The metabolism of sulfur presents a complex and fascinating molecular network whose activities impact many areas of biology, including human disease. We now know that at least four of the enzymes involved in sulfur assimilation in bacteria organize into a multifunctional complex from which new catalytic function (ATP hydrolysis) emerges. Remarkably, this hydrolysis is kinetically and energetically linked, via conformational changes, to turnover of the first enzyme in the cysteine biosynthetic pathway - ATP sulfurylase. We intend to explore the mechanism of this linkage and to determine the composition and organization of the cysteine-metabolome both in vitro and in the environment of a living cell. We have discovered that in Type III sulfate activating complexes (SACs), activated sulfate (APS) travels between the active sites that produce and consume it along a deep 75 E-long groove that opens and closes in response to the position of APS. Using FRET, we will test the hypothesis that the channel closes to form a tubular structure during APS transit. Combining pre-steady state and FRET measurements, we will construct a timeline that interdigitates the events that occur in the catalytic cycle with changes in distance along the length of the channel. Using Brownian Dynamics we will advance a cutting-edge model of how changes in the shape and electrostatics of this remarkable molecular machine are coupled to the movement of the APS within it. Transfer of the sulfuryl- moiety (SO3) from activated sulfate to biological recipients is used widely by the cell to regulate metabolism. Sulfotransferases, which catalyze these transfers, are subject to allosteric substrate inhibition that is not well understood primarily because extensive structural and function work has not identified an allosteric binding pocket. The human estrogen sulfotransferase (EST) exhibits a presteady-state product burst that corresponds to precisely one-half of the active sites in the dimer. If EST is a half-site reactive enzyme, the non-catalytic active site might well function as the allosteric site of inhibition. We will test this hypothesis using the human EST, an enzyme whose activity is tightly and causally linked to cancer in the breast and endometrium.

Public Health Relevance

Transfer of the sulfuryl-group (SO3) from activated sulfate to various metabolic recipients is used widely by the cell to regulate function. Sulfotransferases, which catalyze these reactions, are themselves regulated by allosteric substrate inhibition, the molecular mechanism of which is unknown despite considerable effort to the contrary. We believe we now understand this mechanism, and will prove our mechanistic hypotheses in the upcoming grant period. The actions of these enzymes are tightly, causally linked to numerous human disease conditions, including: hemophilia B, compromised immune systems, androgyny, and breast and endometrial tumors. In a second Aim, we will explore a rare complex found in M. tuberculosis. This complex, which we discovered, is not present in mammals and therefore holds the promise of species-specific inhibition. This cysteine metabolome is comprise of at least four of the six enzymes in the cysteine biosynthetic pathway, and exhibits remarkable catalytic synergies. We will characterize this complex in detail, and for the first time.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function E Study Section (MSFE)
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Jones, Warren
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Albert Einstein College of Medicine
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Wang, Ting; Cook, Ian; Falany, Charles N et al. (2014) Paradigms of sulfotransferase catalysis: the mechanism of SULT2A1. J Biol Chem 289:26474-80
Rodriguez, Sofia B; Leyh, Thomas S (2014) An enzymatic platform for the synthesis of isoprenoid precursors. PLoS One 9:e105594
Cook, Ian; Wang, Ting; Almo, Steven C et al. (2013) The gate that governs sulfotransferase selectivity. Biochemistry 52:415-24
Jing, Chaoran; Cornish, Virginia W (2013) Design, synthesis, and application of the trimethoprim-based chemical tag for live-cell imaging. Curr Protoc Chem Biol 5:131-55
Leyh, Thomas S; Cook, Ian; Wang, Ting (2013) Structure, dynamics and selectivity in the sulfotransferase family. Drug Metab Rev 45:423-30
Pinto, Rachel; Leotta, Lisa; Shanahan, Erin R et al. (2013) Host cell-induced components of the sulfate assimilation pathway are major protective antigens of Mycobacterium tuberculosis. J Infect Dis 207:778-85
Cook, Ian; Wang, Ting; Falany, Charles N et al. (2013) High accuracy in silico sulfotransferase models. J Biol Chem 288:34494-501
Cook, Ian; Wang, Ting; Almo, Steven C et al. (2013) Testing the sulfotransferase molecular pore hypothesis. J Biol Chem 288:8619-26
Cook, Ian; Wang, Ting; Falany, Charles N et al. (2012) A nucleotide-gated molecular pore selects sulfotransferase substrates. Biochemistry 51:5674-83
Rohn, Katie Jo; Cook, Ian T; Leyh, Thomas S et al. (2012) Potent inhibition of human sulfotransferase 1A1 by 17ýý-ethinylestradiol: role of 3'-phosphoadenosine 5'-phosphosulfate binding and structural rearrangements in regulating inhibition and activity. Drug Metab Dispos 40:1588-95

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