1.1 Preface
In the post genomic era, great quantities of sequence information are available and most of them are needed to be interpreted for their functional meanings. For example, as the human genomic project is completed in 2003, approximately 20,000-25,000 genes in human DNA were identified and most of them are either unknown or poorly studied. This is following by structural genomics, a field dedicated to a broad understanding of protein structures and functions in relation to gene sequences. The structure of one or more proteins from each family, for a total of about 10000 protein structures in 10 years, will be determined. With these sequence and structure information available, great opportunities have evolved for us to subsequently elucidate the detailed mechanism of enzyme action.
Sulfonation is a widespread biological reaction. Sulfotransferase that catalyzes the transfer of a sulfuryl group from 3’-phosphoadenosine 5’-phosphosulfate (PAPS) to a substrate acceptor group is responsible for all the known biological sulfonation. Pathological and toxicological evidences indicate that the abnormal sulfonation leads to inflammations, cancers and infectious diseases (1-3). Understanding the enzyme catalytic mechanism is important for drug design and therapeutic strategy consequently. The use of structure-based sequence alignments and three-dimensional quantitative structure-activity relationship (3D-QSAR) techniques were shown to be very useful to screen for the inhibitors as drug targets in high throughput scale (4-5).
In this research we deduce the enzyme catalytic mechanism of human dehydroepiandrosterone sulfotransferase (SULT2A1, DHEA-ST) by the comparison of its amino acid sequences and spatially resolved crystal structures. SULT2A1 mainly catalyzes the sulfonation of various steroids and their derivatives, including hydroxysteroids such as
dehydroepiandrosterone (DHEA), androsterone (ADT), testosterone, estradiol, and many endogenous steroids (6-9). Steroid sulfonation has been recognized as an important process for maintaining steroid hormone levels during their metabolism. In humans, dehydroepiandrosterone sulfate (DHEAS) is the most prevalent steroid precursor, and is one of the major secretory products of both adult and fetal adrenals (10). To know more about the actions of SULT2A1 to convert the DHEA to DHEAS, this research from sequence and structure of SULT2A1 is studied and then the deduced enzyme mechanism is further confirmed by site-directed mutagenesis and enzymatic analysis.
1.2 Sulfotransferase 1.2.1 Sulfonation
It has been known for a long time that sulfonation occurs in a biological system.
Baumann first described sulfonate conjugation as a pathway in biotransformation in 1876 (11).
However, the biochemistry of sulfotransferase enzyme has not been well characterized until the past three decades. Furthermore, the application of molecular biology technology had made it possible to determine the characteristics of these enzymes, whose substrate specificities, regulation, and evolutionary relationships (12). However, the mechanism by which sulfonation takes place was not examined until after the isolation of the active sulfate donor, 3’-phosphoadenosine 5’-phosphosulfate (PAPS), about 80 years later (13).
Sulfotransferases (SULTs) catalyze the transfer of a sulfuryl group from 3’-phosphoadenosine 5’-phosphosulfate (PAPS), the universal sulfonate donor molecule, to a variety of amine and hydroxyl substrates as nucleophiles in a process originally called sulfonation (Fig. 1). There are two classes of sulfotransferases: cytosolic sulfotransferases and membrane-associated sulfotransferases. Cytosolic sulfotransferases sulfonate small endogenous and exogenous compounds, such as drugs, steroid hormones, chemical carcinogens, bile acids, and neurotransmitters (14-19). Membrane-associated sulfotransferases, many of which have been implicated recently in crucial biological processes, sulfonate larger biomolecules, suchas carbohydrates and proteins (20-22).
Because of the biological importance and medical relevance of sulfotransferases, there is intense interest in the exact functions of these enzymes. Much information is available with regard to the structure, substrate specificity, and kinetic mechanism of cytosolic sulfotransferases, but less information is available on the transition-state structure and design of potent and specific inhibitors. The membrane-associated enzymes are not as well understood, particularly because their important roles in biological processes have only
recently been uncovered.
1.2.2 Classification of sulfotransferases
The membrane-associated sulfotransferaes are still without a consistent, universal nomenclature scheme and they are named according to their substrate specificity so far. This nomenclature is clarified as required. On the side of cytosolic sulfotransferases, they had often been named after their substrates. However, since the substrate specificities of different sulfotransferases are overlapping, such names can be misleading. To date 10 or 11 human cytosolic sulfotransferases have been characterized and so far the new nomenclature guidelines were applied to 65 sulfotransferase cDNAs and 18 sulfotransferase genes that were characterized from eukaryotic organisms (23). These sequences were evaluated and named on the basis of encoded amino acid sequence identity. Family members share at least 45% amino acid sequence identity whereas subfamily members are at least 60% identical. cDNAs which encode amino acid sequences of at least 97% identity to each other were assigned identical isoform names (Fig. 2).
1.2.3 Substrate specificity of sulfotransferases
Although the sulfotransferase mainly possess PAPS as the universal sulfonate donor molecule, a number of studies have also been conducted on the specificity of the PAPS-binding site (24-25). Moreover, different sulfotransferases exhibit distinct substrate specificity. For example, sulfonation of carbohydrates, peptides, hormones, steroids and neurotransmitters are all catalyzed by different sulfotransferases. However, within a subfamily, sulfotransferase has a broad substrate spectrum for similar substrates. As can be seen in Table I, the diverse substrate specificity is catalyzed by different sulfotransferases
substrates of sulfotransferase are simple-phenol compounds, such as p-nitrophenol (pNP), dopamine, and thyroid. Sulfotransferase in SULT2 family, however, mainly catalyzes hydroxysteroids, such as cholesterol, androsterone, dehydroepiandrosterone, pregnenolone, and estradiol. Each of the two families, SULT1A1 and SULT2A1, contains diverse substrate binding site and a separate but identical PAPS binding site.
1.2.4 Structure of cytosolic sulfotransferase
Sulfotransferases are a single α/β globular protein with a characteristic five-stranded parallel β-sheet and α-helices flank both sides of the sheet (26-27). Sulfotransferases share a similar structural resemblance to the nucleotide kinases with their secondary structures conserved not only in position but also in connectivity (28).
The strand–loop–helix and strand–turn–helix motifs constitute the core PAPS binding site, providing the majority of the enzyme interactions with the PAP molecule. The PSB-loop interacts with the 5’-phosphate of the PAP molecule, whereas helix 6 of the strand–turn– helix unit that runs parallel to the PSB-loop provides interaction with the 3’-phosphate. Not only the structures, but also the amino acid sequences of the phosphate-binding sites are conserved in all sulfotransferases including both the cytosolic and membrane enzymes. (29).
Furthermore, although the sulfotransferase enzymes display broad substrate specificity, a given enzyme can often be characterized by a specific substrate. The underlying principle that regulates the characteristic substrate specificity is not well developed. Finally, cytosolic sulfotransferases are generally homodimers in their catalytically active forms in solution. The conserved dimerization motif is found in the cytosolic enzymes (30). Multiple amino acid sequence alignments show that the motif consists of ten residues near the C-terminus and is represented by the consensus sequence KXXXTVXXXE. Structural comparisons and mutagenesis studies were undertaken with mouse SUL T1E1 (a monomer) and human
SULT1E1 (a dimer) in an attempt to identify a common structural motif. It was found that the mutations V269E and V260E converted the homodimers SULT1E1 and SULT2A1, respectively, into monomers.
1.3 Dehydroepiandrosterone (DHEA) and dehydroepiandrosterone 3β-sulfate (DHEAS) 1.3.1 Background
Hormones play a plenty of functions physiologically and the abnormal regulation may force the dysfunction and raise the risk, such as the hormone homeostasis and hormone-related disease or metabolism. In this study, I mainly interested in the study of sexual hormone, such as the estradiol, androsterone, testosterone, and dehydroepisterone. For example, the dehydroepisterone (DHEA) plays the profound role and is the precursor of the sexual hormone.The abnormal regulation may cause the irregular secretory amounts of the other hormone as described above. Dehydroepiandrosterone (DHEA) was isolated in urine in 1934, and DHEA 3β-sulfate (DHEAS) was identified 10 years later (31-32). It took another decade to identify DHEA and DHEAS in peripheral blood (33-34). DHEA is one of the hormones produced by the adrenal glands. After being secreted by the adrenal glands, it circulates in the bloodstream as DHEAS and is converted as needed into other hormones.
DHEA and DHEAS are the most abundant steroid hormones in the human bloodstream.
DHEA is known to be a precursor to the numerous steroid sex hormones (including estrogen and testosterone), which serve well-known functions. While DHEA levels reach their peak in the early morning hours, DHEAS levels show no diurnal variation. It is also one of the most significant age-related biomarkers, which predictably declines with age in even the healthiest of people. Blood levels are highest in the developing fetus, drop sharply after birth, begin climbing again at age 6 to 8 (a time of rapid growth) to a peak at age 25 to 30 and then decline to about 10% of the peak level by age 80 (Fig. 3). Abnormally low levels of DHEA have been reported to be related to a number of diseases, including cancer, diabetes, coronary artery disease, and obesity.
1.3.2 Physiological regulation
Little is known about how DHEA works in the body and it often has different effects in men, premenopausal women, and postmenopausal women (35-36). Supplementation with DHEA-S has resulted in increased levels of testosterone and androstenedione (37). The conversion of DHEA into testosterone may account for the fact that low blood levels of DHEA have been reported in some men with erectile dysfunction. The findings of a double-blind trial using 50 mg supplements of DHEA taken daily for six months suggests that DHEA may improve erectile function in some men (38).
1.3.3 DHEA modulates immunity
A group of elderly men with low DHEA levels who were given 50 mg of DHEA per day for 20 weeks experienced a significant activation of immune function (39). Postmenopausal women have also shown increased immune functioning in just three weeks when given DHEA in double-blind research (40). DHEA also regulates the systemic lupus erythematosus (SLE), an autoimmune disease. It has been linked to abnormalities in sex hormone metabolism (41). Supplementation with very large amounts of DHEA (200 mg per day) improved clinical status and reduced the number of exacerbations of SLE in a double-blind trial (42). A preliminary trial has confirmed the benefit of 50-200 mg per day of DHEA for people with SLE (43).
1.3.4 DHEA and cardiopathy
Some reports have suggested that DHEA might reduce the risk of heart disease, perhaps by lowering cholesterol levels. In fact, higher levels of DHEA and DHEAS have been associated with cardiovascular risk factors in women, including high blood pressure and
1.4 Conculsion
DHEA is a multifunctional hormone and plays a plenty of function, such as steroid metabolism, cancer, immunity, and aging or more. The sulfated form of DHEA, DHEAS, is primarily in the adrenals, the liver, and small intestines. However, in blood, most DHEA is found as DHEAS with levels that are about 300 times higher than free DHEA. Orally ingested DHEA is converted to its sulfate when passing through intestines and liver. DHEAS is biologically active only after its sulfate group has been split and it becomes DHEA again.
From a practical point measurement of DHEAS is preferable to DHEA as levels are more stable. The conversion between DHEA and DHEAS is regulated by SULT2A1 which preferred substrate is DHEA and the regulation of this enzyme and catalytic mechanism is still far from known. In this study we deduce the enzyme mechanism and function from its amino acid sequence and the solved crystal structure. Herein we analyze the quaternary structure, wild-type homodimer and monomer mutant. The monomer mutant, V260E, sufficiently interfere the hydrophobic dimerization interface and the molecular weight is confirmed by gel filtration. Further the circular dichroism spectrum, enzyme kinetics, substrate binding affinity, and the thermal and conformational stability reveal the difference between dimer and monomer of SULT2A1. Moreover, the comparisons of SULT2A1 structure complex also facilitate us to infer the substrate inhibition mechanism and further identify the regulatory amino acid residues. Experimental results and the multiple structural alignment all indicate that the involvement of this amino acid residue in substrate inhibition may be a general feature in all cytosolic sulfotransferases.
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Table 1: Substrate specificity of human cytosolic sulfotransferasesa.
a Adapted from Chapman et al. (2004) Angew. Chem. Int. Ed. Engl. 43, 3526-3548.
Figure 1. Sulfuryl group transfer in a biological system.
Figure 2. Cytosolic sulfotransferase protein classification on the basis of primary amino acid sequence. The cladogram depicts the evolutionary relationship of sulfotransferase enzyme superfamily members based on their amino acid sequences. (Adapted from Blanchard et al. (2004) Pharmacogenetics. 14, 199-211).
Figure 3. Natural level of DHEA with age. (adapted from the website http://www.benbest.com/).
CHAPTER 2 Effects of Quaternary Structure on the Activity of Human Dehydroepiandrosterone Sulfotransferase
Human dehydroepiandrosterone sulfotransferase (SULT2A1, DHEA-ST) catalyzes the transfer of the sulfuryl group of 3’-phosphoadenosine 5’-phosphosulfate (PAPS) to hydroxysteroids such as dehydroepiandrosterone (DHEA). Most cytosolic sulfotransferases are generally known to be homodimer in solution and the specific amino acid residues responsible for the dimerization have been identified as the KXXXTVXXXE motif (Petrotchenko et al. (2001) FEBS Lett. 490, 39–43). However, the comparisons of the characteristics and catalytic actions between dimer and monomer of cytosolic sulfotransferases have not been studied in detail so far. To study the effects of quaternary structure on the activity of the enzyme, monomer mutant of SULT2A1 was prepared through site-directed mutagenesis. The mutant V260E of SULT2A1 was sufficient to convert to the monomer by interfering with hydrophobic KTVE motif and confirmed by gel filtration. The circular dichroism spectrum of dimer and monomer mutant reveals the slight conformational change whether in the secondary or tertiary structure. The kinetic constants of dimer and monomer mutant are quite similar whether the PAPS or DHEA are examined, however, the catalytic efficiency, kcat/Km, shows two folds higher in dimer than that in monomer. It indicates that the two subunits in dimer may be both catalytically competent. The binding affinity shows no significantly change whether for PAP in the binary complex (SULT2A1/PAP) or DHEA in the tertiary complex (SULT2A1/PAP/DHEA) between dimer and monomer. Furthermore, the thermal and conformational stability between dimer and monomer mutant all revealed the monomer is more labile than dimer. The effect of the quaternary structure on SULT2A1 may be a model to deduce the other cytosolic sulfotransferases whether the wild type is the homodimer or monomer in solution naturally.
INTRODUCTION
Sulfotransferases (SULTs) are a large family of enzymes that catalyze the transfer of sulfuryl group from the common 3’-phosphoadenosine 5’-phosphosulfate (PAPS) to numerous endogenous and exogenous compounds. Cytosolic sulfotransferases sulfonate small molecules such as drugs, steroid hormones, chemical carcinogens, bile acids, and neurotransmitters (1-6).
Human dehydroepiandrosterone sulfotransferase (SULT2A1, DHEA-ST) catalyzes the sulfonation of various steroids and their derivatives, including hydroxysteroids such as dehydroepiandrosterone (DHEA), androsterone (ADT), testosterone, estradiol, and many endogenous steroids (7-10). Steroid sulfonation has been recognized as an important process for maintaining steroid hormone levels during their metabolism. In humans, dehydroepiandrosterone sulfate (DHEAS) is the most prevalent steroid precursor, and is one of the major secretory products of both adult and fetal adrenals (11).
Cytosolic sulfotransferases consist of around 300 amino acid residues, and most of them
Cytosolic sulfotransferases consist of around 300 amino acid residues, and most of them