Active Site Arginine Residue Research Articles

Role of active site arginine residues in substrate recognition by PPM1A

Reversible protein phosphorylation is a key mechanism for regulating numerous cellular events. The metal-dependent protein phosphatases (PPM) are a family of Ser/Thr phosphatases, which uniquely recognize their substrate as a monomeric enzyme. In the case of PPM1A, it has the capacity to dephosphorylate a variety of substrates containing different sequences, but it is not yet fully understood how it recognizes its substrates. Here we analyzed the role of Arg33 and Arg186, two residues near the active site, on the dephosphorylation activity of PPM1A. The results showed that both Arg residues were critical for enzymatic activity and docking-model analysis revealed that Arg186 is positioned to interact with the substrate phosphate group. In addition, our results suggest that which Arg residue plays a more significant role in the catalysis depends directly on the substrate.

Naturally Occurring Mutations in Creatine Kinase Brain‐Type Exhibit Decreased Tenofovir Activation In Vitro

Pre-exposure prophylaxis regimens contain tenofovir prodrugs that must undergo intracellular cleavage and two phosphorylation steps to become the pharmacologically active metabolite, tenofovir-diphosphate (TFV-DP). TFV-DP is a nucleotide analog that mimics ATP in structure and competitively inhibits HIV reverse transcriptase. It has been shown that creatine kinase muscle-type (CKM) is responsible for the final phosphoryl transfer step in the formation of TFV-DP in colon tissue. A closely related enzyme, creatine kinase brain-type (CKB), is 80% homologous to CKM and is highly expressed in brain, prostate, and the gastrointestinal tract. Thus, we speculate that CKB may be responsible for the activation of tenofovir in these tissues. To investigate this, in vitro activity assays were performed by incubating recombinantly expressed and purified CKB with tenofovir-monophosphate and phosphocreatine. Formation of TFV-DP was detected using ultra-high performance liquid chromatography tandem mass spectrometry. Results showed no significant difference in TFV-DP formation when comparing CKB to CKM. Interestingly, it has been observed that intracellular TFV-DP concentrations are variable among individuals and genetic variation in tenofovir activating enzymes could contribute to this. To examine the impact of genetic variation on CKB activity, fifteen naturally occurring missense mutations wereselected based on their location, predicted impact (SIFT and PolyPhen scores), and residue conservation between creatine kinase enzymes. In vitro activity assays using recombinantly expressed mutant CKB enzymes demonstrated that 10 of the chosen mutations exhibit a statistically significant reduction in the formation of TFV-DP when compared to wild-type CKB. Mutations at active site arginine residues (R132C, R236Q, and R292Q) showed only 1-3% activity. Other notable mutations that exhibited reduced TFV-DP formation include S128R (30.4%) and H296R (5.6%) in the tenofovir-monophosphate binding pocket, R172P (21.3%) at the dimer interface, and C74S (20.1%) and R96P (6.8%) in the phosphocreatine binding pocket. Of the five mutations where no reduction in TFV-DP formation was observed, two are located in the dimer interface and three are located on the exterior of the enzyme. Overall, these data suggest that CKB may be responsible for tenofovir activation in certain tissues and naturally occurring mutations in this enzyme could result in variable intracellular TFV-DP concentrations.

Effects of active site arginine residue on redox behavior of flavins in bifurcating electron transfer flavoprotein

Flavin based electron bifurcation (FBEB) is observed in bifurcating electron transfer flavoproteins (BfEtfs). Electron bifurcation couples endergonic and exergonic electron transfer reactions to exploit lower potential electron transfer reaction step therefore this is considered as a third mode of energy conservation mechanism in biological systems. We study about the bifurcating EtfAB system from Rhodoseudomonas palustris (Rpal). This is a heterodimer containing two flavin adenine dinucleotides (FADs), one in each domain. One flavin accepts two electrons from NADH and bifurcates (Bf-FAD) each electron to higher potential electron transfer FAD (ET-FAD) and lower potential ferredoxin or flavodoxin. ET-FAD likely rests in its anionic semiquinone (ASQ) state and upon accepting an electron from the Bf-FAD it becomes a hydroquinone (HQ). BF-FAD accepts a pair of electrons from NADH to form HQ and distributes the electrons to separate acceptors without accumulating a SQ intermediate. This contrasting redox behavior of the two flavins in this system in turn points a spotlight on whether the conserved active site arginine residues have different effects in the two binding sites. R273 favors the ASQ of ET-FAD, whereas R165 near the Bf-FAD appears not to, possibly due to neutralization of its positive charge by nearby C174. R273 forms a pi- pi stacking interaction with ET-FAD whereas R165 appears to form hydrogen bond interactions with Bf-FAD. We performed site directed mutagenesis to change the size of the positively charged residue by replacing with lysine, neutralize the charge by inserting alanine and make the charge tunable via modulation of pH by introducing a histidine. Amino acid substitutions were made in each of the two flavin-binding sites separately. Based on the Spectro-electrochemical redox titrations, all the variants retained the WT pattern of single electron reactivity at the ET site and pairwise reactivity of the Bf-FAD. Reduction midpoint potential titrations showed 50% less accumulation of ASQ in R273H and R273A, whereas R273K produced similar an amount similar to that observed in WT. This confirms that positive charge helps in stabilization of ASQ. Redox titrations performed at pH 7 and 8 suggest that the substituting H273 residue remains neutral at these pHs. Amino acid replacement in the bifurcating site, R165K did not affect the formation of ASQ in ET-FAD, but removal of residue 165's positive charge in R165H resulted in diminished stability prevented FAD binding in the Bf site at pH8. Therefore, R273 plays a vital role in Bf-ETF by stabilizing the ASQ of the ET-FAD. On the other hand, R165 stabilizes the Bf-FAD that is essential for electron bifurcation in RpalEtf.

Glutarate Hydroxylation by the Carbon Starvation-Induced Protein D: A Computational Study into the Stereo- and Regioselectivities of the Reaction.

The carbon starvation-induced protein D (CsiD) is a recently characterized iron(II)/α-ketoglutarate-dependent oxygenase that activates a glutarate molecule as substrate at the C2 position to exclusively produce (S)-2-hydroxyglutarate products. This selective hydroxylation reaction by CsiD is an important component of the lysine biodegradation pathway in Escherichia coli; however, little is known on the details and the origin of the selectivity of the reaction. So far, experimental studies failed to trap and characterize any short-lived catalytic cycle intermediates. As no computational studies have been reported on this enzyme either, we decided to investigate the chemical reaction mechanism of glutarate activation by an iron(IV)-oxo model of the CsiD enzyme. In this work, we present a density functional theory study on a large active site cluster model of CsiD and investigate the glutarate hydroxylation pathways by a high-valent iron(IV)-oxo species leading to (S)-2-hydroxyglutarate, (R)-2-hydroxyglutarate, and 3-hydroxyglutarate. In agreement with experimental observation, the favorable product channel leads to pro-S C2-H hydrogen atom abstraction to form (S)-2-hydroxyglutarate. The reaction is stepwise with a hydrogen atom abstraction by an iron(IV)-oxo species followed by OH rebound from a radical intermediate. The work presented in this paper shows that despite the fact that the C-H bond strengths at the C2 and C3 positions of glutarate are similar in the gas phase, substrate binding and positioning guide the reaction to an enantioselective reaction process by destabilizing the hydrogen atom abstraction pathways for the pro-R C2-H and C3-H positions. Our studies predict the chemical properties of the iron(IV)-oxo species and its rate constants with glutarate and deuterated-glutarate. Moreover, the work shows little protein motions during the catalytic process, while the substrate entrance into the substrate binding pocket appears to be guided by three active site arginine residues that position the substrate for pro-S C2-H hydrogen atom abstraction. Finally, the calculations show that irrespective of the position of the substrate and what C-H bond is closest to the metal center, the lowest energy pathway is for a selective pro-S C2-H hydrogen atom abstraction.

Characteristics and Prognostic Effects of IDH Mutations across the Age Spectrum in AML: A Collaborative Analysis from COG, SWOG, and ECOG

Characteristics and Prognostic Effects of IDH Mutations across the Age Spectrum in AML: A Collaborative Analysis from COG, SWOG, and ECOG

Naturally‐Occurring Variants of Muscle‐Type Creatine Kinase Exhibit Altered Tenofovir Monophosphate Phosphorylation Activity

Tenofovir (TFV), prescribed as a disoproxil fumarate prodrug, is a component of the only FDA‐approved regimen for the prevention of HIV. TFV is an acyclic nucleotide analog reverse transcriptase inhibitor prodrug that mimics a nucleotide monophosphate in structure and must be phosphorylated twice by intracellular kinases to produce the pharmacologically active TFV‐diphosphate (DP) form. There have been clinical observations of variation in intracellular TFV‐DP levels, along with efficacy of TFV‐based HIV pre‐exposure prophylaxis (PrEP). We hypothesize that interindividual differences in the kinases that phosphorylate TFV may be a cause of these outcomes. We have elucidated the kinases responsible for TFV phosphorylation in tissues important to forming a barrier of protection against HIV infection, including muscle‐type creatine kinase (CKM), which is the primary kinase responsible for the phosphorylation of TFV‐monophosphate (MP) to the active TFV‐DP in colon tissue. In pursuit of understanding interindividual differences in these kinases, we sequenced the CKM gene in over 1000 patient samples and discovered several previously unreported single nucleotide variants, some of which lead to mutations in the CKM amino acid sequence. Using bioinformatic tools, we predicted that several of these mutants may markedly decrease enzyme function toward the endogenous substrate of the enzyme, ADP. We performed molecular docking simulations, using AutoDock, to model the binding of TFV‐MP to CKM and found that TFV‐MP binds to CKM much as does ADP, which TFV‐MP mimics in structure, suggesting that the bioinformatic predictions may also apply to changes in TFV‐MP phosphorylation activity of CKM. Docking results reveal a pocket of active site arginine residues at positions 130, 132, 236, 292, and 320 as likely important to charge stabilization of the phosphates of TFV‐MP. Notably, our sequencing revealed genetic variants producing R130H and R132C mutations that are predicted to decrease enzyme activity. In order to determine the effects of the naturally‐occurring point mutations on CKM activity toward TFV‐MP, wild‐type human CKM and 15 mutants from the genetic variation analysis were recombinantly expressed and purified. Then, in vitro assays were performed by incubating the proteins with TFV‐MP and measuring TFV‐DP formation via ultra‐high performance liquid chromatography tandem mass spectrometry. These results reveal 11 mutations in CKM that significantly decrease TFV‐MP phosphorylation. Three active site mutations, R130H, R132C, and N286I, along with W211R resulted in a decrease in TFV‐DP formation to only 3% of that of wild‐type CKM. Additionally, T35I, R43Q, I92M, H97Y, F169L, and Y173C mutations decreased activity to 14–25% of that of wild‐type. This evidence suggests that CKM genetics may play a role in interindividual variability in TFV activation and potentially the efficacy of TFV in PrEP settings.Support or Funding InformationThis research is funded by NIH R01 AI128781

Naturally Occurring Mutations in Muscle‐Type Creatine Kinase Impact Tenofovir Monophosphate Phosphorylation

Tenofovir (TFV), administered as a tenofovir disoproxil fumarate prodrug, is a nucleotide analog reverse transcriptase inhibitor used in combination for the treatment and prevention of HIV. TFV requires two sequential phosphorylation events in order to become pharmacologically active. We previously found that in colon tissue, a putative site of HIV infection, the muscle‐type isoenzyme of creatine kinase (CKM) is responsible for the second TFV activating phosphorylation step, converting tenofovir monophosphate (TFV‐MP) to the active tenofovir diphosphate (TFV‐DP). Due to the similarity in structure between TFV‐MP and ADP, this is likely analogous to the endogenous activity of CKM, which regenerates ATP from ADP by phosphoryl transfer from phosphocreatine. In addition, we have previously discovered naturally occurring genetic variants of the CKM gene in humans. Some of these single nucleotide variants are predicted to cause single residue mutations in the translated protein, a fraction of which are predicted by bioinformatics programs to have deleterious effects on enzymatic function. In the present study, molecular docking was employed to investigate the potential effects of these mutations on TFV monophosphate (TFV‐MP) binding and phosphorylation by CKM. These results indicate CKM binds TFV‐MP similar to how it binds its natural substrate, ADP. Molecular docking simulation also predict a cluster of active site arginine residues at positions 130, 132, 236, 292, and 320 are likely important in the charge stabilization of the phosphates of TFV‐MP, which may impact substrate binding and phosphorylation. To directly investigate the impact of the aforementioned mutations on TFV‐MP phosphorylation, CKM wild type and mutant proteins were recombinantly expressed and purified. In vitro assays were then performed to compare the ability of the mutant proteins to convert TFV‐MP to TFV‐DP. Enzyme activity was measured by liquid chromatography‐tandem mass spectrometry using a method that separated and detected the phosphorylated tenofovir metabolites concomitantly. It was found that R130H and R132C mutations decreased enzymatic activity to less than 10% of that of the wild type protein. These results indicate that genetic variation in the CKM gene may impact tenofovir activation, potentially implicating a risk to patients possessing these genetic variants.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Molecular remission and response patterns in patients with mutant-IDH2 acute myeloid leukemia treated with enasidenib

AbstractApproximately 8% to 19% of patients with acute myeloid leukemia (AML) have isocitrate dehydrogenase-2 (IDH2) mutations, which occur at active site arginine residues R140 and R172. IDH2 mutations produce an oncometabolite, 2-hydroxyglutarate (2-HG), which leads to DNA and histone hypermethylation and impaired hematopoietic differentiation. Enasidenib is an oral inhibitor of mutant-IDH2 proteins. This first-in-human phase 1/2 study evaluated enasidenib doses of 50 to 650 mg/d, administered in continuous 28-day cycles, in patients with mutant-IDH2 hematologic malignancies. Overall, 214 of 345 patients (62%) with relapsed or refractory (R/R) AML received enasidenib, 100 mg/d. Median age was 68 years. Forty-two patients (19.6%) attained complete remission (CR), 19 patients (10.3%) proceeded to an allogeneic bone marrow transplant, and the overall response rate was 38.8% (95% confidence interval [CI], 32.2-45.7). Median overall survival was 8.8 months (95% CI, 7.7-9.6). Response and survival were comparable among patients with IDH2-R140 or IDH2-R172 mutations. Response rates were similar among patients who, at study entry, were in relapse (37.7%) or were refractory to intensive (37.5%) or nonintensive (43.2%) therapies. Sixty-six (43.1%) red blood cell transfusion–dependent and 53 (40.2%) platelet transfusion–dependent patients achieved transfusion independence. The magnitude of 2-HG reduction on study was associated with CR in IDH2-R172 patients. Clearance of mutant-IDH2 clones was also associated with achievement of CR. Among all 345 patients, the most common grade 3 or 4 treatment-related adverse events were hyperbilirubinemia (10%), thrombocytopenia (7%), and IDH differentiation syndrome (6%). Enasidenib was well tolerated and induced molecular remissions and hematologic responses in patients with AML for whom prior treatments had failed. The study is registered at www.clinicaltrials.gov as #NCT01915498.

Two active site arginines are critical determinants of substrate binding and catalysis in MenD: a thiamine-dependent enzyme in menaquinone biosynthesis.

The bacterial enzyme MenD, or 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC) synthase, catalyzes an essential Stetter reaction in menaquinone (vitamin K2) biosynthesis via thiamine diphosphate (ThDP)-bound tetrahedral post-decarboxylation intermediates. The detailed mechanism of this intermediate chemistry, however, is still poorly understood, but of significant interest given that menaquinone is an essential electron transporter in many pathogenic bacteria. Here, we used site-directed mutagenesis, enzyme kinetic assays, and protein crystallography to reveal an active-inactive intermediate equilibrium in MenD catalysis and its modulation by two conserved active site arginine residues. We observed that these conserved residues play a key role in shifting the equilibrium to the active intermediate by orienting the C2-succinyl group of the intermediates through strong ionic hydrogen bonding. We found that when this interaction is moderately weakened by amino acid substitutions, the resulting proteins are catalytically competent with the C2-succinyl group taking either the active or the inactive orientation in the post-decarboxylation intermediate. When this hydrogen-bonding interaction was strongly weakened, the succinyl group was re-oriented by 180° relative to the native intermediate, resulting in the reversal of the stereochemistry at the reaction center that disabled catalysis. Interestingly, this inactive intermediate was formed with a distinct kinetic behavior, likely as a result of a non-native mode of enzyme-substrate interaction. The mechanistic insights gained from these findings improve our understanding of the new ThDP-dependent catalysis. More importantly, the non-native-binding site of the inactive MenD intermediate uncovered here provides a new target for the development of antibiotics.

Mapping Functional Substrate-Enzyme Interactions in the pol β Active Site through Chemical Biology: Structural Responses to Acidity Modification of Incoming dNTPs.

We report high-resolution crystal structures of DNA polymerase (pol) β in ternary complex with a panel of incoming dNTPs carrying acidity-modified 5'-triphosphate groups. These novel dNTP analogues have a variety of halomethylene substitutions replacing the bridging oxygen between Pβ and Pγ of the incoming dNTP, whereas other analogues have alkaline substitutions at the bridging oxygen. Use of these analogues allows the first systematic comparison of effects of 5'-triphosphate acidity modification on active site structures and the rate constant of DNA synthesis. These ternary complex structures with incoming dATP, dTTP, and dCTP analogues reveal the enzyme's active site is not grossly altered by the acidity modifications of the triphosphate group, yet with analogues of all three incoming dNTP bases, subtle structural differences are apparent in interactions around the nascent base pair and at the guanidinium groups of active site arginine residues. These results are important for understanding how acidity modification of the incoming dNTP's 5'-triphosphate can influence DNA polymerase activity and the significance of interactions at arginines 183 and 149 in the active site.

Crystal structures of an archaeal thymidylate kinase from Sulfolobus tokodaii provide insights into the role of a conserved active site Arginine residue.

Thymidylate kinase (TMK) is a key enzyme that plays an important role in DNA synthesis. Therefore, it serves as an attractive therapeutic target for the development of antibacterial, antiparasitic and anticancer drugs. Herein, we report the biochemical characterization and crystal structure determination of thymidylate kinase from a hyperthermophilic organism Sulfolobus tokodaii (StTMK) in its apo and ADP-bound forms. Our study describes the first three-dimensional structure of an archaeal TMK. StTMK is a thermostable enzyme with optimum activity at 80°C. Despite the overall similarity to homologous TMKs, StTMK structures revealed several residue substitutions at the active site. However, enzyme assays demonstrated specificity to its natural substrates ATP and dTMP. Analysis of the structures also revealed multiple conformational states of Arg93 which is located at the reaction centre and is a part of the highly conserved DRX motif. Only one of these states was found to be suitable for the proper positioning of the α-phosphate group of dTMP at the active site. Computational alanine scanning and MM/PBSA binding energy calculation revealed the importance of Arg93 side chain in substrate binding. Subsequent site directed mutagenesis at this position to an Ala resulted in the loss of activity. Thus, the computational and biochemical studies reveal the importance of Arg93 for enzyme function, while the different conformational states of Arg93 observed in the structural studies imply its regulatory role in the catalytically competent placement of dTMP.

Neisseria meningitidis Translation Elongation Factor P and Its Active-Site Arginine Residue Are Essential for Cell Viability.

Translation elongation factor P (EF-P), a ubiquitous protein over the entire range of bacterial species, rescues ribosomal stalling at consecutive prolines in proteins. In Escherichia coli and Salmonella enterica, the post-translational β-lysyl modification of Lys34 of EF-P is important for the EF-P activity. The β-lysyl EF-P modification pathway is conserved among only 26–28% of bacteria. Recently, it was found that the Shewanella oneidensis and Pseudomonas aeruginosa EF-P proteins, containing an Arg residue at position 32, are modified with rhamnose, which is a novel post-translational modification. In these bacteria, EF-P and its Arg modification are both dispensable for cell viability, similar to the E. coli and S. enterica EF-P proteins and their Lys34 modification. However, in the present study, we found that EF-P and Arg32 are essential for the viability of the human pathogen, Neisseria meningitidis. We therefore analyzed the modification of Arg32 in the N. meningitidis EF-P protein, and identified the same rhamnosyl modification as in the S. oneidensis and P. aeruginosa EF-P proteins. N. meningitidis also has the orthologue of the rhamnosyl modification enzyme (EarP) from S. oneidensis and P. aeruginosa. Therefore, EarP should be a promising target for antibacterial drug development specifically against N. meningitidis. The pair of genes encoding N. meningitidis EF-P and EarP suppressed the slow-growth phenotype of the EF-P-deficient mutant of E. coli, indicating that the activity of N. meningitidis rhamnosyl–EF-P for rescuing the stalled ribosomes at proline stretches is similar to that of E. coli β-lysyl–EF-P. The possible reasons for the unique requirement of rhamnosyl–EF-P for N. meningitidis cells are that more proline stretch-containing proteins are essential and/or the basal ribosomal activity to synthesize proline stretch-containing proteins in the absence of EF-P is lower in this bacterium than in others.

Photoactivation of Mutant Isocitrate Dehydrogenase 2 Reveals Rapid Cancer-Associated Metabolic and Epigenetic Changes.

Isocitrate dehydrogenase is mutated at a key active site arginine residue (Arg172 in IDH2) in many cancers, leading to the synthesis of the oncometabolite (R)-2-hydroxyglutarate (2HG). To investigate the early events following acquisition of this mutation in mammalian cells we created a photoactivatable version of IDH2(R172K), in which K172 is replaced with a photocaged lysine (PCK), via genetic code expansion. Illumination of cells expressing this mutant protein led to a rapid increase in the levels of 2HG, with 2HG levels reaching those measured in patient tumor samples, within 8 h. 2HG accumulation is closely followed by a global decrease in 5-hydroxymethylcytosine (5-hmC) in DNA, demonstrating that perturbations in epigenetic DNA base modifications are an early consequence of mutant IDH2 in cells. Our results provide a paradigm for rapidly and synchronously uncloaking diverse oncogenic mutations in live cells to reveal the sequence of events through which they may ultimately cause transformation.

Functional role of R462 in the degradation of hyaluronan catalyzed by hyaluronate lyase from Streptococcus pneumoniae.

Hyaluronan lyase from Streptococcus pneumoniae can degrade hyaluronic acid, which is one of the major components in the extracellular matrix. Hyaluronan can regulate water balance, osmotic pressure, and act as an ion exchange resin. Followed by our recent work on the catalytic reaction mechanism and substrate binding mode, we in this work further investigate the functional role of active site arginine residue, R462, in the degradation of hyaluronan. The site directed mutagenesis simulation of R462A and R462Q were modeled using a combined quantum mechanical and molecular mechanical method. The overall substrate binding features upon mutations do not have significant changes. The energetic profiles for the reaction processes are essentially the same as that in wild type enzyme, but significant activation barrier height changes can be observed. Both mutants were shown to accelerate the overall enzymatic activity, e.g., R462A can reduce the barrier height by about 2.8 kcal mol(-1), while R462Q reduces the activation energy by about 2.9 kcal mol(-1). Consistent with the active site model calculated using density functional theory, our results can support that the positive charge on R462 guanidino side chain group plays a negative role in the catalysis. Finally, the functional role of R462 was proposed to facilitate the formation of initial enzyme-substrate complex, but not in the subsequent catalytic degradation reaction. Graphical Abstract Degradation of hyaluronan catalyzed by hyaluronate lyase from Streptococcus pneumoniae.

Analyzing the catalytic role of active site residues in the Fe-type nitrile hydratase from Comamonas testosteroni Ni1.

A strictly conserved active site arginine residue (αR157) and two histidine residues (αH80 and αH81) located near the active site of the Fe-type nitrile hydratase from Comamonas testosteroni Ni1 (CtNHase), were mutated. These mutant enzymes were examined for their ability to bind iron and hydrate acrylonitrile. For the αR157A mutant, the residual activity (k cat = 10 ± 2 s(-1)) accounts for less than 1% of the wild-type activity (k cat = 1100 ± 30 s(-1)) while the K m value is nearly unchanged at 205 ± 10 mM. On the other hand, mutation of the active site pocket αH80 and αH81 residues to alanine resulted in enzymes with k cat values of 220 ± 40 and 77 ± 13 s(-1), respectively, and K m values of 187 ± 11 and 179 ± 18 mM. The double mutant (αH80A/αH81A) was also prepared and provided an enzyme with a k cat value of 132 ± 3 s(-1) and a K m value of 213 ± 61 mM. These data indicate that all three residues are catalytically important, but not essential. X-ray crystal structures of the αH80A/αH81A, αH80W/αH81W, and αR157A mutant CtNHase enzymes were solved to 2.0, 2.8, and 2.5 Å resolutions, respectively. In each mutant enzyme, hydrogen-bonding interactions crucial for the catalytic function of the αCys(104)-SOH ligand are disrupted. Disruption of these hydrogen bonding interactions likely alters the nucleophilicity of the sulfenic acid oxygen and the Lewis acidity of the active site Fe(III) ion.

Effects of IDH1 and IDH2 genes mutations on tumors

Isocitrate dehydrogenases (IDHs) are considered as key enzymes in the tricarboxylic acid cycle. Recurrent mutations in the IDH1 and IDH2 genes are recently found in several human cancers. Those point mutations specifically affect IDH1 and IDH2 active site arginine residues and confer a neomorphic enzyme function of directly catalyzing α-ketoglutarate (α-KG) to R-2-hydroxyglutarate (R-2-HG). R-2-HG can competitively inhibits α-KG-dependent enzymes and may therefore contribute to the occurrence and development of tumor. In addition, Mutation status of IDH1 and IDH2 are closely relative to the progress and prognosis of certain tumor. Thus IDH1 and IDH2 are considered to be promising biomarkers for early diagnosis and prognosis and targeted therapy. Key words: Isocitrate dehydrogenase; Methylation; Metabolic diseases; Neoplasms

Abstract 521: Glioma-associated IDH mutation induces miR-34a repression and stem cell-like physiology through enhanced PDGF signaling

Abstract Point mutations in isocitrate dehydrogenase enzymes (IDH1 and IDH2) have been identified in 70-90% of lower-grade gliomas (LGGs; WHO grade II and III) as foundational genomic alterations. Glioma-associated IDH mutations occur in active site arginine residues and confer a neomorphic activity resulting in overproduction of the oncometabolite R(-)-2-hydroxyglutarate (2HG). The affects of 2HG on cellular physiology are widespread and include global shifts in epigenomic profiles. LGGs have also been repeatedly linked with dysregulated platelet-derived growth factor (PDGF) signaling. We have recently demonstrated that miR-34a is downregulated in gliomas in response to dysregulated PDGF signaling and that miR-34a directly targets the PDGF receptor (PDGFRA) in high-grade gliomas. Analyzing a panel of diffuse gliomas, we find that miR-34a is specifically downregulated in LGG in response to IDH mutation. Similarly, in multiple isogenic cell line systems, IDH mutation induces repression of miR-34a. Also in these contexts, IDH mutation induces neurosphere formation and the expression of stem cell-associated genes. miR-34a has been reported to target pluripotency factors such as LIN28, OCT4 and NANOG. Accordingly, we find that murine neuroglial progenitor cells expressing IDH mutant isoform express higher levels of these factors than isogenic controls. The stem cell-like phenotype of IDH mutant-expressing cells is morphologically reversed by restoring miR-34a expression in the cells. Despite findings that IDH mutation correlates with hypermethylation at the MIR34a locus in LGG tumors, we have not been able to demonstrate the functional relevance of this. Instead, we find that IDH mutation is associated with enhanced PDGF signaling, and we demonstrate that inhibition or stimulation of PDGF signaling in IDH mutant cells increases or decreases miR-34a expression, respectively. Our studies support a model in which glioma-associated IDH mutation induces miR-34a repression and stem cell-like physiology through enhanced PDGF signaling. Citation Format: Joachim Silber, Girish Harinath, Prasanna Tamarapu Parthasarathy, Armida W.M. Fabius, Sevin Turcan, Timothy A. Chan, Jason T. Huse. Glioma-associated IDH mutation induces miR-34a repression and stem cell-like physiology through enhanced PDGF signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 521. doi:10.1158/1538-7445.AM2014-521

Abstract 3741: Development and characterization of a cell based assay for the validation of mutant IDH1 inhibitors

Abstract Recurrent isocitrate dehydrogenase (IDH) mutations have recently been identified in several cancer types. These point mutations specifically affect IDH1 and IDH2 active site arginine residues and confer the ability to reduce α-ketoglutarate to R-2-hydroxyglutarate (2HG), an oncometabolite that competitively inhibits α-ketoglutarate-dependent enzymes, such as histone and DNA demethylases. Accumulating evidence indicates that mIDH enzymes are attractive targets for the development of novel cancer therapeutics. To validate potential mIDH inhibitors in a cellular assay, we acquired several cell lines that harbor mIDH1 R132H and R132C: HCT116 mIDH1 (colon cancer, mIDH1 R132H +/-, Horizon Discovery), U87 MG mIDH1 (glioma, m IDH1 R132H +/+, Japan), HT1080 (fibrosarcoma, mIDH1 R132C +/-) and JJ012 (chondrosarcoma, mIDH1 R132C +/-, Rush University Medical Center). Cell lines were sequenced to confirm the presence of mIDH1 and 2HG production was measured in cellular supernatants by GC-MS. We observed that cells harboring mIDH1 R132C produce higher amounts of 2HG compared to lines harboring mIDH1 R132H, in agreement with recent reports. In addition, we show that accumulation of 2HG is time dependent and that its half-life in cellular supernatant is over 48 hours. Moreover, we demonstrate that 2HG is a stable analyte in media (through five sample free-thaw cycles) and that our samples are within linear range, providing a good intra-assay and inter-assay variability (CV <10%). Using this assay we evaluated the impact of five reference compounds obtained from published structures (Agios Pharmaceuticals, Cambridge MA), on the accumulation of 2HG in the supernatant of HT1080. These compounds were reported to have different ranges of activity against mIDH1 R132H and R132C enzymes in in vitro assays. Four of the mIDH reference compounds significantly inhibited 2HG accumulation in HT1080 supernatants in a dose dependent fashion, with potencies that were comparable to enzyme inhibition values. In agreement with previous reports, none of compounds affected HT1080 proliferation over a 48 hours incubation. In conclusion, we have identified mIDH cellular models that produce differing concentrations of 2HG, detectable by a GC/MS assay with sensitivity sufficient to determine levels as low as 0.1μg/ml. We have tested several inhibitors of the mIDH enzyme and confirmed an association between in vitro enzyme inhibition and a decrease in 2HG accumulation; therefore this assay can be used for the cellular evaluation of novel mIDH1 inhibitors. Funded by NCI Contract No. HHSN261200800001E. This research was supported [in part] by the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis of the National Cancer Institute. Citation Format: Nicole D. Fer, Erik Harris, Stephen Fox, Ming Zhou, Catherine Simpson, Jing Liu, Ilia Korboukh, Emily A. Hull-Ryde, William P. Janzen, Stephen V. Frye, Anne Monks, Beverly Teicher, Annamaria Rapisarda. Development and characterization of a cell based assay for the validation of mutant IDH1 inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3741. doi:10.1158/1538-7445.AM2014-3741

Mechanistic and Bioinformatic Investigation of a Conserved Active Site Helix in α-Isopropylmalate Synthase from Mycobacterium tuberculosis, a Member of the DRE-TIM Metallolyase Superfamily

The characterization of functionally diverse enzyme superfamilies provides the opportunity to identify evolutionarily conserved catalytic strategies, as well as amino acid substitutions responsible for the evolution of new functions or specificities. Isopropylmalate synthase (IPMS) belongs to the DRE-TIM metallolyase superfamily. Members of this superfamily share common active site elements, including a conserved active site helix and an HXH divalent metal binding motif, associated with stabilization of a common enolate anion intermediate. These common elements are overlaid by variations in active site architecture resulting in the evolution of a diverse set of reactions that include condensation, lyase/aldolase, and carboxyl transfer activities. Here, using IPMS, an integrated biochemical and bioinformatics approach has been utilized to investigate the catalytic role of residues on an active site helix that is conserved across the superfamily. The construction of a sequence similarity network for the DRE-TIM metallolyase superfamily allows for the biochemical results obtained with IPMS variants to be compared across superfamily members and within other condensation-catalyzing enzymes related to IPMS. A comparison of our results with previous biochemical data indicates an active site arginine residue (R80 in IPMS) is strictly required for activity across the superfamily, suggesting that it plays a key role in catalysis, most likely through enolate stabilization. In contrast, differential results obtained from substitution of the C-terminal residue of the helix (Q84 in IPMS) suggest that this residue plays a role in reaction specificity within the superfamily.

Structural and Computational Studies of the Staphylococcus aureus Sortase B-Substrate Complex Reveal a Substrate-stabilized Oxyanion Hole

Sortase cysteine transpeptidases covalently attach proteins to the bacterial cell wall or assemble fiber-like pili that promote bacterial adhesion. Members of this enzyme superfamily are widely distributed in Gram-positive bacteria that frequently utilize multiple sortases to elaborate their peptidoglycan. Sortases catalyze transpeptidation using a conserved active site His-Cys-Arg triad that joins a sorting signal located at the C terminus of their protein substrate to an amino nucleophile located on the cell surface. However, despite extensive study, the catalytic mechanism and molecular basis of substrate recognition remains poorly understood. Here we report the crystal structure of the Staphylococcus aureus sortase B enzyme in a covalent complex with an analog of its NPQTN sorting signal substrate, revealing the structural basis through which it displays the IsdC protein involved in heme-iron scavenging from human hemoglobin. The results of computational modeling, molecular dynamics simulations, and targeted amino acid mutagenesis indicate that the backbone amide of Glu(224) and the side chain of Arg(233) form an oxyanion hole in sortase B that stabilizes high energy tetrahedral catalytic intermediates. Surprisingly, a highly conserved threonine residue within the bound sorting signal substrate facilitates construction of the oxyanion hole by stabilizing the position of the active site arginine residue via hydrogen bonding. Molecular dynamics simulations and primary sequence conservation suggest that the sorting signal-stabilized oxyanion hole is a universal feature of enzymes within the sortase superfamily.