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.
Highlights
IntroductionThe plethora of biotransformations that together constitute human drug metabolism is subdivided into phase I (functionalization) and phase II (conjugation) reactions
The plethora of biotransformations that together constitute human drug metabolism is subdivided into phase I and phase II reactions
Sulfation activities of the twelve strains expressing human SULT1 or SULT2 enzymes were confirmed by whole-cell biotransformations using the known standard substrates 4-nitrophenol, 1-naphthol, 7-hydroxycoumarin, and DHEA, respectively. These results confirm that the intracellular level of the cofactor phosphoadenosine -phosphosulfate (PAPS) is sufficiently high for sulfation reactions in fission yeast, as was previously demonstrated in similar experiments in baker’s yeast [6]
Summary
The plethora of biotransformations that together constitute human drug metabolism is subdivided into phase I (functionalization) and phase II (conjugation) reactions. Cytosolic sulfotransferases (SULTs) catalyze the transfer of a sulfate group from the universal sulfate donor 3 -phosphoadenosine 5 -phosphosulfate (PAPS) to both endogenous and xenobiotic compounds [2] In these reactions, the SO3− moiety is transferred to hydroxy or amino functions of small molecule substrates. Most of the human SULTs are reported to be functionally expressed in Escherichia coli [5] or in Saccharomyces cerevisiae [6] For two of these enzymes, namely, SULT4A1 and SULT6B1, no endogenous substrate nor activity data were ever reported before this study. We successfully used fission yeast Schizosaccharomyces pombe for the functional expression of orphan cytochrome P450 (CYP) enzymes, such as CYP2A7, CYP4Z1, CYP4A22, and CYP20A1 [13,14,15,16], and UGTs such as UGT1A5 [17]. We aimed to rationalize our experimental results by modeling SULT–substrate complexes using homology modeling and substrate docking experiments
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
