Site-directed mutagenesis of rat hepatic hydroxysteroid sulfotransferases

Homma, Hiroshi; Ogawa, Kohei; Hirono, Keiko; Morioka, Yasunori; Hirota, Masami; Tanahashi, Isamu; Matsui, Masanao

doi:10.1016/0167-4838(96)00065-9

Cited by 15 publications

(8 citation statements)

References 29 publications

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“…The key residues such as Lys48 and His108 are coordinated with the leaving phosphate, vanadate, and water molecules so as to stabilize the transition state of the sulfuryl transfer reaction. This is consistent with the fact that all known mutations of the corresponding lysine and histidine residues abolish the activity of sulfotransferases [18][19][20][45][46][47].…”

Section: ‫מ3‬supporting

confidence: 87%

Crystal structure‐based studies of cytosolic sulfotransferase

Yoshinari

Petrotchenko

Pedersen

et al. 2001

J Biochem & Molecular Tox

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Sulfation is a widely observed biological reaction conserved from bacterium to human that plays a key role in various biological processes such as growth, development, and defense against adversities. Deficiencies due to the lack of the ubiquitous sulfate donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS) are lethal in humans. A large group of enzymes called sulfotransferases catalyze the transfer reaction of sulfuryl group of PAPS to the acceptor group of numerous biochemical and xenochemical substrates. Four X-ray crystal structures of sulfotransferases have now been determined: cytosolic estrogen, hydroxysteroid, aryl sulfotransferases, and a sulfotransferase domain of the Golgi-membrane heparan sulfate N-deacetylase/N-sulfotransferase 1. These have revealed the conserved core structure of the PAPS binding site, a common reaction mechanism, and some information concerning the substrate specificity. These crystal structures introduce a new era of the study of the sulfotransferases.

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Section: ‫מ3‬supporting

confidence: 87%

Crystal structure‐based studies of cytosolic sulfotransferase

Yoshinari

Petrotchenko

Pedersen

et al. 2001

J Biochem & Molecular Tox

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“…Our findings therefore suggest that p-nitrophenol and dopamine binding is dependent on amino acid residues contributed by different regions in the HASTs. This phenomenon has been described previously by Homma et al [26] for rat hydroxysteroid ST binding of dehydroepiandrosterone and cortisol. Further, our findings confirm those of a recent Structural characterization of human sulphotransferases study by Sakakibara et al [24], which indicated that amino acids within Regions A and B determine the substrate preferences of HAST1 and HAST3.…”

Section: St Activities Of Chimaeric Hast Proteins Towards the Substrasupporting

confidence: 80%

Structural characterization of human aryl sulphotransferases

Brix

Duggleby

Gaedigk

et al. 1999

Biochemical Journal

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Human aryl sulphotransferase (HAST) 1, HAST3, HAST4 and HAST4v share greater than 90% sequence identity, but vary markedly in their ability to catalyse the sulphonation of dopamine and p-nitrophenol. In order to investigate the amino acid(s) involved in determining differing substrate specificities of HASTs, a range of chimaeric HAST proteins were constructed. Analysis of chimaeric substrate specificities showed that enzyme affinities are mainly determined within the N-terminal end of each HAST protein, which includes two regions of high sequence divergence, termed Regions A (amino acids 44-107) and B (amino acids 132-164). To investigate the substrate-binding sites of HASTs further, site-directed mutagenesis was performed on HAST1 to change 13 individual residues within these two regions to the HAST3 equivalent. A single amino acid change in HAST1 (A146E) was able to change the specificity for p-nitrophenol to that of HAST3. The substrate specificity of HAST1 towards dopamine could not be converted into that of HAST3 with a single amino acid change. However, compared with wild-type HAST1, a number of the mutations resulted in interference with substrate binding, as shown by elevated Ki values towards the co-substrate 3'-phosphoadenosine 5'-phosphosulphate, and in some cases loss of activity towards dopamine. These findings suggest that a co-ordinated change of multiple amino acids in HAST proteins is needed to alter the substrate specificities of these enzymes towards dopamine, whereas a single amino acid at position 146 determines p-nitrophenol affinity. A HAST1 mutant was constructed to express a protein with four amino acids deleted (P87-P90). These amino acids were hypothesized to correspond to a loop region in close proximity to the substrate-binding pocket. Interestingly, the protein showed substrate specificities more similar to wild-type HAST3 than HAST1 and indicates an important role of these amino acids in substrate binding.

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“…To examine this possibility, His 107 was mutated to asparagine to resemble histidine without the negative charge, and the enzymatic characteristics of the mutant were analyzed. Unlike previous alanine mutants that were unstable (5,21,22), the hEST H107N mutant was expressed as a soluble protein to as high a level as the wild-type enzyme in E. coli cells. The mutant was subjected to enzyme assays for estrogen sulfotransferase and PAPS hydrolysis activities.…”

Section: Role Of Sermentioning

confidence: 99%

Crystal Structure of the Human Estrogen Sulfotransferase-PAPS Complex

Pedersen¹,

Petrotchenko²,

Shevtsov

et al. 2002

Journal of Biological Chemistry

114

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Estrogen sulfotransferase (EST) transfers the sulfate group from 3-phosphoadenosine 5-phosphosulfate (PAPS) to estrogenic steroids. Here we report the crystal structure of human EST (hEST) in the context of the V269E mutant-PAPS complex, which is the first structure containing the active sulfate donor for any sulfotransferase. Superimposing this structure with the crystal structure of hEST in complex with the donor product 3-phosphoadenosine 5-phosphate (PAP) and the acceptor substrate 17␤-estradiol, the ternary structure with the PAPS and estradiol molecule, is modeled.

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Site-directed mutagenesis of rat hepatic hydroxysteroid sulfotransferases

Cited by 15 publications

References 29 publications

Crystal structure‐based studies of cytosolic sulfotransferase

Crystal structure‐based studies of cytosolic sulfotransferase

Structural characterization of human aryl sulphotransferases

Crystal Structure of the Human Estrogen Sulfotransferase-PAPS Complex

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