UbiD domain dynamics underpins aromatic decarboxylation

The widespread UbiD enzyme family utilises the prFMN cofactor to achieve reversible decarboxylation of acrylic and (hetero)aromatic compounds. The reaction with acrylic compounds based on reversible 1,3-dipolar cycloaddition between substrate and prFMN occurs within the confines of the active site. In contrast, during aromatic acid decarboxylation, substantial rearrangement of the substrate aromatic moiety associated with covalent catalysis presents a molecular dynamic challenge. Here we determine the crystal structures of the multi-subunit vanillic acid decarboxylase VdcCD. We demonstrate that the small VdcD subunit acts as an allosteric activator of the UbiD-like VdcC. Comparison of distinct VdcCD structures reveals domain motion of the prFMN-binding domain directly affects active site architecture. Docking of substrate and prFMN-adduct species reveals active site reorganisation coupled to domain motion supports rearrangement of the substrate aromatic moiety. Together with kinetic solvent viscosity effects, this establishes prFMN covalent catalysis of aromatic (de)carboxylation is afforded by UbiD dynamics.

Detoxification of methylglyoxal by the glyoxalase system is required for glutathione availability and virulence activation in Listeria monocytogenes

Listeria monocytogenes is a Gram-positive, food-borne pathogen that lives a biphasic lifestyle, cycling between the environment and as a facultative intracellular pathogen of mammals. Upon entry into host cells, L. monocytogenes upregulates expression of glutathione synthase (GshF) and its product, glutathione (GSH), which is an allosteric activator of the master virulence regulator PrfA. Although gshF mutants are highly attenuated for virulence in mice and form very small plaques in host cell monolayers, these virulence defects can be fully rescued by mutations that lock PrfA in its active conformation, referred to as PrfA*. While PrfA activation can be recapitulated in vitro by the addition of reducing agents, the precise biological cue(s) experienced by L. monocytogenes that lead to PrfA activation are not known. Here we performed a genetic screen to identify additional small-plaque mutants that were rescued by PrfA* and identified gloA, which encodes glyoxalase A, a component of a GSH-dependent methylglyoxal (MG) detoxification system. MG is a toxic byproduct of metabolism produced by both the host and pathogen, which if accumulated, causes DNA damage and protein glycation. As a facultative intracellular pathogen, L. monocytogenes must protect itself from MG produced by its own metabolic processes and that of its host. We report that gloA mutants grow normally in broth, are sensitive to exogenous MG and severely attenuated upon IV infection in mice, but are fully rescued for virulence in a PrfA* background. We demonstrate that transcriptional activation of gshF increased upon MG challenge in vitro, and while this resulted in higher levels of GSH for wild-type L. monocytogenes, the glyoxalase mutants had decreased levels of GSH, presumably due to the accumulation of the GSH-MG hemithioacetal adduct. These data suggest that MG acts as a host cue that leads to GSH production and activation of PrfA.

Structural basis for ligand binding modes of CTP synthase

Cytidine triphosphate synthase (CTPS), which comprises an ammonia ligase domain and a glutamine amidotransferase domain, catalyzes the final step of de novo CTP biosynthesis. The activity of CTPS is regulated by the binding of four nucleotides and glutamine. While glutamine serves as an ammonia donor for the ATP-dependent conversion of UTP to CTP, the fourth nucleotide GTP acts as an allosteric activator. Models have been proposed to explain the mechanisms of action at the active site of the ammonia ligase domain and the conformational changes derived by GTP binding. However, actual GTP/ATP/UTP binding modes and relevant conformational changes have not been revealed fully. Here, we report the discovery of binding modes of four nucleotides and a glutamine analog 6-diazo-5-oxo-L-norleucine in Drosophila CTPS by cryo–electron microscopy with near-atomic resolution. Interactions between GTP and surrounding residues indicate that GTP acts to coordinate reactions at both domains by directly blocking ammonia leakage and stabilizing the ammonia tunnel. Additionally, we observe the ATP-dependent UTP phosphorylation intermediate and determine interacting residues at the ammonia ligase. A noncanonical CTP binding at the ATP binding site suggests another layer of feedback inhibition. Our findings not only delineate the structure of CTPS in the presence of all substrates but also complete our understanding of the underlying mechanisms of the allosteric regulation and CTP synthesis.

Non-coding sequence variants define a novel regulatory element in the first intron of the N-acetylglutamate synthase gene.

N-acetylglutamate synthase deficiency (NAGSD, MIM #237310) is an autosomal recessive urea cycle disorder caused either by decreased expression of the NAGS gene or defective NAGS enzyme resulting in decreased production of N-acetylglutamate (NAG), an allosteric activator of carbamylphosphate synthetase 1 (CPS1). NAGSD is the only urea cycle disorder that can be effectively treated with a single drug, N-carbamylglutamate (NCG), a stable NAG analog, which activates CPS1 to restore ureagenesis. We describe three patients with NAGSD due to four novel sequence variants in the NAGS regulatory regions. All three patients had hyperammonemia that resolved upon treatment with NCG. Sequence variants NM_153006.2:c.-3065A>C and NM_153006.2:c-3098C>T reside in the NAGS enhancer, within known HNF1 and predicted glucocorticoid receptor binding sites, respectively. Sequence variants NM_153006.2:c.426+326G>A and NM_153006.2:c.427-218A>C reside in the first intron of NAGS and define a novel NAGS regulatory element that binds retinoic X receptor α. Reporter gene assays in HepG2 and HuH-7 cells demonstrated that all four substitutions could result in reduced expression of NAGS. These findings show that analyzing non-coding regions of NAGS and other urea cycle genes can reveal molecular causes of disease and identify novel regulators of ureagenesis.

Elucidating the role of GRIM-19 as a substrate and allosteric activator of pro-apoptotic serine protease HtrA2

HtrA2 (High-temperature requirement A2) and GRIM-19 (Gene associated with retinoic and interferon-induced mortality 19 protein) are involved in various biological functions with their deregulation leading to multiple diseases. Although it is known that the interaction between GRIM-19 with HtrA2 promotes pro-apoptotic activity of the latter, the mechanistic details remained elusive till date. Moreover, designing allosteric modulators of HtrA2 remains obscure due to lack of adequate information on the mode of interaction with its natural substrates cum binding partners. Therefore, in this study, we have unfolded the interaction between HtrA2 and GRIM-19 so as to understand its subsequent functional repercussions. Using in silico analyses and biochemical assays, we identified the region in GRIM-19 that is involved in protein-protein interaction with HtrA2. Furthermore, we have presented a comprehensive illustration of HtrA2’s cleavage site specificity. Quantitative analysis using enzyme kinetics underscored the role of GRIM-19 in significant allosteric activation of HtrA2. Overall, this is an extensive study that not only defines HtrA2-GRIM-19 interaction, but also creates a framework for developing strategies toward allosteric regulation of HtrA2 for future therapeutic interventions.

Tyrosine 121 moves revealing a druggable pocket that couples catalysis to ATP-binding in serine racemase

ABSTRACTHuman serine racemase (hSR) catalyses racemisation of L-serine to D-serine, the latter of which is a co-agonist of the NMDA subtype of glutamate receptors that are important in synaptic plasticity, learning and memory. In a ‘closed’ hSR structure containing the allosteric activator ATP, the inhibitor malonate is enclosed between the large and small domains while ATP is distal to the active site, residing at the dimer interface with the Tyr121 hydroxyl group contacting the ATP a-phosphate. In contrast, in ‘open’ hSR structures, Tyr121 sits in the core of the small domain with its hydroxyl contacting the key catalytic residue Ser84. The ability to regulate SR activity by flipping Tyr121 from the core of the small domain to the dimer interface appears to have evolved in animals with a CNS. Multiple X-ray crystallographic enzymefragment structures show that Tyr121 is flipped out of its pocket, suggesting that this pocket is druggable.

Gene delivery corrects N-acetylglutamate synthase deficiency and enables insights in the physiological impact of L-arginine activation of N-acetylglutamate synthase

The urea cycle protects the central nervous system from ammonia toxicity by converting ammonia to urea. N-acetylglutamate synthase (NAGS) catalyzes formation of N-acetylglutamate, an essential allosteric activator of carbamylphosphate synthetase 1. Enzymatic activity of mammalian NAGS doubles in the presence of L-arginine, but the physiological significance of NAGS activation by L-arginine has been unknown. The NAGS knockout (Nags−/−) mouse is an animal model of inducible hyperammonemia, which develops hyperammonemia without N-carbamylglutamate and L-citrulline supplementation (NCG + Cit). We used adeno associated virus (AAV) based gene transfer to correct NAGS deficiency in the Nags−/− mice, established the dose of the vector needed to rescue Nags−/− mice from hyperammonemia and measured expression levels of Nags mRNA and NAGS protein in the livers of rescued animals. This methodology was used to investigate the effect of L-arginine on ureagenesis in vivo by treating Nags−/− mice with AAV vectors encoding either wild-type or E354A mutant mouse NAGS (mNAGS), which is not activated by L-arginine. The Nags−/− mice expressing E354A mNAGS were viable but had elevated plasma ammonia concentration despite similar levels of the E354A and wild-type mNAGS proteins. The corresponding mutation in human NAGS (NP_694551.1:p.E360D) that abolishes binding and activation by L-arginine was identified in a patient with NAGS deficiency. Our results show that NAGS deficiency can be rescued by gene therapy, and suggest that L-arginine binding to the NAGS enzyme is essential for normal ureagenesis.

Oral Administration of FT-4202, an Allosteric Activator of Pyruvate Kinase-R, Has Potent Anti-Sickling Effects in a Sickle Cell Anemia (SCA) Mouse Model, Resulting in Improved RBC Survival and Hemoglobin Levels

Introduction: Sickle cell anemia (SCA) results from a mutant β-globin gene that produces abnormal hemoglobin S (HbS). HbS polymerizes upon deoxygenation, resulting in red blood cell (RBC) sickling and membrane damage, leading to vaso-occlusions and hemolysis. Additionally, sickle RBCs contain less ATP and more 2,3-diphosphoglycerate (2,3-DPG) than normal RBCs; 2,3,DPG allosterically reduces hemoglobin (Hb) oxygen (O2)-affinity [i.e. increases P50], promoting faster unloading of O2, which potentiates HbS polymerization and RBC sickling. FT-4202, a selective and orally bioavailable allosteric activator of RBC pyruvate kinase (PKR), decreases 2,3-DPG and increases ATP in normal human RBCs (Blood, 2019, 134, Supplement 1:616). We hypothesized that oral administration of FT-4202 to SCA mice will increase HbS O2-affinity, and thereby decrease RBC sickling and membrane damage. Methods: Berkeley SCA mice were given 500-1000 mg/kg/day FT-4202 in chow (FT-4202 group) or control chow (control group) in 4 cohorts for 2 weeks (total 17-18 mice/group). In all cohorts, the health status, weight, and average chow consumption of each mouse was determined 3 times/week. Three cohorts were injected with sulfo-NHS-biotin 1 week into treatment (10-11 mice/group), and RBC survival assessed over the next week with serial micro-bleeds while on treatment. The 4th cohort was only bled at 2 week time-point to obtain P50 (Hemox Analyzer) and Hb levels (Hemavet). At experiment termination, all cohorts were terminally bled to determine (a) RBC levels of 2,3-DPG and ATP, (c) plasma levels of FT-4202 by LC-MS/MS, (d) the proportion of irreversibly sickled RBC (ISC) on blood smears (Image-J analysis), (e) the kinetics of experimentally-induced sickling (Lorrca®Oxygenscan) and (f) membrane deformability (Lorrca®Ektacytometry). Results: SCA mice on FT-4202 consumed a similar amount of food, and had similar weights and survival, compared to SCA mice on control chow throughout the 2-week period. As hypothesized, HbS O2 affinity increased, reflected by a decrease in P50 from 29.6 ± 0.62 mmHg (mean ± SEM) in the control group to 27.6 ± 0.58 mmHg in the FT-4202 group (p<0.03). Determinations of 2,3-DPG, ATP and FT-4202 are ongoing and will be presented. As expected, this increased HbS O2-affinity in the FT-4202 group reduced RBC sickling and membrane damage. At 2 weeks, the proportion of ISCs on blood smears was reduced in the FT-4202 group to 2.4 ± 0.3% vs. 5.9 ± 1.4% in the control group (p<0.02). The sickle RBC half-life increased to 1.8 ± 0.07 days in FT-4202 group vs. 1.4 ± 0.1 days in the control group, a 28% increase in RBC survival (p<0.01, Figure 1A). Hence, Hb levels in the FT-4202 group increased from 9.1 ± 0.2 g/dL before treatment, to 10.8 ± 0.3 g/dL 2 weeks after treatment (p<0.001), while Hb levels in the control group remained unchanged (Figure 1B). The reticulocytes remained unchanged in both groups before and after treatment. When sickle RBCs were de-oxygenated from an ambient pO2 of ~150 mmHg to a pO2 of 10-15 mmHg, followed by their re-oxygenation to ambient pO2 at a constant shear stress of 30 Pa (Oxygenscan), the point of sickling (PoS; pO2 level when the EI becomes 95% of the EI at ambient O2) decreased on average from 37% pO2 in the control group, to 30% pO2 in the FT-4202 group (p<0.002, Figure 1C), with a significantly improved Elongation Index at the point of minimum pO2 (EImin), (p<0.05). Next, RBC membrane deformability was measured under ambient pO2 (normoxic conditions), but varying shear stress after the de-oxygenation/re-oxygenation cycle on the Oxygenscan. Sickle RBCs from the FT-4202 group were significantly more deformable [i.e. had a higher Elongation Index (EI)] compared to control sickle RBCs (p<0.01, Figure 1D), as shear stress increased to ≥3 Pa, demonstrating that FT-4202 sickle RBCs sustained significantly less membrane damage following sickling and un-sickling. Conclusion: A 2-week oral FT-4202 administration was well tolerated by SCA mice and demonstrated beneficial biological effects: improved RBC membrane deformability and sickling parameters, with a shift in the PoS to lower pO2, and increased RBC survival and Hb levels. A parallel human phase-I study in healthy subjects and sickle cell disease patients to assess the safety and PK/PD of FT-4202 is ongoing (NCT03815695). Overall, our results suggest that FT-4202 can be a potentially useful orally available agent with significant anti-sickling effect. Disclosures Drake: Forma Therapeutics: Other: Shareholder of Forma Therapeutics. Fulzele:FORMA Therapeutics, Inc: Current Employment, Other: Shareholder of Forma Therapeutics. Guichard:FORMA Therapeutics, Inc: Current Employment, Other: Shareholder of Forma Therapeutics; AstraZeneca: Other: Shareholder. Malik:Aruvant Sciences, Forma Therapeutics, Inc.: Consultancy; Aruvant Sciences, CSL Behring: Patents & Royalties.