(S)-Mandelate dehydrogenase from Pseudomonas putida, a member of the flavin mononucleotide-dependent alpha-hydroxy acid oxidase/dehydrogenase family, oxidizes (S)-mandelate to benzoylformate. The enzyme was purified with a carboxy-terminal histidine tag. Steady-state kinetic parameters indicate that it preferentially binds large substrates. A good correlation was obtained between the kcat, the substrate kinetic isotope effect (KIE), and the pKa of the substrate alpha-proton. The kcat decreased and the KIE increased for substrates whose alpha-protons have pKas higher than that of mandelate. These results support a mechanism involving a carbanion intermediate but are difficult to reconcile with one involving a direct hydride transfer. pH effects on steady-state parameters were determined with (S)-mandelate and a slow substrate, (R,S)-3-phenyllactate. The kcat/Km pH profile shows that two groups with apparent pKas of 5.5 and 8.9 in the free enzyme are important for activity. These pKas are shifted to 5.1 and 9.6 on binding (S)-mandelate, as shown in the kcat pH profile. The pH dependence of the KIEs suggests that the residues with these pKas are involved in the alpha-carbon-hydrogen bond-breaking step. pH dependencies of the inhibition constants for competitive inhibitors identified these residues as histidine 274 and arginine 277. We propose that histidine 274 is the base that abstracts the substrate alpha-proton and arginine 277 is important for substrate binding as well as stabilization of the carbanion/enolate intermediate.

Highlights d The Shigella ubiquitin ligase effector IpaH7.8 blocks pyroptosis in human cells d IpaH7.8 targets human, but not mouse, gasdermin D for proteasomal degradation d IpaH7.8 targets the pore-forming domain of gasdermin D to suppress pyroptosis d Mice lacking both GSDMD and NLRC4 are hyper susceptible to S.

Most current therapies that target plasma membrane receptors function by antagonizing ligand binding or enzymatic activities. However, typical mammalian proteins comprise multiple domains that execute discrete but coordinated activities. Thus, inhibition of one domain often incompletely suppresses the function of a protein. Indeed, targeted protein degradation technologies, including proteolysis-targeting chimeras1 (PROTACs), have highlighted clinically important advantages of target degradation over inhibition2. However, the generation of heterobifunctional compounds binding to two targets with high affinity is complex, particularly when oral bioavailability is required3. Here we describe the development of proteolysis-targeting antibodies (PROTABs) that tether cell-surface E3 ubiquitin ligases to transmembrane proteins, resulting in target degradation both in vitro and in vivo. Focusing on zinc- and ring finger 3 (ZNRF3), a Wnt-responsive ligase, we show that this approach can enable colorectal cancer-specific degradation. Notably, by examining a matrix of additional cell-surface E3 ubiquitin ligases and transmembrane receptors, we demonstrate that this technology is amendable for ‘on-demand’ degradation. Furthermore, we offer insights on the ground rules governing target degradation by engineering optimized antibody formats. In summary, this work describes a strategy for the rapid development of potent, bioavailable and tissue-selective degraders of cell-surface proteins.

(S)-Mandelate dehydrogenase from Pseudomonas putida is an FMN-dependent alpha-hydroxy acid dehydrogenase. Structural studies of two homologous enzymes, glycolate oxidase and flavocytochrome b(2), indicated that a conserved arginine residue (R277 in MDH) interacts with the product carboxylate group [Lindqvist, Y., Branden, C.-I., Mathews, F. S., and Lederer, F. (1991) J. Biol. Chem. 266, 3198-3207]. The catalytic role of R277 was investigated by site-specific mutagenesis together with chemical rescue experiments. The R277K, R277G, R277H, and R277L proteins were generated and purified in active forms. The k(cat) for the charge-conserved mutation, R277K, was only 4-fold lower than wt-MDH, but its K(m) value was 40-fold lower; in contrast, k(cat)s for R277G, R277H, and R277L were 400-1000-fold lower than for wt-MDH and K(m) values were 5-15-fold lower compared to R277K. The K(d)s for negatively charged competitive inhibitors were relatively unaffected in all four R277 mutants. The k(cat) for R277G could be enhanced by the addition of exogenous guanidines or imidazoles; the maximum rescued k(cat) was approximately 70% of the wt-MDH value. Only reagents that were positively charged and could function as hydrogen bond donors were effective rescue agents. Our results indicate that R277 plays a major role in transition state stabilization through its positive charge-consistent with a mechanism involving a carbanion intermediate. The positive charge has a relatively small contribution toward substrate binding. R277 also forms a specific hydrogen bond with both the substrate and the transition state; this interaction contributes significantly to the low K(m) for (S)-mandelate.

Highlights d The immunoglobulin superfamily (IgSF) interactome identifies over 500 receptor pairs d The IgSF interactome reveals receptor-ligand communities dysregulated in cancer d More than 60 receptor-ligand pairs confirmed using orthogonal methods d Interacting protein signatures are correlated with clinical outcome