Biocatalytic sulfation of aromatic and aliphatic alcohols catalyzed by arylsulfate sulfotransferases

Abstract

Many relevant metabolites, as well as chemical commodities, contain at least one sulfate ester group. Consequently, biocatalytic strategies to attach sulfate to a molecule under mild conditions are of high interest. In order to expand the enzymatic toolbox available, five new arylsulfate sulfotransferases (ASSTs) were identified in this study. Overexpression in Escherichia coli and enzyme purification resulted in soluble proteins which catalyzed the sulfate transfer to an acceptor substrate using p-nitrophenyl sulfate (pNPS) as sulfate donor. Optimal reaction conditions were established with respect to temperature and pH, as well as their tolerance to organic co-solvents and melting temperature. Additionally, the kinetic parameters (V_max, K_M, and k_cat) were determined. The substrate scope for the acceptor showed that a structurally diverse spectrum of alcohols is accepted. The substrates included phenolic alcohols with one, two, and three hydroxy groups, linear and cyclic aliphatic alcohols, and amines. The phenolic substrates were accepted reaching activities of up to 154 U/mg purified enzyme. Additionally, also the aliphatic alcohols (both linear and cyclic) were accepted at reduced activity, showing that these enzymes are not limited to phenolic alcohols. Moreover, catalytic activity was detected when using aniline as an acceptor substrate implying their ability to sulfate also amino groups. Finally, the consecutive sulfation of di- and trihydroxy compounds was observed, resulting in the detection of the corresponding disulfated molecules. KEY POINTS: • Five novel arylsulfate sulfotransferases were identified and characterized. • Accepted substrates included aromatic and aliphatic alcohols, as well as aniline. • Disulfation of di- and trihydroxy aromatic compounds was studied and confirmed.

Keywords: Aliphatic alcohol sulfation; Amine sulfation; Arylsulfate sulfotransferase; Disulfated compounds; Enzymatic sulfation.

Fig. 1

Sulfation reaction catalyzed by the two classes of sulfotransferase (ST). a PAPS-dependent ST and b PAPS-independent ST, i.e., ASSTs

Fig. 2

Specific activity displayed by ASSTHeff (white box), ASSTDor (light gray box), ASSTDfor (gray box), ASSTC (dark gray box), and ASSTDdeh (black box) depending on the reaction temperature. The insert shows an amplification of the plot between 0.0 and 0.4 U/mg of protein to facilitate the visualization of the activity from the less active ASSTs. Reactions were carried out in buffer Tris/HCl 50 mM, pH 8.0; phenol 1.0 mM; pNPS 1.0 mM; and purified ASST. Kinetic measures were followed during 30 min at temperatures between 25 and 45 °C

Fig. 3

Relative activity displayed by ASSTHeff, ASSTDor, ASSTDfor, ASSTC, ASSTDdeh depending on the reaction pH. Reactions were carried out with phenol 1.0 mM, pNPS 1.0 mM, and purified ASST. Kinetic measures were followed at 30 °C during 30 min. Reactions with ASSTDor were not carried out at pH above 9.0 due to precipitation of the enzyme in the reaction

Fig. 4

Relative activity displayed by ASSTHeff (white box), ASSTDor (light gray box), ASSTDfor (gray box), ASSTC (dark gray box), ASSTDdeh (black box) depending on the presence of co-solvents (10% v/v). Reactions were carried out in buffer Tris/HCl 50 mM, pH 8.0 with 10% (v/v) DMSO, phenol 1.0 mM, pNPS 1.0 mM, and purified ASST. Kinetic measures were followed during 30 min at temperatures at 30 °C

Fig. 5

Compounds screened as sulfate acceptors for the ASSTs, i.e., phenol (1), catechol (2), resorcinol (3), hydroquinone (4), 1,2,4-benzenetriol (5), 1,3,5-benzenetriol (6), 4,4′-dihydroxybiphenyl (7), 2-naphthol (8), aniline (9), cyclohexanol (10), all-rac-cyclohexanediol (11), all-rac-2-aminocyclohexanol (12), cyclohexylamine (13), uridine (14), N-acetylglucosamine (15), 1-hexanol (16), rac-1,5-hexanediol (17), rac-2-hexanol (18), and all-rac-2-methoxycyclohexanol (19)

Fig. 6

Specific activity of ASSTs towards phenolic compounds (1–8) and pNPS determined by colorimetric assay following pNP formation at 410 nm. Reactions conditions: Tris/HCl buffer (50 mM, pH 8.0), pNPS (1 mM), acceptor substrate (1, 10 or 100 mM), and the purified ASST

Fig. 7

Specific activity towards non-phenolic acceptors (9–19) displayed by the studied ASSTs. Reaction conditions: Tris/HCl buffer (50 mM, pH 8.0), pNPS (1 mM), acceptor substrate (1, 10 or 100 mM), and the purified ASST