Two radical-dependent mechanisms for anaerobic degradation of the globally abundant organosulfur compound dihydroxypropanesulfonate

Jiayi Liua,b,1, Yifeng Weic,1, Lianyun Lina, Lin Tenga, Jinyu Yina, Qiang Lua, Jiawei Chena, Yuchun Zhenga, Yaxin Lia, Runyao Xua, Weixiang Zhaid, Yangping Liud, Yanhong Liue, Peng Caof, Ee Lui Angc, Huimin Zhaoc,g,2, Zhiguang Yuchia,2, and Yan Zhanga,b,2

aTianjin Key Laboratory for Modern Drug Delivery & High-Efficiency, Collaborative Innovation Center of Chemical Science and Engineering, School of Pharmaceutical Science and Technology, Tianjin University, 300072 Tianjin, China; bFrontiers Science Center for Synthetic Biology (Ministry of Education), Tianjin University, 300072 Tianjin, China; cInstitute of Chemical and Engineering Sciences, Agency for Science, Technology and Research (A*STAR), 138669 Singapore, Singapore; dTianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics, School of Pharmacy, Tianjin Medical University, 300070 Tianjin, People’s Republic of China; eTechnical Institute of Physics and Chemistry, Chinese Academy of Sciences, 100190 Beijing, China; fKey Laboratory of Drug Targets and Drug Leads for Degenerative Diseases, Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, 210023 Nanjing, China; and gDepartment of Chemical and Biomolecular Engineering, University of Illinois at Urbana–Champaign, Urbana, IL 61801

Edited by JoAnne Stubbe, Massachusetts Institute of Technology, Cambridge, MA, and approved May 28, 2020 (received for review February 23, 2020) 2(S)-dihydroxypropanesulfonate (DHPS) is a microbial degradation pathway is favored by fermentative bacteria, while the sulfo-ED of 6-deoxy-6-sulfo-d-glucopyranose (sulfoquinovose), a pathway is favored by respiratory bacteria (7). component of plant sulfolipid with an estimated annual production Apart from being a product of sulfoglycolysis, DHPS is also an of 1010 tons. DHPS is also at millimolar levels in highly abundant important organosulfur compound in its own right. It is present marine phytoplankton. Its degradation and sulfur recycling by mi- in up to millimolar intracellular concentrations in eukaryotic crobes, thus, play important roles in the biogeochemical sulfur cycle. marine phytoplankton, including the highly abundant diatoms (2, However, DHPS degradative pathways in the anaerobic biosphere 8), which are estimated to contribute ∼20% of the total global

are not well understood. Here, we report the discovery and charac- primary production (9). These phytoplankton use sulfate, present BIOCHEMISTRY terization of two O2-sensitive glycyl radical that use distinct at ∼30 mM levels in seawater to synthesize a variety of organo- mechanisms for DHPS degradation. DHPS-sulfolyase (HpsG) in – sulfur compounds including DHPS and sulfoquinovose, Secretion sulfate- and sulfite-reducing bacteria catalyzes C S cleavage to or cell lysis makes these compounds available for degradation by release sulfite for use as a terminal electron acceptor in respiration, marine heterotrophic bacteria, accounting for a large component producing H S. DHPS- (HpfG), in fermenting bacteria, 2 of the flux of organic carbon in the surface oceans (8, 10). catalyzes C–O cleavage to generate 3-sulfopropionaldehyde, subse- quently reduced by the NADH-dependent sulfopropionaldehyde re- ductase (HpfD). Both enzymes are present in bacteria from diverse Significance environments including human gut, suggesting the contribution of enzymatic radical chemistry to sulfur flux in various anaerobic niches. DHPS is one of the most abundant organosulfonates on this planet. The mechanisms for DHPS degradation in the anaerobic glycyl radical | sulfoglycolysis | dihydroxypropanesulfonate | biosphere are not well understood. Here, we report the sulfate- and sulfite-reducing bacteria | gut bacteria bioinformatics-aided discovery, biochemical, and structural char- acterizations of two O2-sensitive glycyl radical enzymes that use rganosulfonates are ubiquitous in our environment, our distinct radical-mediated mechanisms for DHPS degradation in Obodies, and the food that we eat. Mechanisms by which they anaerobic bacteria from diverse terrestrial and marine sources as are metabolized by diverse microbes in the human microbiome well as human gut. These enzymes play an important role in the and in the environment are of great relevance to human health biogeochemical sulfur cycle and link dietary sulfonates to micro- and to the biogeochemical sulfur cycle. Two organosulfonates of bial production of H2S, which is a causative agent of chronic special importance are sulfoquinovose and DHPS due to their diseases, such as inflammation and colorectal cancer. production in large volumes globally by photoautotrophs (1, 2). Author contributions: J.L., Y.W., H.Z., Z.Y., and Y. Zhang designed research; J.L., Y.W., L.L., Sulfoquinovose is the polar headgroup of the plant sulfolipid L.T., J.Y., Q.L., J.C., Y. Zheng, Y.L., R.X., W.Z., Yanhong Liu, and Y. Zhang performed sulfoquinovosyl diacylglycerol (3), a component of photosynthetic research; Yangping Liu, Yanhong Liu, P.C., E.L.A., H.Z., Z.Y., and Y. Zhang contributed thylakoid membranes in all vascular plants, mosses, algae, and new reagents/analytic tools; J.L., Y.W., L.L., L.T., J.Y., Q.L., J.C., Y. Zheng, Y.L., R.X., W.Z., most photosynthetic bacteria (1). The annual global production of Yanhong Liu, H.Z., Z.Y., and Y. Zhang analyzed data; and J.L., Y.W., E.L.A., H.Z., Z.Y., and Y. Zhang wrote the paper. sulfoquinovose is estimated to be 1010 tons, making it one of the most abundant organic sulfur compounds in nature (1). Bacterial The authors declare no competing interest. degradation of sulfoquinovose was recently discovered and named This article is a PNAS Direct Submission. sulfoglycolysis due to its resemblance to classical glycolytic path- Published under the PNAS license. ways. The sulfo-Embden–Meyerhof–Parnas (sulfo-EMP) pathway Data deposition: Desulfovibrio vulgaris IseG has been deposited in the Data Bank, was characterized in Escherichia coli K-12 and is used during both www.wwpdb.org (accession no. 5YMR); two homologs of DvIseG have been deposited in UniProt, https://www.uniprot.org/ (accession nos. E5Y378 and E5Y7I4); and the crystallog- aerobic growth (4) and anaerobic mixed acid fermentation (5). In raphy, atomic coordinates, and structure factors have been deposited in the RCSB Protein this pathway, half of the carbon of sulfoquinovose is converted to Data Bank, https://www.rcsb.org (accession no. 6LON). dihydroxyacetonephosphate and used for growth, while the other 1J.L. and Y.W. contributed equally to this work. half is excreted as DHPS, which is, subsequently, metabolized by 2To whom correspondence may be addressed. Email: [email protected], yuchi@tju. other bacteria (5, 6). A second sulfoglycolytic pathway, the sulfo- edu.cn, or [email protected]. Entner–Doudoroff (sulfo-ED) pathway, was characterized in the This article contains supporting information online at https://www.pnas.org/lookup/suppl/ environmental isolate Pseudomonas putida SQ1 (7) and produces doi:10.1073/pnas.2003434117/-/DCSupplemental. 3-sulfolactate instead of DHPS. It is thought that the sulfo-EMP

www.pnas.org/cgi/doi/10.1073/pnas.2003434117 PNAS Latest Articles | 1of10 Downloaded by guest on September 30, 2021 Various pathways for the degradation of DHPS have been iden- tified in aerobic environmental bacteria. In Cupriavidus pinatubonensis (11), (S)-DHPS is racemized in two steps through two DHPS-2- dehydrogenases (HpsO and HpsP). A DHPS-1-dehydrogenase (HpsN) then converts (R)-DHPS into (R)-sulfolactate (SL), followed by C–S cleavage by the enantiospecific (R)-sulfolactate-sulfolyase (SuyAB) (SI Appendix,Fig.S1A). The sulfite released is oxidized by the periplasmic sulfite dehydrogenase and excreted as sulfate. Apart from direct C–S cleavage by SuyAB, two other processes are known for the desulfonation of (R)-SL. In the aerobic marine bacterium Roseovarius nubinhibens ISM (12), SL is converted to cysteate followed by C–S cleavage by cysteate sulfolyase and converted to sulfoacetaldehyde followed by C–Scleavageby sulfoacetaldehyde actyltransferase (SI Appendix,Fig.S1A). In the anaerobic biosphere, which includes environments ranging from ocean sediments to the digestive tracts of marine and terrestrial animals, degradation of DHPS by sulfate- and sulfite-reducing bacteria (SSRB) results in conversion of the sulfonate sulfur to H2S (5). This process is of particular interest in the human gut where fermentation of dietary sulfoquinovose rich in fruits and vegetables produces DHPS, and generation of Scheme 1. Radical-dependent (S)-DHPS degradation pathways involving H2S by SSRB has been linked to diseases, such as inflammation the sulfolyase HpsG and dehydratase HpfG. and colorectal cancer (13). Despite its importance, pathways for the degradation of DHPS in anaerobic bacteria are not well understood. The only organism in which it has been studied is the is (S)-DHPS due to the abundance of this organo- Desulfovibrio sp. DF1, an isolate from sewage sludge (5). In this sulfonate in nature. bacterium, (S)-DHPS is imported into the cell through an ABC To test this hypothesis, we recombinantly produced E5Y7I4 transporter and oxidized sequentially to sulfolactate by two NAD+- and its adjacent activating enzyme E5Y7I3 from B. wadsworthia dependent dehydrogenase DhpA and SlaB (SI Appendix,Fig.S1B). 3_1_6 (SI Appendix, Fig. S3). Biochemical characterization C–S cleavage by SuyAB releases sulfite, which is used as a terminal confirmed that it was, indeed, a DHPS sulfolyase (Fig. 1), and we electron acceptor (TEA), producing H2S as an end product. renamed the GRE and activating enzyme HpsG and HpsH, re- Previously, we (14) and others (15) reported a radical-dependent spectively. HpsG could be activated by anaerobically recon- mechanism for C–S cleavage of 2-hydroxyethylsulfonate (isethionate), stituted HpsH (SI Appendix, Fig. S4), forming 0.04 ± 0.02 (out of which is structurally similar to DHPS, in anaerobic bacteria. The a theoretical maximum of 1) (19) G• per dimer (Fig. 1A). The reaction is catalyzed by the O2-sensitive enzyme IseG, a member low radical yield is an issue with many GREs and requires further of the glycyl radical enzyme (GRE) superfamily (16–18). GREs analysis. Incubation of activated HpsG with racemic DHPS contain an essential O2-sensitive glycyl radical (G•) , resulted in the production of hydroxyacetone (Fig. 1 B–D) and generated by an activating enzyme through chemistry involving sulfite as detected using a colorimetric Fuchsin assay (21) (SI S-adenosylmethionine (SAM) and a [4Fe–4S]1+ cluster (19, 20), Appendix, Fig. S5A). HpsG exhibited Michaelis–Menten kinetics −1 and catalyze diverse radical-dependent reactions. IseG belongs for DHPS desulfonation (kcat = 26 ± 1s , Km = 13 ± 2 mM, kcat/ −1 −1 to a subset of GREs catalyzing 1,2 reactions, including Km = 1.9 ± 0.2 mM s )(SI Appendix, Fig. S5 B and C) with a – – – C O, C N, and C S (16). The substrates of these lyases much lower activity for isethionate desulfonation (kcat = 0.57 ± – −1 −1 −1 contain a C1 OH group and a variable C2 leaving the group. 0.03 s , Km = 34 ± 5 mM, kcat/Km = 0.017 ± 0.003 mM s ) Since DHPS contains two OH groups, radical-dependent cleav- (Fig. 1B and SI Appendix, Fig. S5 D and E). (The kcat reported is age could, in theory, lead to two possible outcomes: C–SorC–O normalized by radical content.) cleavage (Scheme 1). Here, we describe our bioinformatics-aided discovery, biochemical and structural characterizations of two Crystal Structure of HpsG in Complex with (S)-DHPS. To further ex- GREs, a (S)-DHPS sulfolyase (HpsG) and a (S)-DHPS dehy- amine the origin of the different substrate specificities of HpsG, dratase (HpfG). HpsG is present in SSRB, while HpfG is present we determined the crystal structure of HpsG in complex with in fermenting bacteria, and their differing distribution and meta- racemic DHPS at 2.2 Å (Fig. 2 and SI Appendix, Fig. S6 and bolic roles are discussed. Table S1). The C121 crystals contain four monomers per asym- metric unit, each exhibiting a canonical β/α barrel fold common Results to other GREs. The overall structure of HpsG is similar to that Identification and Biochemical Characterization of the DHPS-Sulfolyase of DvIseG with a rmsd of 0.73 Å between 796 Cα atoms (SI HpsG. During our previous crystallographic studies of Desulfovibrio Appendix, Fig. S6A). The well-defined electron density for (S)- vulgaris IseG (DvIseG, [PDB] accession no.: DHPS in the of HpsG enabled unambiguous assignment 5YMR) (14), we noted that some close homologs of DvIseG of the substrate. The C2–OH moiety of the bound (S)-DHPS is contain variations in substrate-interacting residues, suggesting the located at a position similar to the C1–OH of isethionate in IseG possibility that they may accept structurally related sulfonate and is positioned for deprotonation by Glu464 (Fig. 2A). The substrates. We focused on the sulfite-reducing human gut bacte- substrate sulfonate group is also coordinated in a similar fashion rium Bilophila wadsworthia, which encodes two homologs of as in IseG by Arg183, Gln187, and Arg672 of HpsG. Three water DvIseG (UniProt: E5Y378 and E5Y7I4, 60% identity between the molecules contribute to the extensive hydrogen bond network two homologs). Previously, growth of B. wadsworthia RZATAU interacting with the substrate (Fig. 2A). However, the orientation with isethionate as the sole TEA resulted in the induction of only of the C3 methylene group of (S)-DHPS is markedly different E5Y378 but not E5Y7I4 (14). The substrate-interacting residues from that of the corresponding C2 methylene group of isethionate of DvIseG were identical to those of E5Y378 and were highly with a 160° rotation between the C2–C3–S plane of (S)-DHPS similar but not identical to those of E5Y7I4 (SI Appendix,Fig.S2), relative to the C1–C2–S plane of isethionate (SI Appendix,Fig. suggesting a structurally similar substrate. We hypothesized that S6 B and C). The replacement of Phe680 and Thr310 in IseG with

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.2003434117 Liu et al. Downloaded by guest on September 30, 2021 BIOCHEMISTRY

Fig. 1. EPR spectra and enzymatic assays of HpsG. (A) X-band EPR spectrum of HpsG (40 μM) reconstituted with HpsH (80 μM), SAM (1 mM), and reductant Ti (III) citrate (100 μM), acquired at a temperature of 90 K and microwave power of 1 mW. (B) Specific activity of HpsG-catalyzed conversion of DHPS to hydroxyacetone and isethionate to acetaldehyde, measured using NADH-coupled spectrophotometric assays with E. coli glycerol dehydrogenase (GldA) and Saccharomyces cerevisiae alcohol dehydrogenase (ADH1), respectively. Error bars represent the SD of three individual experiments. (C) Detection of the products of HpsG-catalyzed reactions by derivatization with 2,4-dinitrophenylhydrazine (DNPH) and liquid chromatography–mass spectrometry (LC–MS) analysis. (D) Negative ionization mass spectra of DNPH-derivatized reaction products, corresponding to peaks 1 and 2 in C. (Theoretical mass of monoanions, DNPH hydroxyacetone = 253.0, and DNPH acetaldehyde = 223.0).

the less bulky Ile676 and Ala306 in HpsG accommodates the new stereochemistry is thought to be abstracted in all other mecha- orientation of the C3 methylene group and the additional C1 nistically related GREs: CutC, propanediol dehydratase, glycerol hydroxymethyl group of (S)-DHPS (SI Appendix,Fig.S6B and C). dehydratase, and ribonucleotide reductase (16). Binding of DHPS in the active site of HpsG is further established by a water molecule bridging C1–OH of the substrate and the Investigation of DHPS Transporters in SSRB. To identify HpsG in hydroxyl group of Ser305 in HpsG, which replaces a Gly309 in other organisms, we examined the UniRef50 cluster UniRef50_ IseG at the same position. A0A348AJ86 (where each member shares ≥50% sequence identity A dual conformation of the thiyl radical residue Cys462 is and ≥80% overlap with the seed sequence of the cluster) (22), observed in HpsG, suggesting high conformational flexibility of which contains IseG and HpsG sequences. A sequence similarity this residue. The minimal distance between the thiyl radial and network (SSN) was constructed for these , revealing an the Cα of the glycyl radical residue Gly799 is 4.27 Å (chain D), organization into two major clusters (Fig. 3A). The first contained and the minimal distance between the C2 of DHPS and the thiyl 103 proteins, the majority of which have active-site residues iden- radical is 4.09 Å (chain D), which would allow C–H abstraction tical to those of IseG from D. vulgaris or D. bizertensis (SI Appendix, to occur (Fig. 2B and SI Appendix, Fig. S6C). In the structure of Fig. S2). The second cluster contained 14 proteins with active-site DvIseG, the orientation of isethionate suggested that its pro-R residues identical to those of B. wadsworthia HpsG (Fig. 3A and SI hydrogen is abstracted. The C2–Hof(S)-DHPS has the same Appendix,TableS2) and are, thus, assigned as HpsG. orientation, implicating a similar stereochemistry of abstraction To find other associated with DHPS degradation, the (SI Appendix, Fig. S6C). In contrast, the hydrogen of the opposite genome neighborhoods of the HpsG sequences were examined

Liu et al. PNAS Latest Articles | 3of10 Downloaded by guest on September 30, 2021 Fig. S7A) showed enthalpy-driven binding of DHPS with a Kd of 6.7 μM(SI Appendix,Fig.S7B) but no binding of isethionate or 3-hydroxypropane-1-sulfonate (3-HPS) (SI Appendix,Fig.S7C and D), confirming that it is a DHPS TRAP transporter (HpsKLM) (Fig. 3C).

HpsG Is Involved in DHPS Dissimilation in B. wadsworthia. We further studied the ability B. wadsworthia RZATAU to use DHPS as a TEA. When pyruvate is supplied as a carbon and electron source, growth was supported with DHPS as the sole TEA (Fig. 4A), accompanied by the production of H2S as detected using a methylene blue assay (Fig. 4A). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) analysis revealed a prominent pro- tein band with molecular weight of ∼95 kDa (Fig. 4C), present in DHPS—but not thiosulfate-grown cells. Mass spectrometric analysis identified this protein as HpsG (Dataset S3), suggesting its involvement in DHPS dissimilation in this bacterium.

Identification and Biochemical Characterization of the DHPS Dehydratase HpfG. Apart from desulfonation, GREs could, in theory, also catalyze radical-mediated DHPS dehydration, converting it to 3-sulfopropionaldehyde (Scheme 1). Because of the global abun- dance of (S)-DHPS, we hypothesized the existence of a GRE (S)- DHPS dehydratase, which would provide an alternative pathway for DHPS degradation. The catalytic mechanism of GRE diol dehy- dratases has been extensively studied both experimentally (26) and computationally (27). The reaction involves five conserved active- site residues: a Cys thiyl radical involved in substrate radical generation, Glu involved in substrate C1–OH deprotonation, and a triad of two His and one Asp involved in coordination and protonation of the substrate C2–OH leaving group. To search for a GRE (S)-DHPS dehydratase, 674 candidate diol dehydratase sequences containing the five conserved active- site residues were extracted from the 22,000 sequences in the GRE superfamily (InterPro IPR004184). A SSN was constructed for these proteins, revealing an organization into two major clusters (Fig. 5A). The first contained previously characterized Clostridium butyricum glycerol dehydratase (GDH) and Roseburia inulinivorans propanediol dehydratase. The second cluster con- tained 74 GREs of unknown function, corresponding to the UniRef cluster UniRef50_E3H691 (SI Appendix,TableS3). Examination of Fig. 2. HpsG active-site structure. (A) Structure of the HpsG active site in complex with the (S)-DHPS (Chain D). The proposed pathway for hydrogen their genome neighborhoods revealed genes that could be involved atom transfer is indicated by red arrows, and hydrogen bonds are indicated in DHPS degradation (Fig. 5B). Like other diol , this by black dotted lines. 2Fo–Fc electron densities for isethionate are shown at GRE is associated with a metal-dependent alcohol dehydrogenase 1.0σ. The two rotamers observed for Cys462 are shown. (B) Proposed (ADH), which catalyzes the reduction of the aldehyde product. In mechanism of DHPS cleavage by HpsG. The thiyl radical, which is transiently addition, this GRE is associated with a very characteristic generated by the G• cofactor in all GREs, abstracts a hydrogen (shown in red) transporter belonging to the TauE family (PF01925), which from the substrate DHPS, and returns it to form the product hydroxyacetone. includes bacterial sulfite, isethionate, and sulfoacetate exporters, suggesting a link to sulfonate metabolism. We, therefore, hypothesized apathwayinwhich(S)-DHPS is converted to 3-sulfopropionaldehyde – using the enzyme function initiative genome neighborhood tool by the GRE (HpfG), reduced to sulfopropanol by the ADH (HpfD), (Fig. 3B) (23). Two of them (Desulfovibrio litoralis German Collec- and excreted through the exporter (HpfE) (Fig. 5C). tion of Microorganisms [DSM] 11393, uncultured Desulfovibrio sp To test this hypothesis, we recombinantly produced HpfG Tax ID: 167968) were associated with transporters in the MFS (A0A318FL05) and its activating enzyme HpfH (A0A318FEA4) family (Pfam PF07690). These were closely related to the Cupriavidus from Klebsiella oxytoca (SI Appendix,Fig.S8) and HpfD (A0A374P995) pinatubonensis (S)-DHPS transporter HpsU (60% sequence identity) from Hungatella hathewayi (SI Appendix, Fig. S9A). HpfD cata- (11), suggesting that they are DHPS transporters (Fig. 3C). lyzed NAD+-dependent oxidation of 3-HPS (SI Appendix,Fig. Several of the HpsG homologs, including B. wadsworthia S9B) and is presumed to also catalyze the NADH-dependent HpsG, were also associated with a tripartite ATP-independent sulfopropionaldehyde reduction because of the thermodynamic periplasmic (TRAP) transporter, a family of transporters that reversibility of this reaction. HpfG could be activated by anaero- catalyzes the import of carboxylic and sulfonic acids (24) and bically reconstituted HpfH (SI Appendix,Fig.S1A), forming includes the previously characterized isethionate transporter IseKLM 0.03 ± 0.01 (out of a theoretical maximum of 1) (19) G• per dimer from Desulfovibrio piger (14). TRAP transporters are reliant on a (Fig. 6A). The low radical yield is an issue with many GREs, and soluble periplasmic substrate-binding protein (SBP), which is requires further analysis. Incubation of activated HpfG with race- amenable to biochemical characterization by isothermal titration mic DHPS resulted in the production of 3-sulfopropionaldehyde calorimetry (ITC) (14, 25). The recombinant SBP (E5Y7I1) of the (Fig. 6 B–D). HpfG exhibited Michaelis–Menten kinetics for DHPS −1 HpsG-associated TRAP transporter in B. wadsworthia (SI Appendix, dehydration (kcat = 130 ± 4s , Km = 5.0 ± 0.6 mM, and

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.2003434117 Liu et al. Downloaded by guest on September 30, 2021 BIOCHEMISTRY

Fig. 3. SSN and genome neighborhoods of HpsG homologs. (A) SSN for IseG homologs belonging to UniRef50_A0A348AJ86, displayed at the E-value cutoff of 10−360. Each node represents a unique protein sequence, and the most highly related proteins are grouped together in clusters, putatively sharing the same function. Blue, active-site residues identical to D. vulgaris or Desulfovibrio bizertensis IseG; red, active-site residues identical to B. wadsworthia HpsG. (B) Genome neighborhoods of selected homologs in the HpsG cluster. (C) Proposed pathway for DHPS import and HpsG-dependent degradation in B. wadsworthia.

−1 −1 kcat/Km = 26 ± 3mM s )(SI Appendix,Fig.S10). (The kcat sensitive GREs, HpsG and HpfG, described in this study, provide reported is normalized by radical content.) radical-dependent pathways for DHPS degradation in anaerobic bacteria and highlight the diversity of O2-sensitive chemistry oc- Homology Model and Molecular Docking of HpfG. A homology curring in anoxic environments. model of HpfG was constructed using a C. butyricum glycerol The HpsG sequences that we have identified are present in dehydratase structure (PDB 1R9D) as a template (37% se- gram-negative SSRB (SI Appendix,TableS2). Like in B. wadsworthia, quence identity between the two proteins), followed by docking the HpsG clusters appear to be involved in the uptake of (S)- of the DHPS substrate. The position and the pose of (S)-DHPS DHPS, followed by desulfonation, producing sulfite for use as a in the model is similar to that of glycerol in GDH (SI Appendix, TEA (Fig. 3C). The HpfG sequences are present in gram-negative Fig. S11). The positions of the conserved Cys464, Glu466, and Gammaproteobacteria, and gram-positive Clostridia and Lacto- the triad Asp478, His191, and His308 (SI Appendix, Fig. S11) are bacilliales bacteria (SI Appendix,TableS3). Several of the HpfG consistent with the conserved mechanism for these diol dehy- sequences in Gammaproteobacteria are associated with homologs dratases. The substrate sulfonate group interacts with Arg363, of E. coli sulfoglycolysis enzymes, including sulfoquinovose Val677, Tyr481, and Asp478, which are conserved in the mem- (YihS), sulfofructose kinase (YihV), sulfofructose-1- bers of the HpfG cluster. However, given the low sequence phosphate aldolase (YihT), and sulfolactaldehyde reductase identity between HpfG and the used template, further structural (YihU) (Fig. 5B), suggesting the metabolism of (S)-DHPS produced studies are required and are currently underway. internally through sulfoglycolysis (Fig. 5C). HpfG sequences in other bacteria are associated with a protein annotated as a sodium:sulfate Discussion symporter (PF00939), which we hypothesize is a (S)-DHPS importer, DHPS is produced in large volumes globally, and the ability of its suggesting the uptake and metabolism of (S)-DHPS from the two hydroxyl groups to serve as reactive handles for enzymatic environment (Fig. 5C). chemistry provides a wide range of possible pathways for DHPS Many of the HpsGs and HpfGs are from gut bacteria, in- degradation in metabolically diverse microbes. The two O2- cluding strains from the human gut microbiome (SI Appendix,

Liu et al. PNAS Latest Articles | 5of10 Downloaded by guest on September 30, 2021 conduct all anaerobic experiments in an atmosphere of N2 and with the O2 maintained at less than 5 ppm.

Gene Synthesis, Molecular Cloning, and Plasmid Construction. DNA fragments encoding E. coli codon-optimized sequences of B. wadsworthia HpsG (BwHpsG, E5Y7I4), HpsH (BwHpsH,E5Y7I3),HpsK(BwHpsK, E5Y7I1), K. oxytoca HpfG (KoHpfG, A0A318FL05), HpfH (KoHpfH, A0A318FEA4), and H. hathewayi HpfD (HhHpfD, A0A374P995) were synthesized and inserted into plasmids by General Biosystems, Inc. (Anhui, China). Plasmids used in this study are pET-28a(+) and

modified pET28 vectors including HMT (containing, in tandem, a His6-tag, maltose binding protein [MBP], and a tobacco etch virus (TEV) protease cleavage site, followed by the construct of interest), and HT (containing a

His6-tag and a TEV protease cleavage site, followed by the construct of in- terest). The BwHpsG, KoHpsG, and HhHpfD fragments were inserted into HT at the SspI site. For the production of soluble BwHpsH, BwHpsK, and KoHpfH, the respective fragments were inserted into HMT at the SspI site. The N-terminal 22-a.a. (amino acid) signal peptide of BwHpsK was omitted. For the production of high-purity BwHpsG with a N-terminal 20-a.a. trunca- tion for crystallography, the BwHpsG fragment was amplified by PCR using primers 1F and 1R (SI Appendix, Table S4) and inserted into the HMT at the SspI site to form HMT-BwHpsG (-20 a.a.). A surface-entropy reduction mu- tation, changing a.a. 129–132 from TDMQ to AAAA, was introduced by QuikChange site-directed mutagenesis using primers 2F and 2R to form HMT-BwHpsG (-20 a.a. 129TDMQ132-AAAA).

Production of BwHpsGH and KoHpfGH for Enzymatic Assays. BwHpsGH and KoHpfGH were heterologously expressed in E. coli BL21 (DE3) cells harboring the plasmids HT-BwHpsG, HMT-BwHpsH and HT-KoHpfG, HMT-KoHpfH, re- spectively. For BwHpsH, the cells were cotransformed with the plasmid pTf16 Fig. 4. DHPS-dependent growth of B. wadsworthia and H2S formation. (A) Various sulfur containing TEAs support the growth of B. wadsworthia.(B) (TaKaRa) for coexpression of the tig chaperone. Expression was carried out μ Formation of methylene blue by reacting the headspace gas with N,N-di- in LB supplemented with the appropriate antibiotics (50 g/mL kanamycin μ μ methyl-pphenylenediamine dihydrochloride and the absorbance of the re- for BwHpsG, KoHpfG, and MBP-KoHpfH, 50 g/mL kanamycin and 25 g/mL spective reaction mixtures at 670 nm. The assays were performed in triplicate chloramphenicol for MBP-BwHpsH). Starter cultures (5 mL) were inoculated and are presented with SDs. (C) B. wadsworthia grown on pyruvate plus from single colonies and grown overnight at 37 °C followed by dilution into thiosulfate (lane 2), taurine (lane 3), or pyruvate plus DHPS (lane 4). The 1 L of medium. To induce chaperone expression for the MPB-BwHpsH cul- arrows indicate a ∼95-kDa band identified as HpsG. ture, 2.5 mg/mL L-arabinose was added. The cultures were grown in a shaking incubator at 37 °C and 220 rpm, up to an optical density at 600 nm

(OD600)of0.6–0.8. The temperature was then decreased to 18 °C, followed by addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final con- Tables S2 and S3). The production of H2S by human gut SSRB centration of 0.4 mM, and continued shaking for 16 h. The cultures were has been linked to diseases, and HpsG and HpfG may provide centrifuged (8,000 × g for 10 min at 4 °C), and the cell pellets (∼1gwet competing pathways for DHPS degradation in metabolically weight) were suspended in 5 mL of lysis buffer (50 mM Tris/HCl, pH 8.0, distinct bacteria. HpsG and HpfG are also present in environ- 200 mM KCl, 1 mM phenylmethanesulfonyl fluoride (PMSF), 0.2 mg/mL mental bacteria, including marine bacteria, and identification of lysozyme, 0.03% Triton X-100, and 0.02 mg/mL of DNase I), and frozen in their sequences allows further investigation of their roles in the a −80 °C freezer. recycling of abundant marine sulfonates (2, 8). HpsG is present For protein purification, the cells were thawed and incubated at room in Desulfovibrio desulfuricans ND132, isolated from mesohaline temperature (RT, 25 °C) for 50 min, during which cell lysis occurs. Some 5 mM β Chesapeake Bay sediments and Desulfovibrio sp. HK-II, isolated -mercaptoethanol (BME) was added, and nucleic acid was removed by from sediments of a partial salt lake (SI Appendix, Table S2). precipitation with 1% streptomycin sulfate. For BwHpsG and KoHpfG, the protein solution was loaded on to a 5 mL TALON Co2+ column (Clontech HpfG is present in many marine bacteria, including gram-negative Laboratories, Inc.), preequilibrated with buffer A [20 mM Tris/HCl, pH 7.5, Vibrio and Photobacterium species isolated from seawater, sedi- 5 mM BME, and 0.2 M KCl]. The column was washed with 10 column volumes ments, and marine animals and gram-positive Epulopiscum species (CV) of buffer A, and protein was eluted with 5 CV of buffer A containing

found in fish gut (SI Appendix, Table S3), suggesting a role in 150 mM imidazole. The eluted protein was precipitated with solid (NH4)2SO4 marine anaerobic niches. to 70% saturation and isolated by centrifugation (20,000 × g for 10 min at In conclusion, our study has expanded the repertoire of en- 4 °C). The pellet was dissolved in 5 mL of buffer B [20 mM Tris/HCl, pH 7.5, zymes and transporters involved in the biodegradation of DHPS, 100 mM KCl, and 5 mM BME] and desalted using a G25 column (GE, ther- × a highly abundant organosulfonate on this planet. Existing sequence mostat jacket tube XK16/20, packed 15 cm 2 cm, 30 mL), preequilibrated with buffer B. databases reveal the occurrence of these proteins in microbes For BwHpsH and KoHpfH, the protein solution was loaded on to a column inhabiting diverse anaerobic environments, and their identifica- packed with 20 mL amylose resin (New England Biolabs), and protein was tion facilitates further bioinformatics studies of the biological eluted with buffer A containing 10 mM maltose. The purified BwHpsG −1 −1 −1 −1 recycling of this compound. (e280 = 122,000 M cm ), MBP-BwHpsH (e280 = 88,200 M cm ), KoHpfG −1 −1 −1 −1 (e280 = 118,000 M cm ), and MBP-KoHpfH (e280 = 101,000 M cm ) were Materials and Methods examined by SDS/PAGE on a commercial gel (SurePAGE, Bis-Tris, 8–16%). Materials and General Methods. Lysogeny Broth (LB) was prepared with yeast extract and tryptone purchased from Oxoid, UK. For LC–MS, high-purity Production of N-Terminal 20 a.a. Truncated BwHpsG for Protein Crystallization. methanol and acetonitrile from Concord Technology and formic acid from Expression of a MBP-BwHpsG mutant for crystallography was carried out in Merck were used. The ultrapure deionized water used in all experiments was E. coli BL21 (DE3) cells harboring the plasmid pET28-HMT-BwHpsG (-20 a.a. prepared using a Millipore Direct-Q. Synthetic oligonucleotide primers were 129TDMQ132-AAAA). The transformant was grown in LB medium containing purchased from Tsingke, Inc. (Beijing, China). “ÄKTA pure” or “ÄKTA prime kanamycin (50 μg/mL) at 37 °C in flasks in a shaker incubator at 220 rpm and plus” fast protein liquid chromatography machines were used to perform all induced for the expression of MBP-BwHpsG with 0.3 mM IPTG for 16 h at protein purification chromatographic steps. Proteins were quantified by mea- 18 °C. Typically, cells from 6 L culture were harvested by centrifugation suring their absorption at 280 nm, using a BioPhotometer D30 (Eppendorf, (8,000 × g for 10 min) and were suspended in 5 mL (∼1 g wet weight) of buffer Hamburg, Germany). A Lab2000 glovebox (Etelux, Beijing, China) was used to C [50 mM Tris/HCl, pH 8.0, 200 mM KCl, 1 mM PMSF, 0.03% Triton X-100]. Then,

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.2003434117 Liu et al. Downloaded by guest on September 30, 2021 BIOCHEMISTRY

Fig. 5. SSN and genome neighborhoods of HpfG homologs. (A) SSN for putative diol dehydratases in the GRE superfamily, containing conserved residues − thought to be required for , displayed at the E-value cutoff of 10 260. Each node represents a unique protein sequence, and the most highly related proteins are grouped together in clusters, putatively sharing the same function. (B) Genome neighborhoods of selected homologs in the HpfG cluster. (C) Proposed pathways for DHPS import and HpfG-dependent degradation.

the cells were lysed with sonication (Scientz Biotechnology, Ningbo, China). The [Fe-S] Cluster Reconstitution for MBP-HpsH and MBP-HpfH. Reconstitution of lysate was then centrifuged (20,000 × g 30 min at 4 °C) to remove unbroken the [Fe–S] clusters of MBP-HpsH and MBP-HpfH, quantitation of Fe and S cells and cell debris. The protein solution was then applied to a 10 mL TALON contents, and measurement of their absorption spectra, were carried out column. The protein was eluted with buffer A containing 150 mM imidazole. according to previously described procedures (14). The purity of MBP- The eluate was then diluted twofold with buffer A and loaded on a column BwHpsH and MBP-KoHpfH was estimated to be 69% and 73% based on packed with 40 mL amylose resins. The column was then washed with 5 CV densitometry analysis of the Coomassie-stained SDS/PAGE gel using the buffer A and eluted with 5 CV buffer A containing 10 mM maltose. The eluate software ImageJ and used to estimate the Fe and S contents per monomer.

premixed with recombinant His6-tagged TEV protease (15:1) was dialyzed Sequence alignments suggested that HpsH and HpfH, like many other overnight against 2 L buffer A. The dialyzed sample was loaded on a 10 mL previously characterized GRE activating enzymes, contain one [4Fe–4S] TALON column to retain TEV and MBP proteins. The flow through was col- cluster in a radical SAM domain, and two [4Fe–4S] clusters in a ferredoxin lected and dialyzed against 2 L buffer D [20 mM Tris·HCl, pH 7.5, 5 mM BME] for domain (SI Appendix,Fig.S12) (28), giving a theoretical maximum of 12 Fe 3 h before it was applied to a 5 mL Q Sepharose high performance column. The and 12 S per monomer. Anaerobic reconstitution of the [4Fe–4S] clusters column was eluted with 10 CV linear salt gradient from 100 to 500 mM KCl in resulted in 7.9 ± 0.3 Fe and 8.4 ± 0.1 S per HpsH monomer and 5.4 ± 0.2 Fe buffer D. A prominent peak containing BwHpsG (-20 a.a.) was collected and and 5.3 ± 0.4 S per HpfH monomer, suggesting that our protocol gives rise to concentrated to a final volume of 5 mL (4 mg/mL) using a centrifuge concen- incomplete reconstitution. trator (30K MWCO; Millipore). This protein solution was then injected into a The ultraviolet–visible (UV–Vis) absorption spectra exhibited features Superdex200 gel filtration column (300 mL) and eluted with buffer E [20 mM characteristic of [4Fe–4S]2+ clusters, which disappeared upon reduction (SI Tris·HCl, pH 7.5, 0.2 M KCl, 1 mM dithiothreitol (DTT)]. The eluate from gel Appendix, Fig. S4). Correcting for the 69% purity of MBP-BwHpsH and 73% filtration column was reconcentrated and buffer exchanged with the storage purity of MBP-KoHpfH, the extinction coefficients of the reconstituted MBP- buffer [10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid/KOH, pH 7.4, BwHpsH and MBP-KoHpfH [4Fe–4S] clusters were estimated to be 27 and −1 −1 −1 −1 50 mM KCl, 1 mM Tris-(2-carboxyethyl)-phosphine hydrochloride]. The final 23 mM cm . Assuming an approximate e410nm of 15 mM cm per concentration is 10 mg/mL The purified protein was examined by SDS/PAGE gel. cluster (29), we estimate that for MBP-BwHpsH and MBP-KoHpfH, ∼1.8

Liu et al. PNAS Latest Articles | 7of10 Downloaded by guest on September 30, 2021 Fig. 6. EPR spectra and enzymatic assays. (A) X- band EPR spectrum of HpfG (40 μM) reconstituted with HpfH (80 μM), SAM (1 mM), and reductant Ti (III) citrate (100 μM), acquired at a temperature of 90 K and microwave power of 1 mW. (B) Specific activity of HpfG-catalyzed conversion of DHPS to 3-sulfopropionaldehyde, measured using a NADH-coupled spectrophotometric assay with sulfopropionaldehyde reductase (HpfD). Error bars represent the SD of three individual experiments. (C) Detection of HpfG-catalyzed 3-sulfopropionaldehyde formation by derivatization with DNPH and LC–MS analysis. (D) Negative ionization mass spectra of the DNPH-derivatized reaction product, corresponding to peak 1 c. (Theoretical mass of the monoanion of DNPH sulfopropionaldehyde = 317.0).

[4Fe–4S] clusters and 1.5 [4Fe–4S] clusters per monomer, respectively, were out according to previously described procedures (14). A commercial reconstituted consistent with the measured Fe and S contents. hydroxyacetone standard (LabNetwork, Wuxi, China) was also prepared.

Electron Paramagnetic Resonance Detection of Glycyl Radical Formation. Fuchsin Assay for Sulfite Detection. Sulfite was detected using a colorimetric Continuous wave X-band electron paramagnetic resonance (EPR) spectros- Fuchsin assay according to previously described procedures (14). BwHpsG copy was used to characterize the glycyl radical. A 200 μL reaction mixture activation was carried out as described for the EPR experiments except that containing 20 mM Tris, pH 7.5, 0.1 M KCl, 40 μM BwHpsG/KoHpfG, 80 μM glycerol was omitted. A 100 μL reaction mixture containing 10 μM activated reconstituted MBP-BwHpsH/MBP-KoHpfH, 1 mM SAM, 100 μM Ti(III) citrate BwHpsG, 20 mM DHPS or isethionate was incubated at 30 °C for 1 h in the was incubated at RT for 60 min/15 min in the glovebox. Then, 10% glycerol glovebox. A negative control omitting substrate was also performed. While

for HpsG, or 10% D-sorbitol for HpfG was added into the reactions. Samples the reaction incubated, stock solution A (0.8 M H2SO4, 0.08% Fuchsin, and were loaded into EPR tubes with 4-mm outer diameter and 8-in. length 1.6% formaldehyde, mixed 7:2:1) was freshly prepared. A 50 μL portion per (Wilmad Lab-Glass, 734-LPV-7), sealed with a rubber stopper, removed from reaction sample was mixed with 950 μL of solution A, incubated for 10 min, the glovebox, and frozen in liquid nitrogen prior to EPR analysis. The ex- and the UV–Vis absorption spectra were collected. perimental spectra for the glycyl radical were modeled with Bruker Xepr spin fit to obtain g values, hyperfine coupling constants, and linewidths. Spectrophotometric Activity Assay for H. hathewayi HpfD. A 200 μL reaction Double integration of the simulated spectra was used to measure spin mixture containing 20 mM Tris·HCl buffer, pH 9.5, 100 mM KCl, 0.6 ng concentration (14). For both HpsG and HpfG enzymes, EPR quantitation was HhHpfD, 2 mM NAD+, and 100 mM 3-HPS (Aladdin, Shanghai, China) was performed with triplicate biological samples. The perpendicular mode incubated at RT in a 96-well plate, and the absorbance at 340 nm was X-band EPR spectra were recorded using a Bruker E500 EPR spectrometer. monitored using a plate reader (Tecan M200, Männedorf, Switzerland). Data acquisition was performed with Xepr software (Bruker). The EPR spectra represent an average of 30 scans and were recorded under the fol- Enzymatic Assay for Hydroxyacetone Formation by HpsG. A 200 μL reaction lowing conditions: temperature, 90 K; center field, 3,370 G; range, 200 G; mixture containing 20 mM Tris·HCl, pH 7.5, 0.1 M KCl, 1 μM activated microwave power, 1 mW; microwave frequency, 9.43 GHz; modulation BwHpsG, 10 μM E. coli GldA, 0.4 mM NADH, and 50 mM DHPS was incubated amplitude, 2 mT; modulation frequency, 100 kHz; time constant, 20.48 ms; at RT for 20 min in the glovebox. The mixture was removed from the glo- conversion time, 40 ms; scan time, 81.92 s; and receiver gain, 23 dB. vebox, and its absorbance at 340 nm was measured using a plate reader (Tecan M200). Control assays omitting either SAM or BwHpsG were also LC–MS/MS Assay for Hydroxyacetone and 3-Sulfopropionaldehyde Detection. performed. To investigate the substrate specificity of BwHpsG, assays BwHpsG and KoHpfG activation was carried out as described for the EPR substituting DHPS and GldA with 50 mM isethionate and excess S. cerevisiae experiments except that glycerol/sorbitol was omitted. For BwHpsG, a 100 μL ADH1 were also performed as previously described for IseG (14). reaction mixture containing 10 μM activated BwHpsG, 0.1 mM Ti(III) citrate and 50 mM DHPS (AA blocks) was incubated at 30 °C for 30 min in the Coupled Spectrophotometric Activity Assays for HpsG. A 100 μL reaction glovebox. Two negative controls omitting SAM or DHPS were also per- mixture containing 20 mM Tris·HCl, pH 7.5, 0.1 M KCl, and typically, 1 μM formed. Isethionate (Adamas) added instead of DHPS was also performed. activated BwHpsG, 10 μM GldA, 0.2 mM NADH, and 100 mM DHPS was in- For KoHpfG, a 100 μL reaction mixture containing 10 μM activated KoHpfG, cubated at RT in a 1 cm Eppendorf cuvette in the glovebox. The absorbance 0.1 mM Ti(III) citrate, and 20 mM DHPS was incubated at 30 °C for 30 min in at 340 nm was monitored at 4 s intervals using the cuvette mode of a Thermo the glovebox. Three negative controls omitting KoHpfG, SAM, or DHPS were Scientific Nanodrop OneC in the glovebox. To obtain the Michaelis–Menten also performed. Derivatization with DNPH and LC–MS analysis were carried kinetic parameters, DHPS concentration was varied while keeping a fixed

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.2003434117 Liu et al. Downloaded by guest on September 30, 2021 enzyme concentration of 1 μM BwHpsG. Assays substituting DHPS and GldA Growth of B. wadsworthia with Different TEAs. B. wadsworthia strain RZATAU with isethionate and ADH1 were also prepared, and the absorbance 340 nm (DSM 11045) was purchased from DSMZ. Cells were first inoculated into ABB was monitored at 15 s intervals. These assays contained, typically, a saturating medium supplemented with 5 mM taurine and cultivated anaerobically at substrate concentration of 500 mM isethionateand 5 μM BwHpsG. The kinetic 30 °C for 3–7 d. Then, 100 μL portions of the starter culture were transferred data presented were obtained from a HpsG sample with 0.06 G• per protein into three anaerobic bottles containing 5 mL modified DSM 503 medium, dimer. GraphPad Prism6 was used for data analysis. omitting taurine, and supplemented with 60 mM Na-formate and 200 μg/ L

1,4-naphthochinone. Different TEAs (20 mM) were added: 1) Na2S2O3 (with Enzymatic Assay for 3-Sulfopropionaldehyde Formation by HpfG. A 100 μL 20 mM sodium pyruvate was added as a carbon and electron source), 2) reaction mixture containing 20 mM Tris·HCl, pH 7.5, 0.1 M KCl, 0.5 μMac- taurine, and 3) DHPS (with 20 mM sodium pyruvate was added as a carbon tivated KoHpfG, 10 μM sulfopropionaldehyde reductase (HhHpfD), 0.2 mM and electron source). After 3–7 d incubation at 30 °C, all three cultures be- NADH, and 100 mM DHPS was incubated at RT for 2 min in the glovebox. came turbid and contained a black precipitate. H2S in the headspace gas was Additionally, then its absorbance at 340 nm was measured using the cuvette detected using a methylene blue assay as previously described (14). The mode of a Nanophotometer NP80 Mobile in the glovebox. Control assays samples were diluted prior to absorbance measurement to ensure that they omitting either DHPS or KoHpfG were also performed. fall within the linear region of the spectrometer.

Coupled Spectrophotometric Activity Assays for HpfG. A100μL reaction Protein Identification by SDS/PAGE and Mass Spectrometry. Cells were har- mixture containing 20 mM Tris·HCl, pH 7.5, 0.1 M KCl, and, typically, 0.5 μM vested by centrifugation, lysed by boiling in Laemmli loading buffer, and activated KoHpfG, 10 μM HpfD, 0.2 mM NADH, and 100 mM DHPS was in- analyzed on a 10% SDS/PAGE gel. Prominent protein bands induced by cubated at RT in a 1 cm Eppendorf cuvette in the glovebox. The absorbance growth on sulfonate substrates were manually excised. After in-gel digestion at 340 nm was monitored at 5 s intervals using the cuvette mode of a and extraction, the peptide mixtures were loaded onto Orbitrap Fusion Nanophotometer NP80 Mobile in the glovebox. To obtain the Michaelis– LUMOS MS. The MS/MS spectra from each LC–MS/MS run were searched Menten kinetic parameters, DHPS concentration was varied while keeping a against the B. wadsworthia protein database GCF_000185705.2 (Bilo_- fixed enzyme concentration of 500 nM KoHpfG. The kinetic data presented wads_3_1_6_V2) from UniProt (release date of March 19, 2014; 68,406 en- were obtained from a HpfG sample with 0.04 G• per protein dimer. tries) using an in-house Proteome Discoverer (Version PD1.4, Thermo-Fisher GraphPad Prism6 was used for data analysis. Scientific). Protein identifications were performed based on Sequest HT (34). Source data underlying Fig. 4C are provided as in Dataset S1. X-ray Crystal Structure of BwHpsG. Initial screening of BwHpsG crystals was performed using an automated liquid handling robotic system (Gryphon, Art ITC Assays for BwHpsK. ITC experiments were carried out on a 50 μM solution Robbins) in 96-well format by the sitting-drop vapor diffusion method. The of BwHpsK in buffer containing 20 mM Tris·HCl, pH 7.5 and 200 mM KCl with screens were set up at 295 K using various sparse matrix crystal screening kits 1 mM DTT using a PEAQ-ITC instrument (Malvern). The samples were titrated from Hampton Research and Molecular Dimensions. Several crystallization with 19 injections of 2 μL of 1 mM DHPS, isethionate, or 3-HPS at 25 °C and a conditions gave thin plate-shape crystals. After further optimization using stirring speed of 750 rpm. Deionized water was used for the reference cell. BIOCHEMISTRY the hanging-drop vapor-diffusion method in 24-well plates, we obtained Background titrations were obtained using protein-free buffer, and sub- crystals large enough for single crystal X-ray diffraction studies. The best tracted from the raw titrations. The data were fitted to a single-site binding condition yielding large platelike crystals was 0.2 M sodium acetate and model using the MicroCal PEAQ-ITC software to estimate the stoichiometry, 0.1 M Bis–Tris propane, pH 6.5, 16% (wt/vol) PEG3350 plus 500 mM DHPS. binding affinity, and changes in enthalpy (ΔH) and entropy (ΔS). A Kd of 6.7 Crystals were flash cooled in liquid nitrogen using reservoir solution μM was measured for DHPS with a N value of 2.2. No interaction was ob- containing 25% glycerol as a cryoprotectant. Diffraction data were collected served for isethionate and 3-HPS. on a local Rigaku X-ray diffractor (XtaLAB P200 MM007HF, Tokyo, Japan) to a resolution of 2.20 Å. The data set was indexed, integrated, and scaled Data Availability. Desulfovibrio vulgaris IseG deposited in the Protein Data using HKL3000 suite (30). Molecular replacement was performed by PHENIX Bank, www.wwpdb.org (accession no. 5YMR), two homologs of DvIseG in (31) using the crystal structure of IseG (PDB 5YMR). The structure was UniProt, https://www.uniprot.org/ (accession nos. E5Y378 and E5Y7I4), and manually built according to the modified experimental electron density coordinates and structure factors of HpsG in complex with (S)-DHPS have using Coot (32) and further refined by PHENIX (31) in iterative cycles before been deposited in the RCSB Protein Data Bank, https://www.rcsb.org (ac- it was deposited in the RCSB Protein Data Bank (accession no. 6LON). The SI cession no. 6LON). All other data required to evaluate the paper’s conclu- Appendix, Table S1 contains the statistics for data collection and final re- sions are present in the paper and/or the SI Appendix. finement. All structural figures were generated with UCSF Chimera (33). ACKNOWLEDGMENTS. We thank the instrument analytical center of School Homology Modeling and Docking. A homology model of HpfG was created by of Pharmaceutical Science and Technology at Tianjin University for providing Prime module of Schrödinger software (Schrödinger LLC, New York, NY) the LC–MS analysis, Zhi Li, and Drs. Xinghua Jin, Yan Gao, and Xiangyang using C. butyricum glycerol dehydratase structure (PDB 1R9D) as a template. Zhang for helpful discussion. This work was supported by the National Key The sequence identity between the HpfG and the template protein is ∼37%. R&D Program of China (Grant 2019YFA0905700), the National Natural Science Foundation of China (Grant 31870049) (Y. Zhang), National Key R&D Program The ligand (S)-DHPS was sketched in Chem-DrawUltra 13.0 and, sub- of China (Grant 2017YFD0201400, Grant 2017YFD0201403), National Natural sequently, prepared using the LigPrep module of Schrödinger software. (S)- Science Foundation of China (Grant NSFC31972287), the State Key Laboratory DHPS was docked by Glide module of Schrödinger using an extra precision of Ecological Pest Control for Fujian and Taiwan Crops (Grant SKL2019001, mode to a reference position in the of HpfG similar as glycerol in Grant SKL2019003) (Z.Y.), and the Agency for Science, Research and Technol- the template structure (PDB 1R9D). ogy of Singapore Visiting Investigator Program Grant 1535j00137 (H.Z.).

1. E. D. Goddard-Borger, S. J. Williams, Sulfoquinovose in the biosphere: Occurrence, 8. B. P. Durham et al., Sulfonate-based networks between eukaryotic phytoplankton metabolism and functions. Biochem. J. 474, 827–849 (2017). and heterotrophic bacteria in the surface ocean. Nat. Microbiol. 4, 1706–1715 2. M. A. Moran, B. P. Durham, Sulfur metabolites in the pelagic ocean. Nat. Rev. Microbiol. (2019). 17,665–678 (2019). 9. S. Malviya et al., Insights into global diatom distribution and diversity in the world’s 3. A. A. Benson, H. Daniel, R. Wiser, A sulfolipid in plants. Proc. Natl. Acad. Sci. U.S.A. 45, ocean. Proc. Natl. Acad. Sci. U.S.A. 113, E1516–E1525 (2016). 1582–1587 (1959). 10. E. Celik et al., Metabolism of 2,3-dihydroxypropane-1-sulfonate by marine bacteria. 4. K. Denger et al., Sulphoglycolysis in Escherichia coli K-12 closes a gap in the bio- Org. Biomol. Chem. 15, 2919–2922 (2017). geochemical sulphur cycle. Nature 507, 114–117 (2014). 11. J. Mayer et al., 2,3-Dihydroxypropane-1-sulfonate degraded by Cupriavidus pinatubonensis 5. A. Burrichter et al., Anaerobic degradation of the plant sugar sulfoquinovose con- JMP134: Purification of dihydroxypropanesulfonate 3-dehydrogenase. Microbiology

comitant with H2S production: Escherichia coli K-12 and Desulfovibrio sp. strain DF1 as 156,1556–1564 (2010). co-culture model. Front. Microbiol. 9, 2792 (2018). 12. K. Denger et al., Bifurcated degradative pathway of 3-sulfolactate in Roseovarius 6. K. Denger, T. Huhn, K. Hollemeyer, D. Schleheck, A. M. Cook, Sulfoquinovose nubinhibens ISM via sulfoacetaldehyde acetyltransferase and (S)-cysteate sulfolyase. degraded by pure cultures of bacteria with release of C3-organosulfonates: J. Bacteriol. 191, 5648–5656 (2009). Complete degradation in two-member communities. FEMS Microbiol. Lett. 328, 13. F. Carbonero, A. C. Benefiel, A. H. Alizadeh-Ghamsari, H. R. Gaskins, Microbial 39–45 (2012). pathways in colonic and links with health and disease. Front. 7. A.-K. Felux, D. Spiteller, J. Klebensberger, D. Schleheck, Entner-Doudoroff pathway Physiol. 3, 448 (2012). for sulfoquinovose degradation in Pseudomonas putida SQ1. Proc. Natl. Acad. Sci. 14. M. Xing et al., Radical-mediated C-S bond cleavage in C2 sulfonate degradation by U.S.A. 112, E4298–E4305 (2015). anaerobic bacteria. Nat. Commun. 10, 1609 (2019).

Liu et al. PNAS Latest Articles | 9of10 Downloaded by guest on September 30, 2021 15. S. C. Peck et al., A glycyl radical enzyme enables hydrogen sulfide production by the 25. M. W. Vetting et al., Experimental strategies for functional annotation and metab- human intestinal bacterium Bilophila wadsworthia. Proc. Natl. Acad. Sci. U.S.A. 116, olism discovery: Targeted screening of solute binding proteins and unbiased panning 3171–3176 (2019). of metabolomes. Biochemistry 54, 909–931 (2015). 16. L. R. F. Backman, M. A. Funk, C. D. Dawson, C. L. Drennan, New tricks for the glycyl 26. B. J. Levin, E. P. Balskus, Characterization of 1,2-propanediol dehydratases reveals distinct – radical enzyme family. Crit. Rev. Biochem. Mol. Biol. 52, 674–695 (2017). mechanisms for B12-dependent and glycyl radical enzymes. Biochemistry 57, 3222 3226 (2018). ˇ  17. H. R. Beller et al., Discovery of enzymes for toluene synthesis from anoxic microbial 27. B. Kovacevic et al., Computational tale of two enzymes: Glycerol dehydration with or – communities. Nat. Chem. Biol. 14, 451–457 (2018). without B12. J. Am. Chem. Soc. 140, 8487 8496 (2018). 18. D. Liu et al., Indoleacetate decarboxylase is a glycyl radical enzyme catalysing the 28. B. Selvaraj, W. Buckel, B. T. Golding, G. M. Ullmann, B. M. Martins, Structure and formation of malodorant skatole. Nat. Commun. 9, 4224 (2018). function of 4-hydroxyphenylacetate decarboxylase and its cognate activating enzyme. – 19. K. A. Shisler, J. B. Broderick, Glycyl radical activating enzymes: Structure, mechanism, J. Mol. Microbiol. Biotechnol. 26,76 91 (2016). 29. G. Shen et al., SufR coordinates two [4Fe-4S]2+, 1+ clusters and functions as a tran- and substrate interactions. Arch. Biochem. Biophys. 546,64–71 (2014). scriptional repressor of the sufBCDS operon and an autoregulator of sufR in cyano- 20. J. L. Vey et al., Structural basis for glycyl radical formation by pyruvate formate-lyase bacteria. J. Biol. Chem. 282, 31909–31919 (2007). activating enzyme. Proc. Natl. Acad. Sci. U.S.A. 105, 16137–16141 (2008). 30. W. Minor, M. Cymborowski, Z. Otwinowski, M. Chruszcz, HKL-3000: The integration 21. K. Denger, J. Ruff, U. Rein, A. M. Cook, Sulphoacetaldehyde sulpho-lyase (EC 4.4.1.12) of data reduction and structure solution–from diffraction images to an initial model from Desulfonispora thiosulfatigenes: Purification, properties and primary sequence. in minutes. Acta Crystallogr. D Biol. Crystallogr. 62, 859–866 (2006). – Biochem. J. 357, 581 586 (2001). 31. P. D. Adams et al., PHENIX: A comprehensive Python-based system for macromolec- 22. B. E. Suzek, Y. Wang, H. Huang, P. B. McGarvey, C. H. Wu; UniProt Consortium, UniRef ular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010). clusters: A comprehensive and scalable alternative for improving sequence similarity 32. P. Emsley, K. Cowtan, Coot: Model-building tools for molecular graphics. Acta Crystallogr. – searches. Bioinformatics 31, 926 932 (2015). D Biol. Crystallogr. 60,2126–2132 (2004). 23. S. Zhao et al., Prediction and characterization of enzymatic activities guided by se- 33. E. F. Pettersen et al., UCSF Chimera–A visualization system for exploratory research quence similarity and genome neighborhood networks. eLife 3, e03275 (2014). and analysis. J. Comput. Chem. 25, 1605–1612 (2004). 24. C. Mulligan, M. Fischer, G. H. Thomas, Tripartite ATP-independent periplasmic 34. J. K. Eng, A. L. McCormack, J. R. Yates, An approach to correlate tandem mass spectral (TRAP) transporters in bacteria and archaea. FEMS Microbiol. Rev. 35,68–86 data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass (2011). Spectrom. 5, 976–989 (1994).

10 of 10 | www.pnas.org/cgi/doi/10.1073/pnas.2003434117 Liu et al. Downloaded by guest on September 30, 2021