Thomas S. Leyh

Chemical potential is coupled to cellular processes by the flow of metabolites through catalytic networks known collectively as metabolism. Here we describe an extensive new class of energy-coupling catalysts that act to interconnect metabolic network pathways and their potentials. Members of the class are defined by a common mechanism — half-site reactivity. The well-established sequential subunit turnover of half-site enzymes suggests that the potentials of reactions occurring at the separate subunits are coupled to one another. Here this hypothesis is tested and validated using promiscuous half-site enzymes from two catalytically distinct enzyme families, each with broad metabolic penetrance. Fundamental catalytic parameters (V _max and K _m ) and reaction endpoints are predicted and shown to change dramatically when reaction potentials are coupled — for example, the catalytic efficiency (V _max /K _m ) and endpoint of the retinol oxidation reaction (the rate-limiting step in vitamin A synthesis) are shown to increase 900- and 3,400-fold, respectively, when the reaction is coupled to the more favorable oxidation of ethanol. For the first time it is clear that metabolism has the flexibility to react to changes in the metabolic state of the cell by redistributing chemical potential among the many metabolic pathways interconnected by half-site enzymes. Significance: The findings herein reveal the existence of an extensive, catalytically diverse network of enzymes that distributes chemical potential within and across the pathways of small-molecule metabolism. Members of the network are identified on the basis of a shared mechanistic trait — half-site reactivity. These energy-coupling catalysts allow reactions to proceed orders of magnitude further and more efficiently than their intrinsic potentials allow by coupling them to more favorable reactions. The work offers a raison d’etre for the half-site mechanism and powerful new strategies for de novo metabolic pathway design.

The primary function of human sulfotransferase 2B1b (SULT2B1b) is to sulfonate cholesterol and closely related sterols. SULT2B1b sterols perform a number of essential cellular functions. Many are signaling molecules whose activities are redefined by sulfonation—allosteric properties are switched “on” or “off,” agonists are transformed into antagonists, and vice versa. Sterol sulfonation is tightly coupled to cholesterol homeostasis, and sulfonation imbalances are causally linked to cholesterol-related diseases including certain cancers, Alzheimer disease, and recessive X-linked ichthyosis—an orphan skin disease. Numerous studies link SULT2B1b activity to disease-relevant molecular processes. Here, these multifaceted processes are integrated into metabolic maps that highlight their interdependence and how their actions are regulated and coordinated by SULT2B1b oxysterol sulfonation. The maps help explain why SULT2B1b inhibition arrests the growth of certain cancers and make the novel prediction that SULT2B1b inhibition will suppress production of amyloid β (Aβ) plaques and tau fibrils while simultaneously stimulating Aβ plaque phagocytosis. SULT2B1b harbors a sterol-selective allosteric site whose structure is discussed as a template for creating inhibitors to regulate SULT2B1b and its associated biology. Significance Statement Human sulfotransferase 2B1b (SULT2B1b) produces sterol-sulfate signaling molecules that maintain the homeostasis of otherwise pro-disease processes in cancer, Alzheimer disease, and X-linked ichthyosis—an orphan skin disease. The functions of sterol sulfates in each disease are considered and codified into metabolic maps that explain the interdependencies of the sterol-regulated networks and their coordinate regulation by SULT2B1b. The structure of the SULT2B1b sterol-sensing allosteric site is discussed as a means of controlling sterol sulfate biology.

Among human cytosolic sulfotransferases, SULT2B1b is highly specific for oxysterols — oxidized cholesterol derivatives, including nuclear-receptor ligands that are causally linked to cancer, atherosclerosis, and skin and neurodegenerative diseases. Sulfonation of signaling oxysterols redirects their receptor-binding functions, and controlling these functions is expected to prove valuable in disease prevention and treatment. SULT2B1b is distinct among the human SULT2 isoforms by virtue of its atypically long N-terminus, which extends 18-residues beyond the next longest N-terminus in the family. Here, in-silico studies are used to predict that the N-terminal extension forms an allosteric pocket and to identify potential allosteres. One such allostere, quercetin, is used to confirm the existence of the pocket and to demonstrate that allostere binding inhibits turnover. The structure of the pocket is obtained by positioning quercetin on the enzyme, using spin-label-triangulation NMR, followed by NMR-distance constrained molecular dynamics docking. The model is confirmed using a combination of site-directed mutagenesis and initial-rate studies. Stopped-flow ligand-binding studies demonstrate that inhibition is achieved by stabilizing the closed form of the enzyme active-site cap, which encapsulates the nucleotide, slowing its release. Finally, endogenous oxysterol metabolites are shown bind the site in a highly selective fashion — one of two immediate biosynthetic precursors of cholesterol (7-dehydrocholesterol) is an inhibitor, the other (24-dehydrocholesterol) is not. These findings provide insight into the allosteric dialogue in which SULT2B1b participates in in vivo, and establishes a template against which to develop isoform-specific inhibitors to control SULT2B1b biology.

The 20 uridine diphosphate glycosyl-transferases (UGTs) encoded in the human genome form an essential homeostatic network of overlapping catalytic functions that surveil and regulate the activity and clearance of scores of small molecule metabolites. Biochemical and biophysical UGT studies have been hampered by the inability to purify these membrane-bound proteins. Here, using cell-free expression and nanodisc technology, we assemble and purify to homogeneity the first UGT nanodisc—the human UGT2B7•nanodisc. The complex is readily isolated in milligram quantities. It is stable and its initial-rate parameters are identical within error to those associated with UGT2B7 in microsomal preparations (i.e., Supersomes). The high purity of the nanodisc preparation simplifies UGT assays, which allows complexities traditionally associated with microsomal assays (latency and the albumin effect) to be circumvented. Each nanodisc is shown to harbor a single UGT2B7 monomer. The methods described herein should be widely applicable to UGTs, and these findings are expected to set the stage for experimentalists to more freely explore the structure, function, and biology of this important area of phase II metabolism. SIGNIFICANCE STATEMENT Lack of access to pure, catalytically competent human uridine diphosphate glucuronosyl-transferases (UGTs) has long been an impediment to biochemical and biophysical studies of this disease-relevant enzyme family. Here, we demonstrate this barrier can be removed using nanodisc technology—a human UGT2B7•nanodisc is assembled, purified to homogeneity, and shown to have activity comparable to microsomal UGT2B7.