Two Radical-Dependent Mechanisms for Anaerobic Degradation of the Globally Abundant Organosulfur Compound Dihydroxypropanesulfonate
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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 product 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 enzymes 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-dehydratase (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 enzyme | 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 Protein 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 First published June 22, 2020. www.pnas.org/cgi/doi/10.1073/pnas.2003434117 PNAS | July 7, 2020 | vol. 117 | no. 27 | 15599–15608 Downloaded by guest on October 3, 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 substrate 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