The Structure of Human SULT1A1 Crystallized with Estradiol

Abstract

Human SULT1A1 belongs to the supergene family of sulfotransferases (SULTs) involved in the sulfonation of xeno-and endobiotics. The enzyme is also one of the SULTs responsible for metabolic activation of mutagenic and carcinogenic compounds and therefore is implicated in various cancer forms. Further, it is not well understood how substrate inhibition takes place with rigid fused multiring substrates such as 17␤-estradiol (E2) at high substrate concentrations when subcellular fractions or recombinant enzymes are used. To investigate how estradiol binds to SULT1A1, we co-crystallized SULT1A1 with sulfated estradiol and the cofactor product, PAP (3-phosphoadenosine 5-phosphate). The crystal structure of SULT1A1 that we present here has PAP and one molecule of E2 bound in a nonproductive mode in the active site. The structure reveals how the SULT1A1 binding site undergoes conformational changes to accept fused ring substrates such as steroids. In agreement with previous reports, the enzyme shows partial substrate inhibition at high concentrations of E2. A model to explain these kinetics is developed based on the formation of an enzyme⅐PAP⅐E2 dead-end complex during catalysis. This model provides a very good quantitative description of the rate versus the [E2] curve. This dead-end complex is proposed to be that described by the observed structure, where E2 is bound in a nonproductive mode. Cytosolic sulfotransferases (SULTs)2 are involved in the phase II metabolism of numerous xeno-and endobiotics such as drugs, neurotransmitters, bile acids, and hormones (1, 2). SULTs utilize the cofactor 3Ј-phosphoadenosine 5Ј-phosphosulfate (PAPS) as the sulfonate (SO 3 Ϫ1 ) donor in such reactions that generally lead to the detoxification of substrates by making them more water-soluble and thereby readily excretable via the kidneys. However, in the case of mutagenic and carcinogenic N-hydroxyarylamines and heterocyclic amines and benzylic alcohols of polycyclic aromatic hydrocarbons, sulfonation is a critical step in their metabolic activation to electrophiles that can bind tissue macromolecules such as DNA (3-6). Further, sulfonation has been shown to activate the antihypertensive and hair growth stimulant minoxidil (7). The overall role that sulfonation plays in disease states such as neurodegeneration and cancer is currently under active investigation (8 -10). SULT1A1 has a broad tissue distribution and has been shown to metabolize a wide range of xenobiotics and play a significant role in the metabolism of estrogens and iodothyronines. In resolving the crystal structure (11) of SULT1A1 we showed that it contains a very hydrophobic L-shaped substrate-binding region, which explains how it can accommodate small planar compounds as well as larger L-shaped aromatics such as iodothyronines. However, in those studies we could not explain how extended fused ring systems, such as 17␤-estradiol (E2), can act as substrates of SULT1A1. In addition, no real information has been provided as to why the metabolism of E2 is inhibited at high...

Structural and Chemical Profiling of the Human Cytosolic Sulfotransferases

“…Recent progress in the structural biology and characterization of the catalytic mechanism of hSULTs has established that many family members have distinct, but overlapping, substrate specificities and that the enzymes have a sequential catalytic mechanism that is susceptible to substrate inhibition [6,7]. Nevertheless, only a few of the human enzymes have been subjected to detailed structural and mechanistic studies [6,8–16], and there are no reports of a systematic comparison among all the hSULTs.…”

“…Nevertheless, only a few of the human enzymes have been subjected to detailed structural and mechanistic studies [6,8–16], and there are no reports of a systematic comparison among all the hSULTs. Understanding the structural and mechanistic basis for specificity among hSULTs is essential to elucidate their role in the metabolism of regulatory hormones, drugs, and carcinogens, and may assist in chemical risk assessment and the design of more-effective therapeutics.…”

Removal of Substrate Inhibition and Increase in Maximal Velocity in the Short Chain Dehydrogenase/Reductase Salutaridine Reductase Involved in Morphine Biosynthesis

“…2A (39). For sulfotransferases, the crystal structure showed that the substrate adopts a different conformation and binds closer to the cofactor binding site after the cofactor has been desulfated (40,41). In lactate dehydrogenase, substrate inhibition has been partially linked with positioning of the NAD ϩ cofactor rather than to substrate binding (42).…”

Structure-Based Mutational Studies of Substrate Inhibition of Betaine Aldehyde Dehydrogenase BetB from Staphylococcus aureus

“…Since YdcW exhibits no substrate inhibition by BA, we propose calling this binding mode the productive binding mode, whereas the second type of BA coordination (with the H bond to the Asn157 side chain) is the nonproductive binding mode. The productive and nonproductive substrate binding modes have also been described for the human sulfotransferases (SULT1A, SULT1E, and SULT2A1) and for salutaridine reductase SalR from Papaver bracteatum (13,46,47).…”