig. 2.
Structural alignments of SfmD and TyrH with members of HDAO superfamily. Superpositions were conducted with A chains in PDB entries of 6VDQ, 7KQR, 2NW8, 6E46, and 2X68 for SfmD, TyrH, TDO, IDO, and PrnB. (A) SfmD (green) with TDO (Left), IDO (Middle), and PrnB (Right). (B) TyrH (purple) with TDO (Left), IDO (Middle), and PrnB (Right). (C) TyrH (purple) with SfmD (green). Cartoons are colored from light to deep, representing the transition from N to C terminus. (D) Heme prosthetic groups and substrates/ligands of SfmD (green) and TyrH (purple); and TyrH (purple), TDO (blue), IDO (red), and PrnB (orange) in superposed structures. SfmD structure is the substrate-free form.
LmbB2 and Orf13 are heme-dependent TyrH found in the biosynthetic pathway of lincomycin and anthramycin, promoting the formation of 3,4-dihydroxy-ʟ-phenylalanine (ʟ-DOPA) from ʟ-tyrosine (4951). A homolog from a thermophilic bacterium Streptomyces sclerotialus (SsTyrH) has been functionally and structurally characterized recently (19). SsTyrH shows structural homology with the members of the TDO superfamily (Fig. 2B). The crystal structure of SsTyrH superposes SfmD (Fig. 2C). Despite the fact that SfmD and TyrH enzymes catalyze very similar hydroxylation reactions on tyrosine metabolites, the SsTyrH crystal structure reveals a b-type histidine-ligated heme, in contrast to the c-type bis-histidyl−ligated heme in SfmD. It has also been noted that the heme position of SsTyrH aligns with those of the TDO superfamily members but differs from the heme position of the resting state structure of SfmD (Fig. 2D).
Like MarE, SfmD and TyrH can utilize molecular oxygen as the oxidant in the presence of ascorbate, even though the in vitro activities are approximately two orders of magnitude slower compared to the reaction with hydrogen peroxide: SfmD, kcat = 32.4 ± 1.6 min−1 with H2O2, kcat = 0.029 ± 0.001 mM−1min−1 with ascorbate and O2; Orf13 (TyrH), kobs = 34 ± 4 min−1 with H2O2, kobs = 0.58 ± 0.01 min−1 with ascorbate and O2 (18, 50). It is noteworthy that these enzymes are not designated to operate rapidly in cells because they are not laid in a metabolic pathway of energy extraction or cell proliferation but rather for synthesizing natural products with antimicrobial properties. If those enzymes were highly efficient, the essential aromatic amino acid nutrients, ʟ-tryptophan and ʟ-tyrosine, would be depleted quickly, thereby impairing cell survival. Considering the tight control of hydrogen peroxide concentration in the cell context, the activities of these aromatic acid-oxidizing enzymes may be restricted by the limited availability of hydrogen peroxide and a slower dioxygen utilization.

Endogenous Ligand Dissociation

SfmD is an outlier because of its unusual hexacoordinated and monocovalently attached heme (18). The substrate-induced reversible intramolecular coordination by a histidine (His274) is not a common characteristic of heme proteins involved in oxygen binding (Fig. 3). The heme ligand dissociation is accompanied by an active site reorganization in the distal pocket and the heme prosthetic group (18). The most closely related endogenous heme ligand dissociation is described in a growing number of proteins such as bis-histidine−ligated hemoglobin (52), neuroglobin (53), and ascorbate peroxidase W41A variant (54). The ligand dissociation during catalysis is an interesting observation to the bioinorganic community.
Fig. 3.
(A) redox-linked, or organic substrate-induced non−redox-linked, dissociation of the endogenous heme ligand (His274) in SfmD. (B) The heme active site of TyrH in the 1.58-Å-resolution crystal structure of the ferric heme-bound hydroperoxo (compound 0) intermediate (PDB entry 7KQU) (19). The substrate is shown in wheat color. Interactions between 3-fluoro-ʟ-tyrosine and TyrH are shown with yellow dotted lines. Hydrogen bonding networks are presented with dark gray dotted lines. Since SfmD catalyzes a similar type of chemistry, an analogous substrate binding mode is expected in SfmD. It requires breaking the bis-His locked heme and dissociation of His274 to allow the binding of an oxidant, that is, O2 in the ferrous heme state and H2O2 in the ferric state.
The metal oxidation state is an essential factor for bonding interactions between ligand orbitals and the d orbitals. A redox-induced conformational change is a regulatory strategy common in biology. Such a phenomenon is often observed in sensor systems, such as human Pirin (55). The ligand dissociation in responding to oxidation enables Pirin to interact with NF-κB to turn on the transcription of redox-related genes, thereby acting as a redox-sensing molecular switch.
The redox-induced endogenous heme ligand dissociation or switching is also known in enzymatic systems. It has been reported that the b-type heme-dependent 15-cis-ζ-carotene isomerase has axial ligand switching between His266 and Cys263 with a fixed proximal ligand His150 in the ferric state. In the reduced state, NO/CO binding is observed, indicating the displacement of the axial ligand (56). Cytochrome cd1, nitrite reductase, has His200/Tyr25 ligation in the d1 heme site and His69/His17 ligation in the c heme site. Upon reduction, Tyr25 dissociates for substrate binding, and the axial ligand switching from His17 to Met106 occurs due to the concomitant refolding of the cytochrome c domain (57). Cytochrome c peroxidase furnishes a high-potential heme bound through a His/Met pair and a low-potential heme ligated with bis-His. Upon reducing the high-potential heme, one of the His ligands of the low-potential heme swings off to the surface to open the spot for peroxide binding (58). Thiosulfate dehydrogenase has c-type diheme ligated with His53/Cys96 and His64/Lys208 pairs. Reduction causes the dissociation of Cys96 for substrate binding and the replacement of Lys208 with Met209 (59).
It is worth mentioning that redox-linked ligand dynamics is not limited to hemoproteins. It has also been reported in nonheme diiron proteins. For example, cyanide-insensitive alternative oxidase (AOX) is a parasite terminal oxidase protein from Trypanosoma brucei, which catalyzes the four-electron reduction of dioxygen to water in a mammalian host’s bloodstream. The histidine ligands of the diiron center in trypanosomal AOX dissociate and reassociate during the oxidation−reduction cycle (60).
While the redox-linked ligand dissociation is easier to understand from a bioinorganic chemistry perspective, the organic substrate-triggered ligand dynamics is more challenging to interpret at the molecular and submolecular levels. His274, rotating off from the ferric heme center in SfmD upon 3-methyl-ʟ-tyrosine binding (Fig. 3A), is an interesting phenomenon. Here, the primary substrate binding induces an endogenous ligand dissociation, but it does not directly coordinate the heme iron. It is more thought-provoking to explore the driving force of ligand dissociation.
A well-documented case is found in a “maquette” protein that contains a bis-histidine−ligated heme in a helix bundle. One histidine ligand dissociates from the heme iron upon O2 or CO binding (61). The de novo designed metalloproteins have provided additional insights. This ligand dissociation is explained by helical rotation, thereby imposing a helical strain that weakens the histidine ligation and allows competition from other ligands such as O2 and CO. In nonheme iron proteins, it has long been known that the tyrosine ligand (Tyr447) in an intradiol aromatic ring-cleaving dioxygenase dissociates and is replaced by the protocatechuate substrate (62). The endogenous ligand dissociation results from the direct displacement by a substrate hydroxyl moiety, and a requirement to retain a constant charge at the metal center is the likely driving force. Another related example is a de novo designed four-helix bundle “Due Ferri” (diiron) protein intended to stabilize the semiquinone (SQ) free radical form of 3,5-di-tert-butylcatechol, SQ (63). A de novo designed [DFsc-Zn(II)2] possesses redox-inert transition metal Zn(II) in place of diferric ions, preventing two-electron chemistry of quinone (Q) from its reduced form (QH2) to the oxidized state and stabilizing SQ instead. The histidine ligand, His107 in the starting pentavalent Zn(II), rotates out to create two open coordination spots for SQ upon SQ binding and forms a tetravalent Zn(II) intermediate, which then acquires SQ to become six-coordinated metal ion (63). In these cases, a probable driving force is that substrate binding induces an entatic state (64), which weakens metal−ligand interactions to create an open spot(s) for direct substrate ligation.
In contrast, substrate binding-triggered ligand dissociation in SfmD must have a different mechanism because neither ligand displacement nor a redox state shift of the metal ion is required. One observation is that this event leads to a substantial change of the protein structures, so much so that a new crystallization condition must be pursued for the ES complex. We hypothesize that substrate binding to SfmD induces a shift of the ligand geometry or heme orientation to mimic what has been found in TyrH (Fig. 3B) (19), thus imposing a leaving force on His274. Indeed, substantial modification of the angular orientation of the His274 ligand is observed along with heme orientation shift in the partially reduced crystal structures of SfmD (18). Similar angular orientation change of the His ligand in the ligand binding and dissociation has been described in CO-bound nitric oxide reductase during photolysis (65). Likewise, heme orientation modulated histidine dissociation has been shown in neuroglobin (53).
The amine group of the ʟ-tyrosine bound in TyrH has no direct contact with a protein residue but forms H-bond interactions with a heme propionate group and surrounding protein backbone through a water molecule (Fig. 3B). ʟ-tyrosine is a known alternate substrate of SfmD, and the reaction product is the same as in the TyrH reaction. Considering the very same hydroxylation chemistry on ʟ-tyrosine and similar protein fold of the enzymes, the substrate-bound form of SfmD is expected to have a similar binding mode to ʟ-tyrosine with TyrH (Fig. 3B). Substrate recognition through the ionic interactions of carboxylate with a corresponding arginine residue and H-bond networks of the substrate amine with protein backbone by mediating water molecules would propagate to the heme center locked with bis-histidine in the resting state, thereby opening up the sixth coordination site. Such a feature of the heme ligand dissociation and active site reorganization in SfmD prevents unwanted reactions. Additionally, the methyl group of 3-methyl-ʟ-tyrosine provides another layer of protection for ʟ-tyrosine, as 3-methyl-ʟ-tyrosine is a more efficient substrate than ʟ-tyrosine itself (48).

Bioinformatics Evidence Suggests That the Current TDO Superfamily Is a More Sizeable Protein Group

We analyzed the primary sequences of MarE, SfmD, and TyrH in the phylogenetic context to obtain the evolutionary relationship between the dioxygenases and monooxygenases in the superfamily. The phylogenetic tree is consistent with these proteins belonging to the currently known tryptophan dioxygenase superfamily (Fig. 4A). The phylogenetic results show that TDO proteins are grouped in three clades on the left side of the tree: prokaryotic TDOs, eukaryotic TDOs, and uncharacterized putative TDOs. On the right side, IDO proteins form a distinct clade. Human IDO1 is found on the branch of myoglobin proteins from abalone (or vice versa) (66). SfmD belongs to a clade between TDO and IDO. The clade to its immediate left contains MarE (21). The clade residing to the right side of SfmD is the TyrH group, three of which have been functionally validated, that is, LmbB2, Orf13, and SsTyrH (19, 49, 50). Next to IDO proteins, PrnB and its homologs represent a small and isolated clade. The status quo as it pertains to the function of PrnB is that it catalyzes a monooxygenation reaction of 7-chloro-ʟ-tryptophan, followed by an indole rearrangement and decarboxylation (20). However, it remains possible that the reaction involves an additional enzyme for the chemistry after monooxygenation (67). The sequence identity between the representative members is summarized in SI Appendix, Table S1. The UniProt entries for building up the phylogenetic tree are listed in SI Appendix, Table S2.
Fig. 4.
Bioinformatic analyses reveal a structure-based heme-dependent oxygenase superfamily. (A) Phylogenetic tree analysis of the current HDAO superfamily. From the left, the blue clade is prokaryotic TDOs. Sky blue clade is eukaryotic TDOs. A purple cluster is for unidentified TDOs. The green, light green, and yellow clades are MarE-like proteins, SfmD and its homologs, and TyrH proteins, respectively. The orange clade is for PrnB proteins. The red clade is for IDOs. (B) Sequence similarity network for SfmD and TyrH in the context of the superfamily (IPR037217). An E value of 1 × 10−4 was used for edge generation, and sequences with 50% identity were clustered into single nodes. Red dots are IDO proteins: 1, hIDO2; 2, rIDO2 and mIDO2; 3, hIDO1, mIDO1, and rIDO1. Orange dots are PrnB proteins: 4, PrnB. Yellow dots are TyrH proteins: 5, Orf13; 6, LmbB2; 7, the protein sharing 87% sequence identity with SsTyrH (A0A2U3BXY4). Light green dots are SfmD and its homologs: 8, SfmD; 9, SfcD. Dark green dots are MarE and its homologs: 10, MarE; 11, ACPL_6188. Purple dots are unidentified TDOs in the tree diagram. Sky blue dots are eukaryotic TDOs: 12, hTDO; 13, DmTDO. Dark blue dots are prokaryotic TDO proteins: 14, CmTDO; 15, XcTDO. The isolated group at the bottom of IDOs includes some proteins annotated as putative 15-hydroxyprostaglandin dehydrogenase. The isolated clusters at the top of TDOs contain proteins annotated as HSPRO1/HSPRO2 and domain-containing proteins.
To explore the protein association with IDO/TDO-like proteins, we constructed sequence similarity networks for SfmD and TyrH using the Enzyme Function Initiative–Enzyme Similarity Tool (EFI-EST web server) (68) with the InterPro family number of IDO/TDO-like homologous superfamily, IPR037217 (Fig. 4B). In the network, the TDO superfamily forms a body in which IDO proteins are located in one direction (bottom right in the figure), and TDO proteins are situated in the opposite direction (top left). Some of the known IDO/TDO proteins are marked with blue (prokaryotic TDO), light blue (eukaryotic TDO), and red dots (IDO) with black circles in Fig. 4B. Uncharacterized putative TDO proteins in the tree diagram (Fig. 4A) are also found in the TDO cluster (purple dots). PrnB-like proteins form a separate cluster from IDO proteins (orange dots, bottom left). SfmD and its homologs form a distinct cluster connected the TDO cluster with several edges, and IDO cluster with a single connection. The primary sequence of SfmD is thus closely connected to that of the IDO/TDO proteins (light green dots). TyrH, including the representative members, Orf13, LmbB2, and SsTyrH, locates next to SfmD proteins forming an isolated group (yellow dots). MarE and its homologs are found at the rim of the TDO cluster (dark green dots). Taking into consideration the distant relationship between IDO and TDO in the phylogenetic and sequence similarity network (SSN) analyses, the presence of PrnB, which has been the only functional outlier, encourages filling the gap with new members with functional diversity. The phylogenetic and SSN analyses are consistent with the structural advances of the members of this group of proteins. Together, they suggest that the monooxygenase and dioxygenase members share the same ancestor.

The Heme-Dependent Dioxygenase Superfamily Is Functionally More Diverse

A protein superfamily is established based on similarities of protein sequence, structure, and mechanism. Common ancestry can be inferred from structural alignment and mechanistic similarity, although sequence similarity is not evident. SfmD, TyrH, and their homologs show less than 20% sequence identity with other members of the current TDO superfamily. These enzymes might be easily neglected in the relationship with the superfamily. However, the crystal structures of SfmD and SsTyrH determined in the recent studies enabled a structure-based alignment. The current advances unambiguously assign SfmD and TyrH to the TDO superfamily. In particular, not merely global structural similarity but also the heme position of TyrH match with those of the known members of the superfamily. Since the substrate-free form of SfmD structure is the only outlier regarding the heme position, it is experimentally established with induced ligand dissociation and active site reorganization, including the heme cofactor. Further investigation is needed for the catalytically relevant heme position in SfmD from a substrate-bound structure.
Previous studies have shown that dioxygenation mediated by IDO/TDO is an overall outcome of two consecutive monooxygenation reactions (31, 33). MarE, SfmD, and TyrH mediate only one O-atom transfer to the respective primary substrate, while the second oxygen is possibly reduced to a water molecule under the assistance of ascorbate. PrnB also shares the primary monooxygenation but diverges to decarboxylation and indole rearrangement. This intriguing and complex process requires further studies with isolated protein. Therefore, monooxygenation presumably underlies the early steps of the shared mechanism of the superfamily. In the presence of ʟ-tryptophan, the ferric TDO can be reactivated with H2O2 via a ferryl intermediate (69). The reported peroxygenation activity with IDO by converting indole into oxindoles with H2O2 suggests that the utilization of H2O2 is an intrinsic attribute of the HDAO superfamily analogous to the catalytic shunt of the P450 enzymes (70). A clade of the proteins is tentatively labeled as “unidentified TDO” (Fig. 4A). Future characterization of the members of this subgroup may reveal activities that further expand the functional diversity of the superfamily.

O-Atom Transfer Reactions by a Histidine-Ligated Heme Center

The ability of O-atom transfer to an organic substrate with a histidine-ligated heme center by this enzyme superfamily prompted a new view of heme-based chemistry. The governing factor for the reaction outcome is not merely dependent on the type of the proximal axial ligand. We envision that the chemical structure (all are aromatics thus far for this superfamily) and positioning of the substrate and the surrounding protein environment rendered by second coordination sphere residues play crucial roles in dictating the reaction. A histidine residue, His88, in the distal heme pocket, has been identified as catalytically essential for the monooxygenation mediated by TyrH (Fig. 3B) (19). To date, there is no report of the midpoint reduction potentials for cpd I and II in the HDAO superfamily. Whether or not the second coordination sphere promotes the formation of a more powerful oxidant than a typical histidine-ligated heme remains to be investigated.
It is interesting to compare the approximate strength of C−H bonds activated by these His-ligated heme proteins versus the strength of the C−H bonds activated by cytochromes P450. Because the C–F bond is stronger than the C–H bond regardless of the hybridization of the carbon, and the C–F bond is stronger in sp2 than in sp3 carbons (71), one indicator is how these enzymes activate substrates containing a C−F bond in sp2 carbons. Regarding aromatic C−F bond activation, His-ligated heme in HDAOs is sufficient to achieve the same goal of oxygen atom transfer chemistry as thiolate-ligated heme, at first glance (19, 49). However, their substrates are, so far, limited to fluorine substitution next to the hydroxyl group of a phenol, that is, 2-fluorophenol and 2,6-difluorophenol. In contrast, cytochromes P450, such as 1A2 and 3A4 (72), can activate monofluorobenzene type of substrates (73). This difference indicates the thiolate-ligated heme centers can generate more powerful high-valent ferryl-based intermediates than those in HDAOs and, hence, are more widely used in O-atom transfer reactions.
Nevertheless, some P450 enzymes are outliers and mediate peroxidase type of reactions. A peroxidase-like activity has been reported on bacterial cytochromes P450, such as CYP119A1, CYP102A1, CYP152A1, and CYP101A1 (74). The most recently characterized example is CYP121, which generates free radicals on its substrate for a C−C bond coupling reaction (75, 76). The oxygenation reaction is not the desired outcome, although the enzyme is shown to be capable of oxidizing a methyl group when a methoxylated substrate is in place of the native dityrosine substrate (77). Hence, the chemical identity of the proximal ligand is a primary factor, but the substrate structure, positioning at the enzyme active site, and the protein matrix, especially the second coordination sphere, often also play critical roles in determining the reaction outcomes, such as showcased in CYP121 and the bifunctional enzyme KatG (7880). In the catalytic cycle of TDO, the amino group of the substrate has been experimentally demonstrated to play a role in assisting the second O-atom transfer step (33). It is worth noting that there are multiple dioxygenase members in the superfamily for ʟ-Trp oxidation, but no dioxygenase members for oxidation of ʟ-Tyr or analogs have emerged.


Aromatic amino acid−derived compounds are prevalent and play crucial roles in biology. A group of hemoproteins using tryptophan, tyrosine, or their metabolites as the native substrates produces oxygenated compounds in amino acid metabolism or biosynthetic pathways. The current concept of the heme-dependent dioxygenase superfamily emphasizes dioxygenation but undermines other functions. The addition of MarE, SfmD, TyrH, and a currently unannotated clade of proteins revealed from the bioinformatics study, vide supra, indicates that the members of this structure-based superfamily utilize a histidine-ligated heme for mediating dioxygenation, monooxygenation, and possibly other types of reactions after the initial monooxygenation. Thus, recent advances established a pressing demand to revise the current version of the heme-dependent dioxygenase superfamily. We suggest revising the current tryptophan dioxygenase superfamily to a histidine-ligated heme-dependent aromatic oxygenase (HDAO) superfamily. The revised name of the superfamily would better define its position in the heme-dependent enzymes with other superfamilies, including histidine-ligated heme-dependent peroxidases, thiol-ligated heme-dependent peroxygenases, and cytochromes P450. This update emphasizes that hemoproteins might not be as strict with their choice of protein-derived axial ligand for achieving certain types of functions, including H-atom abstraction and O-atom transfer. The chemical structure of the substrate and the need for the desired catalytic outcome also plays a role in the choice of the axial ligand.


The details of building the phylogenetic tree diagram and the sequence similarity network are described in SI Appendix.

Data Availability

All study data are included in the article and SI Appendix.


This article was supported by the Lutcher Brown Distinguished Chair endowment fund. Our research work in the related subject is supported by NIH Grant GM108988.

Supporting Information

Appendix 01 (PDF)


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Published in

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Proceedings of the National Academy of Sciences
Vol. 118 | No. 43
October 26, 2021
PubMed: 34667125


Data Availability

All study data are included in the article and SI Appendix.

Submission history

Accepted: August 31, 2021
Published online: October 19, 2021
Published in issue: October 26, 2021


  1. heme
  2. dioxygenase
  3. hydroxylase
  4. axial ligand
  5. superfamily


This article was supported by the Lutcher Brown Distinguished Chair endowment fund. Our research work in the related subject is supported by NIH Grant GM108988.


This article is a PNAS Direct Submission.



Department of Chemistry, The University of Texas at San Antonio, San Antonio, TX 78249
Department of Chemistry, The University of Texas at San Antonio, San Antonio, TX 78249
Present address: Department of Chemistry, University of Georgia, Athens, GA 30602.
Department of Chemistry, The University of Texas at San Antonio, San Antonio, TX 78249


To whom correspondence may be addressed. Email: [email protected].
Author contributions: A.L. designed research; I.S. performed research; I.S. and Y.W. analyzed data; and I.S., Y.W., and A.L. wrote the paper.

Competing Interests

The authors declare no competing interest.

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    A new regime of heme-dependent aromatic oxygenase superfamily
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