A Rhodanese‐Like Enzyme that Catalyzes Desulfination of Ergothioneine Sulfinic Acid

Many actinobacterial species contain structural genes for iron‐dependent enzymes that consume ergothioneine by way of O2‐dependent dioxygenation. The resulting product ergothioneine sulfinic acid is stable under physiological conditions unless cleavage to sulfur dioxide and trimethyl histidine is catalyzed by a dedicated desulfinase. This report documents that two types of ergothioneine sulfinic desulfinases have evolved by convergent evolution. One type is related to metal‐dependent decarboxylases while the other belongs to the superfamily of rhodanese‐like enzymes. Pairs of ergothioneine dioxygenases (ETDO) and ergothioneine sulfinic acid desulfinase (ETSD) occur in thousands of sequenced actinobacteria, suggesting that oxidative ergothioneine degradation is a common activity in this phylum.


Introduction
Ergothioneine (1, Figure 1) is a ubiquitous sulfur-containing natural product that emerges from a remarkable diversity of bacterial and fungal biosynthetic activities. [1]Most plants and animals cannot produce 1, but instead, absorb this compound from their nutrition/environment via specific transporters and accumulate it in specific tissues. [2]Because 1 readily reacts with toxic oxygen species, including hydrogen peroxide or singlet oxygen, this compound may participate in cellular protection against oxidative stress. [3]In contrast to these higher eukaryotes in which cellular 1 is consumed predominantly by uncatalyzed and unspecific redox reactions, a broad range of bacteria have evolved specific enzymes that convert 1 to glutamate, formic acid, hydrogen sulfide, and ammonia via a redox neutral pathway. [4]Most bacteria hosting these catabolic enzymes do not produce 1 and therefore depend on ABC-type transporters for uptake from the environment. [5]More recent work revealed that many actinobacterial species contain iron-dependent ergothioneine dioxygenase (ETDO) that specifically oxidizes 1 to ergothioneine sulfinic acid (2, Figure 1). [6]The discovery of this oxidative degradation pathway is remarkable because almost all actinobacteria are also able to produce 1.Hence, these species contain a fully integrated metabolic cycle allowing autonomous and active regulation of the cellular concentration of 1.
In vitro characterization of ETDOs also revealed that the product of this enzyme -2 -is far more stable at neutral pH than previously thought. [7]Indeed, we showed that some actinobacteria encode a metal-dependent ergothioneine sulfinic acid desulfinase (ETSD) that catalyzes the cleavage of 2 into sulfur dioxide (SO 2 ) and Nα-trimethyl histidine (3, Figure 1). [6]ETSDs are usually encoded in the same locus as ETDOs, adding evidence to the idea that the two enzymes cooperate in the degradation of 1. Upon further inspection of more actinobacterial genomes, however, we realized that genes coding for ETDOs are far more common than genes for ETSDs.Hence, we surmised that either a) not all ETDO homologs produce sulfinic acids that require desulfination, or b) desulfination may also be catalyzed by a different type of enzyme.In this report we describe evidence in support of the second explanation: the majority of actinobacterial ETDOs are ergothioneine-specific dioxygenases that are either fused to or co-encoded with a single-domain rhodanese-like protein that catalyzes desulfination of 2. This desulfinase is a rare example of a rhodanese-like enzyme that catalyzes a lyase reaction instead of a transferase reaction.

Results and Discussion
We have previously described the activity of the ETDO homologs of Thermocatellispora tengchongensis (TtETDO) and Nocardioides sp.(NoETDO). [6]Both species also encode a metaldependent ETSD.In contrast, the majority of the 5000 closest homologs of TtETDO or NoETDO occur in species that do not encode putative metal-dependent ETSDs.Instead, 39 of these 5000 ETDO homologs are fused to a C-terminal 120-residue rhodanese-like domain with a conserved Cys residue (Table S1).An inspection of 30 additional genomes from this set revealed that similar rhodanese-like proteins are often co-encoded with ETDO homologs (Table S2), suggesting that these rhodaneselike proteins may play the same role as the metal-dependent ETSDs in oxidative degradation of 1.Alternatively, because ETDOs that are associated with rhodanese-like proteins and ETDOs associated with metal-dependent ETSDs form phylogenetically distinct groups (A, Figure 2), it is also possible that the two families of EDTOs may have different functions.
To distinguish between these two possibilities, we examined two exemplary ETDO homologs from organisms that do not encode recognizable homologs of metal-dependent ETSDs: ETDO from the acidobacterium Occallatibacter savannae (OsET-DO, WP 109487074.1)and from the actinobacterium Lentzea atacamensis (LaETDO, A0 A316IGH2).OsETDO is a 318-residue protein with a C-terminal rhodanese-like protein.LaETDO contains only 192 residues but is encoded next to a 129-residue rhodanese-like protein (A0 A316I8G8).Despite the limited similarity of TtETDO (< 38 % sequence identity) OsETDO and LaETDO contain the same active site residues that have been found essential for the activity of TtETDO. [6]The most important active site residues are the three His residues that form the iron-binding site (His88, 80, and 134 in TtETDO), the catalytic residues His147 and Tyr149 that promote O 2 activation, and residues Arg61, Trp75, and Tyr63 that mediate specific recognition of 1 as substrate (B, Figure 2).To measure their in vitro activities, we produced OsETDO and LaEDTO in Escherichia coli.SDS-PAGE analysis of the recombinant proteins after purification by Ni II -agarose affinity chromatography revealed high homogeneity of LaEDTO (> 95 %) and moderate homogeneity of OsETDO (> 60 %, Figure S1).The identity of both proteins was confirmed by high-resolution electrospray ionization mass spectrometry (HR-ESI-MS, Table S3).The catalytic activity was assessed by monitoring the rate at which 1 was consumed using an HPLC assay (Table 1). [6]Under these conditions, LaETDO catalyzes dioxygenation of 1 with similar efficiency as the previously characterized homologs TtETDO and NoETDO (Table 1, Figures S2 and S3). [6]The efficiency of OsETDO, on the other hand, is at least six-fold lower than that of the other ETDOs.As the previously characterized ETDOs, OsETDO does not consume 2-thiohistidine or 2-mercapto imidazole, suggesting that the betaine moiety of 1 is essential for binding to the Figure 2. A: phylogenetic tree of representative actinobacterial ETDOs, including 3-mercaptopropionic acid dioxygenase from Azotobacter vinelandii (PDB code: 6XB9), [8] 3-mercaptopropionate dioxygenase from Pseudomonas aeruginosa (4TLF), [9] and rat cysteine dioxygenase (6 U4S) [10] as outgroup.Green: ETDOs associated with metal-dependent ETSDs; red: ETDOs associated with rhodanese-like ETSDs.Gray: The probability of main inferred branches to be true (aLRT).Sequence alignment: MUSCLE; [11] Phylogenetic tree calculation: PhyML 3.1/3.0aLRT (approximate likelihood-ratio test); [12] Phylogenetic tree presentation: TreeDyn. [13]B: Sequence alignment of characterized ETDO homologs.Residue numbering is according to TtETDO.The involvement of residues Arg61, His147, and Tyr149 has been confirmed by site-directed mutagenesis. [6]C: Proposed substrate binding mode inferred from the crystal structure of TtETDO.
active site (Figure S4). [6]These observed specific activities, combined with the conserved pattern of active site residues are consistent with the idea that OsETDO and LaETDO are active as ergothioneine dioxygenases, and hence, that ETDOs from the two phylogenetic branches are functionally equivalent (Figure 2).
Despite their similar dioxygenase activity, NoETDO and OsETDO produce different accumulating products.NoETDOcatalyzed consumption of 1 leads to the accumulation of 2, as inferred by 1 H NMR (Figure S5).By contrast, the bifunctional OsETDO converted 1 directly to 3, suggesting that the Cterminal rhodanese-like domain mediates the cleavage of 2 to 3 and SO 2 .Indeed, OsETDO-catalyzed desulfination of 2 is characterized by a turnover frequency of 0.33 s À 1 and a catalytic efficiency of 300 M À 1 s À 1 (Table 1, Figure S6).To examine as to whether these enzymes can degrade 1 in vivo we cultivated NoETDO or OsETDO-producing BL21 E. coli cells in LB medium supplemented with 50 mg/L of kanamycin, 1 mM of 1, and 1 mM isopropyl ß-D-1-thiogalactopyranoside (IPTG) for 20 h at 20 °C.The washed and pelleted cells were extracted with an acidic mixture of D 2 O and MeOD.HR-ESI-MS analysis of these extracts revealed that NoETDO-producing cells contained 3 and deuterated 3 in a 1 : 3 ratio, indicating that these cells have absorbed and oxidized 1 to 2. Some of this sulfinic acid decayed to 3 during the 20 h incubation, but most of 2 was desulfinated during the acidic extraction leading to the observed ratio of deuterated (3-d) and nondeuterated 3.In contrast, the extracts from cells containing OsETDO contained 3 but no detectable 3d, suggesting that 2 was completely consumed by the desulfinase during the 20 h incubation.In contrast, extracts from cells containing a variant of OsETDO with a mutated active site Cys (OsETDO C274A ) contained a 1 : 1 mixture of 3-d and 3, suggesting that the desulfinase activity of this enzyme is deficient (Figure 3).This experiment confirms that a) ETDOs are active in E. coli, b) that the sulfinic acid 2 can accumulate under physiological conditions, and c) that Cys274 is critical for the desulfination activity of OsETDO.
Consistent with a functional role of Cys274, this residue is strictly conserved among ETDO-associated rhodanese-like enzymes.Comparison of the α-fold model of the C-terminal module of OsETDO with the X-ray structures of related proteins shows that this residue is indeed located in the typical active site of rhodanese-like enzymes (Figure S7).In previously characterized sulfur transferases and phosphatases with this fold the active site Cys makes direct contact with the guanidinium side chain of an equally conserved Arg at the of an active site loop (Figure S7).This interaction is important to stabilize the anionic form of Cys before nucleophilic attack onto electrophilic sulfur or phosphor-centers, and stabilizing resulting anionic persulfen-  a) The values given in this table are averages from at least two independent measurements.Catalytic activities of OsEDTO and LaEDTO were determined at 25 °C, in phosphate-buffered solution (50 mM, pH 7.4), in the presence of 0.2 mM ascorbate, 10 μM FeSO 4 , 1 μM enzyme, and 0. 08-6.5 mM 1. Timedependent product formation was quantified by ion exchange high-pressure liquid chromatography (IE-HPLC).The desulfinase activity of OsEDTO was determined in the same buffer, in the presence of 0.1-1.3mM 2. Time-dependent product formation was quantified by UV-Vis at 254 nm.The corresponding Michaelis-Menten plots are shown in the supporting information (Figures S2, S3, and S6).b) Data from Ref [6].ate or thiophosphate intermediates. [14]In ETDO-associated rhodanese-like enzymes, this Arg is replaced by Ala, Ser, or Thr.Among the 5000 most related homologs of the C-terminal module of OsETDO, more than 95 % contain a conserved CÀ X-(E/D)-G-(Y/F/W)-(A/S/T)-S motif (OsETDO: CSEGYAS).Indeed, the presence of this motif in a rhodanese-like protein reliably predicts the presence of an ETDO gene in the same genome neighborhood or the same genome.Based on this search motif we can infer that the vast majority of ETDO-associated rhodanese-like ETSDs are encoded in actinobacteria with the notable exceptions of the OsETDO-like bifunctional enzymes which occur predominantly in Acidobacteria (Table S1).
The unusual active site motif in rhodanese-like ETSDs may be related to their unusual activity.Catalytic desulfination is unprecedented for rhodanese-like enzymes which usually catalyze either sulfur-, selenium-or phosphate transfer or arsenate reduction. [15]There are only four other enzyme types known to catalyzed elimination of SO 2 from a carbon scaffold: 2'-hydroxybiphenyl-2-sulfinate desulfinase (EC 3.13.1.3), [16]3sulfinopropanoyl-CoA desulfinase (EC 3.13.1.4), [17]aspartate βdecarboxylases which catalyze SO 2 elimination from cysteine sulfinic acid as a side activity, [18] and metal-dependent ETSDs.The latter enzyme type is related to enzymes that catalyze decarboxylation of aryl carboxylates such as 5-carboxyvanillate (4, Figure 4). [19]19b,20] A similar mechanism is conceivable for ETSD-catalyzed desulfination of 2. There is no such precedence for rhodanese-like desulfinases.However, it is possible that the active site Cys -in protonated form -may act as a catalytic acid to initiate substrate protonation (Figure 4).The idea that enzyme-catalyzed desulfination of 2 is assisted by a general acid is supported by the observation that the same reaction is also accelerated by specific acid catalysis. [6]

Conclusions
We have discovered a family of bacterial rhodanese-like enzymes that catalyze SO 2 -elimination from ergothioneine sulfinic acid (2).This enzyme, in combination with an ergothioneine dioxygenase (ETDO), enables actinobacterial and acidobacterial species to degrade ergothioneine (1) via an oxidative pathway.Rhodanese-like ETSDs assume the same function as the previously reported metal-dependent ETSDs, [6] suggesting that the two enzyme types emerged by convergent evolution.Our discovery that rhodanese-like ETSDs can catalyze desulfination of an aromatic sulfinic acid adds a new entry to the short list of elementary reactions known to be catalyzed by members of the rhodanese-like protein superfamily. [15]B: Proposed mechanism of the metal-dependent ETSD.C: Proposed mechanism of rhodanese-like ETSD.