Functional Expression of All Human Sulfotransferases in Fission Yeast, Assay Development, and Structural Models for Isoforms SULT4A1 and SULT6B1

Abstract

Cytosolic sulfotransferases (SULTs) catalyze phase II (conjugation) reactions of drugs and endogenous compounds. A complete set of recombinant fission yeast strains each expressing one of the 14 human SULTs was generated, including SULT4A1 and SULT6B1. Sulfation of test substrates by whole-cell biotransformation was successfully demonstrated for all enzymes for which substrates were previously known. The results proved that the intracellular production of the cofactor 3′-phosphoadenosine 5′-phosphosulfate (PAPS) necessary for SULT activity in fission yeast is sufficiently high to support metabolite production. A modified variant of sulfotransferase assay was also developed that employs permeabilized fission yeast cells (enzyme bags). Using this approach, SULT4A1-dependent sulfation of 1-naphthol was observed. Additionally, a new and convenient SULT activity assay is presented. It is based on the sulfation of a proluciferin compound, which was catalyzed by SULT1E1, SULT2A1, SULT4A1, and SULT6B1. For the latter two enzymes this study represents the first demonstration of their enzymatic functionality. Furthermore, the first catalytically competent homology models for SULT4A1 and SULT6B1 in complex with PAPS are reported. Through mechanistic molecular modeling driven by substrate docking, we pinned down the increased activity levels of these two isoforms to optimized substrate binding.

Sulfotransferase 4A1 activity facilitates sulfate-dependent cellular protection to oxidative stress

“…Although, SULT4A1 contains the highly conserved SULT-catalytic Lys-Lys-His residues, no sulfation activity by SULT4A1 for small (physiological) compounds has been reported 1 , 3 , 4 , 25 . A recent report suggested that SULT4A1 is able to sulfonate 1-napthol using the S. pombe ‘enzyme’ bag assay, yet this observation has not been reproduced by other laboratories 32 . Fission yeast and budding yeast do not possess any endogenous SULT-activity.…”

Human Sulfotransferase Assays With PAPS Production in situ

“…The recombined fission yeast strains YN3, YN4, YN20, and YN25 used in this project were described before (Sun et al, 2020). The preparation of media and basic manipulation methods of S. pombe were carried out as described (Alfa et al, 1993).…”

“…This was essentially done as described before (Sun et al, 2020) with slight modifications. Briefly, fission yeast strains were grown in 10 ml liquid culture of EMM at 30 °C and 230 rpm for 24 h. Incubation of main cultures in 250 ml Erlenmeyer flasks was performed subsequently.…”

“…Initially, a sulfonation assay of 4HP (for reaction schemes see Supplementary Figure S1) by SULT1A3 (strain YN20) with external PAPS (100 µM) was carried out as described (Sun et al, 2020). For substitution of the cofactor PAPS by ATP and SO 4 2and optimization of the product yields multifactorial screening supported by Minitab software was started with five factors: ATP concentration, MgCl 2 concentration, number of cells, reaction incubation time, and time of preincubation without substrate.…”

“…While this has yet to be demonstrated experimentally, PAPS synthesis in S. pombe as such has already been reported (Song and Roe, 2008). Previously, all 14 human SULTs have been functionally expressed in S. pombe and, moreover, using this microbial host the functionality of SULT4A1 and SULT6B1 was demonstrated for the first time (Sun et al, 2020). In comparison to whole-cell biotransformation, sulfonation of drugs with permeabilized recombinant fission yeast cells (enzyme bags) provided higher sensitivity and shorter reaction times.…”