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Introduction: The potential role of accumulation of chondroitin sulfates (CSs) in the pathobiology of COVID-19 has not been examined. Accumulation may occur by increased synthesis or by decline in activity of the enzyme arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase) which requires oxygen for activity. Methods: Immunostaining of lung tissue from 28 patients who died due to COVID-19 infection was performed for CS, ARSB, and carbohydrate sulfotransferase (CHST)15. Measurements of mRNA expression of CHST15 and CHST11, sulfotransferase activity, and total sulfated glycosaminoglycans (GAGs) were determined in human vascular smooth muscle cells following angiotensin (Ang) II treatment. Results: CS immunostaining showed increase in intensity and distribution, and immunostaining of ARSB was diminished in COVID-19 compared to normal lung tissue. CHST15 immunostaining was prominent in vascular smooth muscle cells associated with diffuse alveolar damage due to COVID-19 or other causes. Expression of CHST15 and CHST11 which are required for synthesis of CSE and chondroitin 4-sulfate, total sulfated GAGs, and sulfotransferase activity was significantly increased following AngII exposure in vascular smooth muscle cells. Expression of Interleukin-6 (IL-6), a mediator of cytokine storm in COVID-19, was inversely associated with ARSB expression. Discussion/Conclusion: Decline in ARSB and resulting increases in CS may contribute to the pathobiology of COVID-19, as IL-6 does. Increased expression of CHSTs following activation of Ang-converting enzyme 2 may lead to buildup of CSs.

Heparan sulfate (HS) and heparin contain imprinted "sulfation codes", which dictate their diverse physiological and pathological functions. A group of orchestrated biosynthetic enzymes cooperate in polymerizing and modifying HS chains. The biotechnological development of enzymes that can recreate this sulfation pattern on synthetic heparin is challenging, primarily due to the paucity of quantitative data for sulfotransferase enzymes. Herein, we identified critical structural characteristics that determine substrate specificity and shed light on the catalytic mechanism of sugar sulfation of two HS sulfotransferases, 2-O-sulfotransferase (HS2ST) and 6-O-sulfotransferase (HS6ST). Two sets of molecular clamps in HS2ST recognize appropriate substrates; these clamps flank the acceptor binding site on opposite sides. The hexuronic epimers, and not their puckers, have a critical influence on HS2ST selectivity. In contrast, HS6ST recognizes a broader range of substrates. This promiscuity is granted by a conserved tryptophan residue, W210, that positions the acceptor within the active site for catalysis by means of strong electrostatic interactions. Lysines K131 and K132 act in concert with a second tryptophan, W153, shedding water molecules from within the active site, thus providing HS6ST with a binding preference toward 2-O-sulfated substrates. QM/MM calculations provided valuable mechanistic insights into the catalytic process, identifying that the sulfation of both HS2ST and HS6ST follows a SN2-like mechanism. When they are taken together, our findings reveal the molecular basis of how these enzymes recognize different substrates and catalyze sugar sulfation, enabling the generation of enzymes that could create specific heparin epitopes.

Dotinurad, a novel selective urate reabsorption inhibitor, is used to treat hyperuricemia. In humans, orally administered dotinurad is excreted mainly as glucuronide and sulfate conjugates in urine. To identify the isoforms of UDP-glucuronosyltransferase (UGT) and sulfotransferase (SULT) involved in dotinurad glucuronidation and sulfation, microsome and cytosol fractions of liver, intestine, kidney, and lung tissues (cytosol only) were analyzed along with recombinant human UGT and SULT isoforms. Dotinurad was mainly metabolized to its glucuronide conjugate by human liver microsomes (HLMs), and the glucuronidation followed the two-enzyme Michaelis-Menten equation. Among the recombinant human UGT isoforms expressed in the liver, UGT1A1, UGT1A3, UGT1A9, and UGT2B7 catalyzed dotinurad glucuronidation. Based on inhibition analysis using HLMs, bilirubin, imipramine, and diflunisal decreased glucuronosyltransferase activities by 45.5%, 22.3%, and 22.2%, respectively. Diflunisal and 3′-azido-3′-deoxythymidine, in the presence of 1% bovine serum albumin, decreased glucuronosyltransferase activities by 21.1% and 13.4%, respectively. Dotinurad was metabolized to its sulfate conjugate by human liver cytosol (HLC) and human intestinal cytosol (HIC) samples, with the sulfation reaction in HLC samples following the two-enzyme Michaelis-Menten equation and that in HIC samples following the Michaelis-Menten equation. All eight recombinant human SULT isoforms used herein catalyzed dotinurad sulfation. Gavestinel decreased sulfotransferase activity by 15.3% in HLC samples, and salbutamol decreased sulfotransferase activity by 68.4% in HIC samples. These results suggest that dotinurad glucuronidation is catalyzed mainly by UGT1A1, UGT1A3, UGT1A9, and UGT2B7, whereas its sulfation is catalyzed by many SULT isoforms, including SULT1B1 and SULT1A3. SIGNIFICANCE STATEMENT The identification of enzymes involved in drug metabolism is important to predicting drug-drug interactions (DDIs) and interindividual variability for safe drug use. The present study revealed that dotinurad glucuronidation is catalyzed mainly by UGT1A1, UGT1A3, UGT1A9, and UGT2B7 and that its sulfation is catalyzed by many SULT isoforms, including SULT1B1 and SULT1A3. Therefore, dotinurad, a selective urate reabsorption inhibitor, is considered safe for use with a small risk of DDIs and low interindividual variability.

Cancer is related to the cellular proliferative state. Increase in cell-cycle regulatory function augments cellular folate pool. This pathway is therapeutically targeted. A number of drugs influences this metabolism, that is, folic acid, folinic acid, nolatrexed, and methotrexate. Our previous study showed methotrexate influences on rat/human sulfotransferases. Present study explains the effect of nolatrexed (widely used in different cancers) and some micronutrients on the expressions of rat/human sulfotransferases. Female Sprague-Dawley rats were treated with nolatrexed (01-100 mg/kg) and rats of both sexes were treated to folic acid (100, 200, or 400 mg/kg) for 2-weeks and their aryl sulfotransferase-IV (AST-IV; β-napthol sulfation) and sulfotransferase (STa; DHEA sulfation) activities, protein expression (western blot) and mRNA expression (RT-PCR) were tested. In human-cultured hepatocarcinoma (HepG2) cells nolatrexed (1 nM-1.2 mM) or folinic acid (10 nM-10 μM) were applied for 10 days. Folic acid (0-10 μM) was treated to HepG2 cells. PPST (phenol catalyzing), MPST (dopamine and monoamine), DHEAST (dehydroepiandrosterone and DHEA), and EST (estradiol sulfating) protein expressions (western-blot) were tested in HepG2 cells. Present results suggest that nolatrexed significantly increased sulfotransferases expressions in rat (protein, STa, F = 4.87, P

Heparan sulfate (HS) 3-O-sulfotransferase isoform 4 (3-OST-4) is a specialized carbohydrate sulfotransferase participating in the biosynthesis of heparan sulfate. Here, we report the expression and purification of the recombinant 3-OST-4 enzyme and use it for the synthesis of a library of 3-O-sulfated hexasaccharides and 3-O-sulfated octasaccharides. The unique structural feature of the library is that each oligosaccharide contains a disaccharide domain with a 2-O-sulfated glucuronic acid (GlcA2S) and 3-O-sulfated glucosamine (GlcNS3S). By rearranging the order of the enzymatic modification steps, we demonstrate the synthesis of oligosaccharides with different saccharide sequences. The structural characterization was completed by electrospray ionization mass spectrometry and NMR. These 3-O-sulfated oligosaccharides show weak to very weak anti-Factor Xa activity, a measurement of anticoagulant activity. We discovered that HSoligo 7 (HS oligosaccharide 7), a 3-O-sulfated octasaccharide, binds to high mobility group box 1 protein (HMGB1) and tau protein, both believed to be involved in the process of inflammation. Access to the recombinant 3-OST-4 expands the capability of the chemoenzymatic method to synthesize novel 3-O-sulfated oligosaccharides. The oligosaccharides will become valuable reagents to probe the biological functions of 3-O-sulfated HS and to develop HS-based therapeutic agents.

Bile acids play essential roles in facilitating the intestinal absorption of lipophilic nutrients as well as regulation of glucose, lipid, and energy homeostasis via activation of some receptors. Bile acids are cytotoxic, and consequently their concentrations are tightly controlled. A critical pathway for bile acid elimination and detoxification is sulfation. The pattern of bile acid sulfation differs by species. Sulfation preferentially occurs at the 3α-OH of bile acids in humans, but at the 7α-OH in mice. A recent study identified mouse cytosolic sulfotransferase 2A8 (mSULT2A8) as the major hepatic 7α-hydroxyl bile acid-sulfating enzyme. To elucidate the 7α-OH specific sulfation mechanism of mSULT2A8, instead of 3α-OH specific sulfation in humans, we determined a crystal structure of mSULT2A8 in complex with cholic acid, a major bile acid, and 3'-phosphoadenosine-5'-phosphate, the sulfate donor product. Our study shows that bile acid-binding mode of mSULT2A8 and how the enzyme holds the 7α-OH group of bile acids at the catalytic center, revealing that the mechanism underlying 7α-OH specific sulfation. The structure shows the substrate binds to mSULT2A8 in an orientation perpendicular to that of human 3α-hydroxyl bile acid-sulfotransferase (hSULT2A1). The structure of the complex provides new insight into species different bile acid metabolism.

Heparosan is used as the starting polysaccharide sulfated using sulfotransferase to generate fully elaborate heparin, a widely used clinical drug. However, the preparation of heparosan and enzymes was considered tedious since such material must be prepared in separate fermentation batches. In this study, a commonly admitted probiotic, Escherichia coli strain Nissle 1917 (EcN), was engineered to intracellularly express sulfotransferases and, simultaneously, secreting heparosan into the culture medium. The engineered strain EcN::T7M, carrying the λDE3 region of BL21(DE3) encoding T7 RNA polymerase, expressed the sulfotransferase domain (NST) of human N-deacetylase/N-sulfotransferase-1 (NDST-1) and the catalytic domain of mouse 3-O-sulfotransferase-1 (3-OST-1) in a flask. The fed-batch fermentation of EcN::T7M carrying the plasmid expressing NST was carried out, which brought the yield of NST to 0.21 g/L and the yield of heparosan to 0.85 g/L, respectively. Furthermore, the heparosan was purified, characterized by 1H nuclear magnetic resonance (NMR), and sulfated by NST using 3′-phosphoadenosine-5′-phosphosulfate (PAPS) as the sulfo donor. The analysis of element composition showed that over 80% of disaccharide repeats of heparosan were N-sulfated. These results indicate that EcN::T7M is capable of preparing sulfotransferase and heparosan at the same time. The EcN::T7M strain is also a suitable host for expressing exogenous proteins driven by tac promoter and T7 promoter.

Aims: To study the effects of blood glucose regulating compounds on human and rat sulfotransferases (SULTs) expressions. Background: Phase-II enzymes, sulfotransferases catalyze the sulfuryl-group-transfer to endogenous/exogenous compounds. The alteration of expressions of SULTs may have influence on the sulfation of its substrate and other biomolecules. Objectives: The influence of the altered biotransformation might alter different biochemical events, drug-drug interactions and bioaccumulation or excretion pattern of certain drug. Methods: In this brief study, diabetes-inducing drug streptozotocin (STZ; 10 or 50 mg/kg to male Sprague Dawley rat for 2 weeks) or hyperglycemia controlling drug tolbutamide (TLB 0.1 or 10μM to human hepato-carcinoma cells, HepG2 for 10 days) was applied and the SULTs expressions were verified. Extensive protein-protein (STa, SULT2A1/DHEAST) interactions were studied by the STRING (Search-Tool-for-the-Retrieval-of-Interacting Genes/Proteins) Bioinformatics-software. Results: Present result suggests that while STZ increased the STa (in rat) (dehydroepiandrosterone catalyzing SULT; DHEAST in human HepG2), tolbutamide decreased PPST (phenol catalyzing SULT) and DHEAST activity in human HepG2 cells. Moderate decreases of MPST (monoamine catalyzing SULT) and EST (estrogen catalyzing) activities are noticed in this case. STa/DHEAST was found to be highly interactive to SHBG/- sex-hormone-binding-globulin; PPARα/lipid-metabolism-regulator; FABP1/fatty-acid-binding-protein. Conclusions: Streptozotocin and tolbutamide, these two glycaemia-modifying drugs demonstrated regulation of rat and human SULTs activities. The reciprocal nature of these two drugs on SULTs expression may be associated with their contrasting abilities in influencing glucose-homeostasis. Possible association of certain SULT-isoform with hepatic fat-regulations may indicate an unfocused link between calorie-metabolism and the glycemic-state of an individual. Explorations of this work may uncover the role of sulfation metabolism of specific biomolecule on cellular glycemic regulation.

Heparan sulfate (HS) is a linear and complex polysaccharide that modulates the biological activities through protein recognition and interaction. Evidence indicates that protein-binding properties of HS are largely dependent on distinctive sulfation and epimerization patterns that are modified by a series of Golgi-localized enzymes. In particular, the glucuronyl C5-epimerase (Hsepi) converts D-glucuronic acid (GlcA) residues to L-iduronic acid (IdoA) and 2-O-sulfotransferase (2OST) catalyzes sulfation at C2 position of IdoA and rarely GlcA residues. Mice lacking both Hsepi and 2OST display multiple development defects, indicating the importance of IdoA in HS. Here, to gain greater insights of HS structure–function relationships, as well as a better understanding of the regulatory mechanisms of Hsepi and 2OST, the fine structure and cellular signaling functions of HS were investigated after restoration of Hsepi in the mutant mouse embryonic fibroblast (MEF) cells. Introduction of Hsepi into the Hsepi mutant MEF cells led to robustly increased proportion of IdoA residues, which rescued the cell signaling in response to fibroblast growth factor 2. However, we found that Hsepi knockout had no influence on either cellular transport or enzymatic activity of 2OST in the MEF cells, which is not in accord with the findings suggesting that the enzymatic activity and cellular transport of 2OST and Hsepi might be differently regulated.

The sulfation at the 3-OH position of a glucosamine saccharide is a rare modification, but is critically important for the biological activities of heparan sulfate polysaccharides. Heparan sulfate 3-O-sulfotransferase (3-OST), the enzyme responsible for completing this modification, is present in seven different isoforms in humans. Individual isoforms display substrate selectivity to uniquely sulfated saccharide sequences present in heparan sulfate polysaccharides. Here, we report two ternary crystal structures of heparan sulfate 3-OST isoform 3 (3-OST-3) with PAP (3'-phosphoadenosine 5'-phosphate) and two octasaccharide substrates: non 6-O-sulfated octasaccharide (8-mer 1) and 6-O-sulfated octasaccharide (8-mer 3). The 8-mer 1 is a known favorable substrate for 3-OST-3, whereas the 8-mer 3 is an unfavorable one. Unlike the 8-mer 1, we discovered that the 8-mer 3 displays two binding orientations to the enzyme: productive binding and non-productive binding. Results from the enzyme activity studies demonstrate that 8-mer 3 can contribute to either substrate or product inhibition, possibly attributed to a non-productive binding mode. Our results suggest that heparan sulfate substrates interact with the 3-OST-3 enzyme in more than one orientation, which may regulate the activity of the enzyme. Our findings also suggest that different binding orientations between polysaccharides and their protein binding partners could influence biological outcomes.

In Cypridina (Vargula) hilgendorfii, Cypridina luciferin is converted from Cypridina luciferyl sulfate by a sulfotransferase with adenosine 3′, 5′-diphosphate (PAP), and is used for the luminescence reaction of Cypridina luciferase. We found that the luminescence activity of crude extracts of C. hilgendorfii was significantly stimulated by the addition of acetic acid. This stimulation may be explained by an efficient supply of PAP from 3′-phosphoadenosine 5′-phosphosulfate (PAPS) catalyzed by a sulfotransferase. Thus, acetic acid acts as a sulfate acceptor from PAPS, followed by forming acetyl sulfate and PAP. The structure of acetyl sulfate was identified using mass spectrometry and it spontaneously decomposed to acetic acid and free sulfate ion in aqueous solutions. This enzymatic conversion from Cypridina luciferyl sulfate to Cypridina luciferin could be coupled with acetic acid and PAPS by a sulfotransferase.

Mutations in multiple genes required for proper O-mannosylation of α-dystroglycan are causal for congenital/limb-girdle muscular dystrophies and abnormal brain development in mammals. Previously, we and others further elucidated the functional O-mannose glycan structure that is terminated by matriglycan, [(-GlcA-β3-Xyl-α3-)n]. This repeating disaccharide serves as a receptor for proteins in the extracellular matrix. Here, we demonstrate in vitro that HNK-1 sulfotransferase (HNK-1ST/carbohydrate sulfotransferase) sulfates terminal glucuronyl residues of matriglycan at the 3-hydroxyl and prevents further matriglycan polymerization by the LARGE1 glycosyltransferase. While α-dystroglycan isolated from mouse heart and kidney is susceptible to exoglycosidase digestion of matriglycan, the functional, lower molecular weight α-dystroglycan detected in brain, where HNK-1ST expression is elevated, is resistant. Removal of the sulfate cap by a sulfatase facilitated dual-glycosidase digestion. Our data strongly support a tissue specific mechanism in which HNK-1ST regulates polymer length by competing with LARGE for the 3-position on the nonreducing GlcA of matriglycan.

Sulfotransferases constitute a ubiquitous class of enzyme which are poorly understood due to the lack of a convenient tool for screening their activity. These enzymes use the anion PAPS (adenosine-3’-phosphate-5’-phosphosulfate) as a donor for a broad range of acceptor substrates, including carbohydrates, producing sulfated compounds and PAP (adenosine-3’,5’-diphosphate) as a side product. We present a europium(III)-based probe that binds reversibly to both PAPS and PAP, producing a larger luminescence enhancement with the latter anion. We exploit this greater emission enhancement with PAP to demonstrate the first direct real-time assay of a heparan sulfate sulfotransferase using multi-well plate format. The selective response of our probe towards structurally similar nucleoside phosphate anions, and over other anions, is investigated and discussed. This work opens the possibility of investigating more fully the roles played by this enzyme class in health and disease, including operationally simple inhibitor screening.

Sulfation catalyzed by sulfotransferase (SULT) is an important part of phase II metabolism. Similar to uridine 5′-diphospho-glucuronosyltransferases (UGTs), SULTs can metabolize certain drug-like molecules, and reactive metabolites may also form as a result. This chapter provides an overview of the SULT family, several medicinal chemistry strategies to reduce or eliminate sulfation and selected examples from the literature.

Polychlorinated biphenyls (PCBs) are persistent environmental contaminants that continue to be of concern due to their varied toxicities. Upon human exposure, many PCBs with lower numbers of chlorine atoms are metabolized to hydroxylated derivatives (OH-PCBs), and cytosolic sulfotransferases can subsequently catalyze the formation of PCB sulfates. Recent studies have indicated that PCB sulfates bind reversibly with a high affinity to human serum proteins, and that they are also taken up by cells and tissues. Since PCB sulfates might be hydrolyzed to the more toxic OH-PCBs, we have investigated the ability of human hepatic microsomal sulfatase to catalyze this reaction. Twelve congeners of PCB sulfates were substrates for the microsomal sulfatase with catalytic rates exceeding that of dehydroepiandrosterone sulfate as a comparison substrate for steroid sulfatase (STS). These results are consistent with an intracellular mechanism for sulfation and de-sulfation that may contribute to retention and increased time of exposure to OH-PCBs.

Heparan sulfate (HS) with various sulfation patterns is one of important modulators of cancer cell fate through interacting with numerous growth factors. Here we found HS 2-O sulfotransferase 1 (HS2ST1) was downregulated at both mRNA and protein levels during granulocytic differentiation of SKM-1 leukemia cells and also HS (glucosamine) 3-O sulfotransferase 3A (HS3ST3A) in epithelial-mesenchymal transition (EMT) of A549 lung cancer cells, meanwhile, cell-surface HS recognized by anti-HS antibody was also changed in both cancer cell lines. Further, HS3ST3A was negatively correlated with the in vitro cell metastasis capability of A549 cells confirmed by RNA interference technology, wound-healing assay and in vitro Matrigel invasion assay. Together, specific HS sulfotransferases may serve as molecular markers and targets for cancer treatment.

Pyrones comprise a structurally diverse class of compounds. Although they are widespread in nature, their specific physiological functions remain unknown in most cases. We recently described that triketide pyrones mediate the sulfotransfer in caprazamycin biosynthesis. Herein, we report the identification of conexipyrones A-C, three previously unrecognized tetra-substituted α-pyrones, from the soil actinobacterium Conexibacter woesei. Insights into their biosynthesis via a type III polyketide synthase were obtained by feeding studies using isotope-enriched precursors. In vitro assays employing the genetically associated 3'-phosphoadenosine-5'-phosphosulfate (PAPS)-dependent sulfotransferase CwoeST revealed conexipyrones as the enzymes' genuine sulfate acceptor substrates. Furthermore, conexipyrones were determined to function as sulfate shuttles in a two-enzyme assay, because their sulfated derivatives were accepted as donor molecules by the PAPS-independent arylsulfate sulfotransferase (ASST) Cpz4 to yield sulfated caprazamycin intermediates.

Sodium taurocholate cotransporting polypeptide (NTCP, encoded by Slc10a1/SLC10A1) deficiency can result in hypercholanemia but no obvious symptoms in both mice and humans. However, the consequence of and response to long-term hypercholanemia caused by NTCP deficiency remain largely unexplored. Here, we analyzed lifelong dynamics of serum total bile acid (TBA) levels in Slc10a1(−/−) mice, and we also assessed changes of TBA levels in 33 young individuals with SLC10A1 loss-of-function variant p.Ser267Phe. We found that overall serum TBA levels tended to decrease gradually with age in both Slc10a1(−/−) mice and p.Ser267Phe individuals. Liver mRNA profiling revealed notable transcription alterations in hypercholanemic Slc10a1(−/−) mice, including inhibition of bile acid (BA) synthesis, enhancement of BA detoxification, and altered BA transport. Members of the sulfotransferase (SULT) family showed the most dramatic increases in livers of hypercholanemic Slc10a1(−/−) mice, and one of their BA sulfates, taurolithocholic acid 3-sulfate, significantly increased. Importantly, consistent with the mouse studies, comprehensive profiling of 58 BA species in sera of p.Ser267Phe individuals revealed a markedly increased level of BA sulfates. Together, our findings indicate that the enhanced BA sulfation is a major mechanism for BA detoxification and elimination in both mice and humans with Slc10a1/SLC10A1 deficiency.

Background Non-opioid and opioid analgesics, as over-the-counter or prescribed medications, are widely used for the management of a diverse array of pathophysiological conditions. Previous studies have demonstrated the involvement of human cytosolic sulfotransferase (SULT) SULT1A1 in the sulfation of acetaminophen, O-desmethylnaproxen (O-DMN), and tapentadol. The current study was designed to investigate the impact of single nucleotide polymorphisms (SNPs) of the human SULT1A1 gene on the sulfation of these analgesic compounds by SULT1A1 allozymes. Methods Human SULT1A1 genotypes were identified by database search. cDNAs corresponding to nine SULT1A1 nonsynonymous missense coding SNPs (cSNPs) were generated by site-directed mutagenesis. Recombinant wild-type and SULT1A1 allozymes were bacterially expressed and affinity-purified. Purified SULT1A1 allozymes were analyzed for sulfation activity using an established assay procedure. Results Compared with the wild-type enzyme, SULT1A1 allozymes were shown to display differential sulfating activities toward three analgesic compounds, acetaminophen, O-desmethylnaproxen (O-DMN), and tapentadol, as well as the prototype substrate 4NP. Conclusion Results obtained indicated clearly the impact of genetic polymorphisms on the drug-sulfation activity of SULT1A1 allozymes. Such information may contribute to a better understanding about the differential metabolism of acetaminophen, O-DMN, and tapentadol in individuals with different SULT1A1 genotypes.

Sulfation is an important way for detoxifying xenobiotics and endobiotics including catechols. Enzymatic sulfation occurs usually with high chemo- and/or regioselectivity under mild reaction conditions. In this study, a two-step p-NPS-4-AAP screening system for laboratory evolution of aryl sulfotransferase B (ASTB) was developed in 96-well microtiter plates to improve the sulfate transfer efficiency toward catechols. Increased transfer efficiency and improved sulfation stoichiometry are achieved through the two-step screening procedure in a one-pot reaction. In the first step, the p-NPS assay is used (detection of the colorimetric by-product, p-nitrophenol) to determine the apparent ASTB activity. The sulfated product, 3-chlorocatechol-1-monosulfate, is quantified by the 4-aminoantipyrine (4-AAP) assay in the second step. Comparison of product formation to p-NPS consumption ensures successful directed evolution campaigns of ASTB. Optimization yielded a coefficient of variation below 15% for the two-step screening system (p-NPS-4-AAP). In total, 1760 clones from an ASTB-SeSaM library were screened toward the improved sulfation activity of 3-chlorocatechol. The turnover number (kcat = 41 ± 2 s-1) and catalytic efficiency (kcat/KM = 0.41 μM-1 s-1) of the final variant ASTB-M5 were improved 2.4- and 2.3-fold compared with ASTB-WT. HPLC analysis confirmed the improved sulfate stoichiometry of ASTB-M5 with a conversion of 58% (ASTB-WT 29%; two-fold improvement). Mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) confirmed the chemo- and regioselectivity, which yielded exclusively 3-chlorocatechol-1-monosulfate. For all five additionally investigated catechols, the variant ASTB-M5 achieved an improved kcat value of up to 4.5-fold and sulfate transfer efficiency was also increased (up to 2.3-fold).

2,3-Dehydrosilybin A and 2,3-dehydrosilybin B are a pair of enantiomers formed by the oxidation of the natural flavonolignans silybin A and silybin B, respectively. However, the antioxidant activity of 2,3-dehydrosilybin molecules is much stronger than that of their precursors. Here, we investigated the biotransformation of pure 2,3-dehydrosilybin A and 2,3-dehydrosilybin B in isolated human hepatocytes, and we also aimed to identify human UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) with activity toward their respective enantiomers. After incubation with hepatocytes, both 2,3-dehydrosilybin A and 2,3-dehydrosilybin B were converted to hydroxyl derivatives, methylated hydroxyl derivatives, methyl derivatives, sulfates, and glucuronides. The products of direct conjugations predominated over those of oxidative metabolism, and glucuronides were the most abundant metabolites. Furthermore, we found that recombinant human UGTs 1A1, 1A3, 1A7, 1A8, 1A9, and 1A10 were capable of catalyzing the glucuronidation of both 2,3-dehydrosilybin A and 2,3-dehydrosilybin B. UGTs 1A1 and 1A7 showed the highest activity toward 2,3-dehydrosilybin A, and UGT1A9 showed the highest activity toward 2,3-dehydrosilybin B. The sulfation of 2,3-dehydrosilybin A and B was catalyzed by SULTs 1A1*1, 1A1*2, 1A2, 1A3, 1B1, 1C2, 1C4, and 1E1, of which SULT1A3 exhibited the highest activity toward both enantiomers. We conclude that 2,3-dehydrosilybin A and B are preferentially metabolized by conjugation reactions, and that several human UGT and SULT enzymes may play a role in these conjugations.

When human influenza viruses are isolated and passaged in chicken embryos, variants with amino acid substitutions around the receptor binding site of hemagglutinin (HA) are selected; however, the mechanisms that underlie this phenomenon have yet to be elucidated. Here, we analyzed the receptor structures that contributed to propagation of egg-passaged human H1N1 viruses. The analysis included seasonal and 2009 pandemic strains, both of which have amino acid substitutions of HA found in strains isolated or passaged in eggs. These viruses exhibited high binding to sulfated glycans containing NeuAcα2-3Gal. In MDCK cells overexpressing the sulfotransferase that synthesize Galβ1-4(SO3--6)GlcNAc, production of human H1N1 viruses was increased up to 90-fold. Furthermore, these sulfated glycans were expressed on the allantoic and amniotic membranes of chicken embryos. These results suggest that 6-sulfo sialyl Lewis X and/or NeuAcα2-3Galβ1-4(SO3--6)GlcNAc are involved in efficient propagation of human H1N1 viruses in chicken embryos.

Sulfated glycosaminoglycans (GAGs) are a class of important biologics that are currently manufactured by extraction from animal tissues. Although such methods are unsustainable and prone to contamination, animal-free production methods have not emerged as competitive alternatives due to complexities in scale-up, requirement for multiple stages and cost of co-factors and purification. Here, we demonstrate the development of single microbial cell factories capable of complete, one-step biosynthesis of chondroitin sulfate (CS), a type of GAG. We engineer E. coli to produce all three required components for CS production–chondroitin, sulfate donor and sulfotransferase. In this way, we achieve intracellular CS production of ~27 μg/g dry-cell-weight with about 96% of the disaccharides sulfated. We further explore four different factors that can affect the sulfation levels of this microbial product. Overall, this is a demonstration of simple, one-step microbial production of a sulfated GAG and marks an important step in the animal-free production of these molecules., Chondroitin sulfate (CS) is a type of sulfated glycosaminoglycan that is manufactured by extraction from animal tissues for the treatment of osteoarthritis and in drug delivery applications. Here, the authors report the development of single microbial cell factories capable of compete, one-step biosynthesis of animal-free CS production in E. coli.

Radix Bupleuri is one of the most widely used herbal medicines in China for the treatment of fever, pain, and/or chronic inflammation. Quercitrin, epicatechin, and rutin, the flavonoids present in Radix Bupleuri, have been reported to display anti-inflammatory, antitumor, and antioxidant biological activities among others. Sulfation has been reported to play an important role in the metabolism of flavonoids. In this study, we aimed to systematically identify the human cytosolic sulfotransferase enzymes that are capable of catalyzing the sulfation of quercitrin, epicatechin, and rutin. Of the thirteen known human cytosolic sulfotransferases, three (cytosolic sulfotransferase 1A1, cytosolic sulfotransferase 1C2, and cytosolic sulfotransferase 1C4) displayed sulfating activity toward quercitrin, three (cytosolic sulfotransferase 1A1, cytosolic sulfotransferase 1A3, and cytosolic sulfotransferase 1C4) displayed sulfating activity toward epicatechin, and six (cytosolic sulfotransferase 1A1, cytosolic sulfotransferase 1A2, cytosolic sulfotransferase 1A3, cytosolic sulfotransferase 1B1, cytosolic sulfotransferase 1C4, and cytosolic sulfotransferase 1E1) displayed sulfating activity toward rutin. The kinetic parameters of the cytosolic sulfotransferases that showed the strongest sulfating activities were determined. To investigate the effects of genetic polymorphisms on the sulfation of quercitrin, epicatechin, and rutin, individual panels of cytosolic sulfotransferase allozymes previously prepared were analyzed and shown to display differential sulfating activities toward each of the three flavonoids. Taken together, these results provided a biochemical basis underlying the metabolism of quercitrin, epicatechin, and rutin through sulfation in humans.

Doxorubicin is a chemotherapeutic drug widely utilized in cancer treatment. An enzyme critical to doxorubicin metabolism is the cytosolic sulfotransferase (SULT) SULT1C4. This study investigated the functional impact of SULT1C4 single nucleotide polymorphisms (SNPs) on the sulfation of doxorubicin by SULT1C4 allozymes. A comprehensive database search was performed to identify various SULT1C4 SNPs. Ten nonsynonymous SULT1C4 SNPs were selected, and the corresponding cDNAs, packaged in pGEX-2TK expression vector, were generated via site-directed mutagenesis. Respective SULT1C4 allozymes were bacterially expressed and purified by affinity chromatography. Purified SULT1C4 allozymes, in comparison with the wild-type enzyme, were analysed for sulphating activities towards doxorubicin and 4-nitrophenol, a prototype substrate. Results obtained showed clearly differential doxorubicin-sulphating activity of SULT1C4 allozymes, implying differential metabolism of doxorubicin through sulfation in individuals with distinct SULT1C4 genotypes.