Homologous Enzymes Research Articles

Conserved conformational dynamics determine enzyme activity.

Homologous enzymes often exhibit different catalytic rates despite a fully conserved active site. The canonical view is that an enzyme sequence defines its structure and function and, more recently, that intrinsic protein dynamics at different time scales enable and/or promote catalytic activity. Here, we show that, using the protein tyrosine phosphatase PTP1B, residues surrounding the PTP1B active site promote dynamically coordinated chemistry necessary for PTP1B function. However, residues distant to the active site also undergo distinct intermediate time scale dynamics and these dynamics are correlated with its catalytic activity and thus allow for different catalytic rates in this enzyme family. We identify these previously undetected motions using coevolutionary coupling analysis and nuclear magnetic resonance spectroscopy. Our findings strongly indicate that conserved dynamics drives the enzymatic activity of the PTP family. Characterization of these conserved dynamics allows for the identification of novel regulatory elements (therapeutic binding pockets) that can be leveraged for the control of enzymes.

Investigating Peptidoglycan Recycling Pathways in Tannerella forsythia with N-Acetylmuramic Acid Bioorthogonal Probes.

The human oral microbiome is the second largest microbial community in humans, harboring over 700 bacterial species, which aid in digestion and protect from growth of disease-causing pathogens. One such oral pathogen, Tannerella forsythia, along with other species, contributes to the pathogenesis of periodontitis. T. forsythia is unable to produce its own N-acetylmuramic acid (NAM) sugar, essential for peptidoglycan biosynthesis and therefore must scavenge NAM from other species with which it cohabitates. Here, we explore the recycling potential of T. forsythia for NAM uptake with a bioorthogonal modification into its peptidoglycan, allowing for click-chemistry-based visualization of the cell wall structure. Additionally, we identified NAM recycling enzyme homologues in T. forsythia that are similar to the enzymes found in Pseudomonas putida. These homologues were then genetically transformed into a laboratory safe Escherichia coli strain, resulting in the efficient incorporation of unnatural NAM analogues into the peptidoglycan backbone and its visualization, alone or in the presence of human macrophages. This strain will be useful in further studies to probe NAM recycling and peptidoglycan scavenging pathways of T. forsythia and other cohabiting bacteria.

Unusual β1-4-galactosidase activity of an α1-6-mannosidase from Xanthomonas manihotis in the processing of branched hybrid and complex glycans

Mannosidases are a diverse group of glycoside hydrolases that play crucial roles in mannose trimming of oligomannose glycans, glycoconjugates, and glycoproteins involved in numerous cellular processes, such as glycan biosynthesis and metabolism, structure regulation, cellular recognition, and cell–pathogen interactions. Exomannosidases and endomannosidases cleave specific glycosidic bonds of mannoside linkages in glycans and can be used in enzyme-based methods for sequencing of isomeric glycan structures. α1-6-mannosidase from Xanthomonas manihotis is known as a highly specific exoglycosidase that removes unbranched α1-6 linked mannose residues from oligosaccharides. However, we discovered that this α1-6-mannosidase also possesses an unexpected β1-4-galactosidase activity in the processing of branched hybrid and complex glycans through our use of enzymatic reactions, high performance anion-exchange chromatography, and liquid chromatography mass spectrometric sequencing. Our docking simulation of the α1-6-mannosidase with glycan substrates reveals potential interacting residues in a relatively shallow pocket slightly differing from its homologous enzymes in the glycoside hydrolase 125 family, which may be responsible for the observed higher promiscuity in substrate binding and subsequent terminal glycan hydrolysis. This observation of novel β1-4-galactosidase activity of the α1-6-mannosidase provides unique insights into its bifunctional activity on the substrate structure-dependent processing of terminal α1-6-mannose of unbranched glycans and terminal β1-4-galactose of hybrid and complex glycans. The finding thus suggests the dual glycosidase specificity of this α1-6-mannosidase and the need for careful consideration when used for the structural elucidation of glycan isomers.

Multiple Substrate Binding Mode-Guided Engineering of a Thermophilic PET Hydrolase.

Thermophilic polyester hydrolases (PES-H) have recently enabled biocatalytic recycling of the mass-produced synthetic polyester polyethylene terephthalate (PET), which has found widespread use in the packaging and textile industries. The growing demand for efficient PET hydrolases prompted us to solve high-resolution crystal structures of two metagenome-derived enzymes (PES-H1 and PES-H2) and notably also in complex with various PET substrate analogues. Structural analyses and computational modeling using molecular dynamics simulations provided an understanding of how product inhibition and multiple substrate binding modes influence key mechanistic steps of enzymatic PET hydrolysis. Key residues involved in substrate-binding and those identified previously as mutational hotspots in homologous enzymes were subjected to mutagenesis. At 72 °C, the L92F/Q94Y variant of PES-H1 exhibited 2.3-fold and 3.4-fold improved hydrolytic activity against amorphous PET films and pretreated real-world PET waste, respectively. The R204C/S250C variant of PES-H1 had a 6.4 °C higher melting temperature than the wild-type enzyme but retained similar hydrolytic activity. Under optimal reaction conditions, the L92F/Q94Y variant of PES-H1 hydrolyzed low-crystallinity PET materials 2.2-fold more efficiently than LCC ICCG, which was previously the most active PET hydrolase reported in the literature. This property makes the L92F/Q94Y variant of PES-H1 a good candidate for future applications in industrial plastic recycling processes.

Temperature- and light stress adaptations in Zygnematophyceae: The challenges of a semi-terrestrial lifestyle.

Streptophyte green algae comprise the origin of land plants and therefore life on earth as we know it today. While terrestrialization opened new habitats, leaving the aquatic environment brought additional abiotic stresses. More-drastic temperature shifts and high light levels are major abiotic stresses in semi-terrestrial habitats, in addition to desiccation, which has been reviewed elsewhere. Zygnematophyceae, a species-rich class of streptophyte green algae, is considered a sister-group to embryophytes. They have developed a variety of avoidance and adaptation mechanisms to protect against temperature extremes and high radiation in the form of photosynthetically active and ultraviolet radiation (UV) radiation occurring on land. Recently, knowledge of transcriptomic and metabolomic changes as consequences of these stresses has become available. Land-plant stress-signaling pathways producing homologs of key enzymes have been described in Zygnematophyceae. An efficient adaptation strategy is their mat-like growth habit, which provides self-shading and protects lower layers from harmful radiation. Additionally, Zygnematophyceae possess phenolic compounds with UV-screening ability. Resting stages such as vegetative pre-akinetes tolerate freezing to a much higher extent than do young cells. Sexual reproduction occurs by conjugation without the formation of flagellated male gametes, which can be seen as an advantage in water-deficient habitats. The resulting zygospores possess a multilayer cell wall, contributing to their resistance to terrestrial conditions. Especially in the context of global change, understanding temperature and light tolerance is crucial.

Development, Molecular Docking, and In Silico ADME Evaluation of Selective ALR2 Inhibitors for the Treatment of Diabetic Complications via Suppression of the Polyol Pathway.

Diabetic complications are associated with overexpression of aldose reductase, an enzyme that catalyzes the first step of the polyol pathway. Osmotic stress in the hyperglycemic state is linked with the intracellular accumulation of sorbitol along with the depletion of NADPH and eventually leads to oxidative stress via formation of reactive oxygen species and advanced glycation end products (AGEs). These kinds of mechanisms cause the development of various diabetic complications including neuropathy, nephropathy, retinopathy, and atherosclerotic plaque formation. Various aldose reductase inhibitors have been developed to date for the treatment of diabetic complications, but all have failed in different stages of clinical trials due to toxicity and poor pharmacokinetic profiles. This toxicity is rooted in a nonselective inhibition of both ALR2 and ALR1, homologous enzymes involved in the metabolism of toxic aldehydes such as methylglyoxal and 3-oxyglucosazone. In the present study, we developed a series of thiosemicarbazone derivatives as selective inhibitors of ALR2 with both antioxidant and antiglycation potential. Among the synthesized compounds, 3c exhibited strong and selective inhibition of ALR2 (IC50 1.42 μM) along with good antioxidant and antiglycative properties. The binding mode of 3c was assessed through molecular docking and cluster analysis via MD simulations, while in silico ADME evaluation studies predicted the compounds’ druglike properties. Therefore, we report 3c as a drug candidate with promising antioxidant and antiglycative properties that may be useful for the treatment of diabetic complications through selective inhibition of ALR2.

Generation of a ceramide synthase 6 mouse lacking the DDRSDIE C-terminal motif.

The important membrane lipid, ceramide, is generated by a family of homologous enzymes, the ceramide synthases (CerSs), multi-spanning membrane proteins located in the endoplasmic reticulum. Six CerS isoforms exist in mammals with each using a subset of acyl-CoAs for (dihydro)ceramide synthesis. A number of mice have been generated in which one or other CerS has been genetically manipulated, including complete knock-outs, with each displaying phenotypes concomitant with the expression levels of the CerS in question and the presumed biological function of the ceramide species that it generates. We recently described a short C-terminal motif in the CerS which is involved in CerS dimer formation; deleting this motif had no effect on the ability of the CerS to synthesize ceramide in vitro. In the current study, we generated a CerS6 mouse using CRISPR-Cas9, in which the DDRSDIE motif was replaced by ADAAAIA. While levels of CerS6ADAAAIA expression were unaffected in the CerS6ADAAAIA mouse, and CerS6ADAAAIA was able to generate C16-ceramide in vitro, ceramide levels were significantly reduced in the CerS6ADAAAIA mouse, suggesting that replacing this motif affects an as-yet unknown mechanism of regulation of ceramide synthesis via the DDRSDIE motif in vivo. Crossing CerS6ADAAAIA mice with CerS5 null mice led to generation of viable mice in which C16-ceramide levels were reduced by up to 90%, suggesting that depletion of C16-ceramide levels is compensated for by other ceramide species with different acyl chain lengths.

Discovery of Selective Nanomolar Inhibitors for Insulin-Regulated Aminopeptidase Based on α-Hydroxy-β-amino Acid Derivatives of Bestatin.

The oxytocinase subfamily of M1 zinc aminopeptidases comprises emerging drug targets, including the ER-resident aminopeptidases 1 and 2 (ERAP1 and ERAP2) and insulin-regulated aminopeptidase (IRAP); however, reports on clinically relevant inhibitors are limited. Here we report a new synthetic approach of high diastereo- and regioselectivity for functionalization of the α-hydroxy-β-amino acid scaffold of bestatin. Stereochemistry and mechanism of inhibition were investigated by a high-resolution X-ray crystal structure of ERAP1 in complex with a micromolar inhibitor. By exploring the P1 side-chain functionalities, we achieve significant potency and selectivity, and we report a cell-active, low-nanomolar inhibitor of IRAP with >120-fold selectivity over homologous enzymes. X-ray crystallographic analysis of IRAP in complex with this inhibitor suggest that interactions with the GAMEN loop is an unappreciated key determinant for potency and selectivity. Overall, our results suggest that α-hydroxy-β-amino acid derivatives may constitute useful chemical tools and drug leads for this group of aminopeptidases.

Discovery of Novel Phosphoinositide-3-Kinase α Inhibitors with High Selectivity, Excellent Bioavailability, and Long-Acting Efficacy for Gastric Cancer.

Phosphoinositide-3-kinase (PI3K) overexpressed in many tumors is a promising target for cancer therapy. However, due to toxicity from the ubiquitous expression of PI3K in many tissues, the development of PI3K inhibitors with high selectivity and low toxicity has become an urgent need for tumor treatment. Herein, based on the HipHop, we designed and synthesized a series of 6-(4,6-dimorpholino-1,3,5-triazin-2-yl)benzo[d]oxazol-2-amine derivatives as potent, selective, and long-acting PI3Kα inhibitors. Compound 27 was determined with potent PI3Kα inhibitory activity (IC50 = 4.4 nM), which exhibited excellent selectivity for homologous PI3K enzymes and a 370 kinome panel. Meanwhile, 27 featured favorable stability (T1/2 > 10 h) and high bioavailability (130%). Importantly, compound 27 exerted great antigastric cancer activity in vivo when combined with taxol. Collectively, these characteristics suggested 27 to be a promising PI3K agent for cancer treatment.

Discovery of glycosylated naphthacemycins and elucidation of the glycosylation

Two glycosylated naphthacemycins (naphthacemycins D1 and D2) were identified in Streptomyces sp. N12W1565. These two compounds not only showed antimicrobial potential against bacteria but also exhibited more aqueous solubility than naphthacemycins. Furthermore, the whole genome of Streptomyces sp. N12W1565 has been sequenced, the natY gene, located outside the biosynthetic gene cluster encoding a D-glucose glycosyltransferase, was identified to mediate glycosylation in the phenolic hydroxyl of the naphthacemycin core scaffold. Glycosyltransferase was elucidated in vitro by using a homologous enzyme, which showed potential as a biocatalyst.

Structural dynamics and in silico design of pyrazolopyran-based inhibitors against Plasmodium serine hydroxymethyltransferases

The clinical efficacy of antimalarial drugs has been reduced due to resistance spreading over many parts of the world. Target-based approaches on attractive drug targets, such as Plasmodium serine hydroxymethyltransferases (SHMTs) exhibiting distinct structure and function as well as kinetic mechanisms from the human enzyme homologue, are highly useful methods to be used for bypassing the present resistance in the field. Herein, 500-ns molecular dynamics (MD) simulations were carried out to investigate the mode of action of pyrazolopyran(+)-85 and pyrazolopyran(+)-86 with the most attractive inhibition efficiency in Plasmodium falciparum and P. vivax SHMTs (in the Schiff base form of PLP-L-serine (PLS) bound enzyme). The binding free energy results indicated the binding affinity of pyrazolopyran(+)-86 to Plasmodium SHMTs that is more favorable than pyrazolopyran(+)-85 by ∼ 2 kcal⋅mol−1, supported by the stronger ligand–protein hydrogen bonding and the lower solvent accessibility within the enzyme active site. According to the per-residue decomposition free energy analysis, residues L124, G128, H129, L130, K139, N356, and T357 are essential for inhibitors binding. By the rational structure-based drug design, the isopropyl moiety on the pyrazolopyran core should be changed to the negatively charged group (e.g., carboxylate group) for interacting with the positively charged residue R371. Alternatively, the phenolic compounds could be substituted with a phenyl or piperidine ring to promote hydrogen bond formation with the surrounding residues. Therefore, our findings presented here provide insights into the mode of inhibition of pyrazolopyran-based inhibitors and rational ideas for designing novel antimalarial drugs targeting Plasmodium SHMTs.

PGE2 inhibits neutrophil phagocytosis through the EP2R-cAMP-PTEN pathway.

Prostaglandin E2 (PGE2) is a potent lipid mediator of inflammation that modulates immune cell function by binding to unique G protein‐coupled receptors (EP receptors). PGE2 production increases during microbial infection and inflammation. In this study, we assessed the effect of PGE2 on the phagocytosis of bacteria by neutrophils, which are key players during infection and inflammation. We also looked for specific EP receptor signaling pathways that contributed to the neutrophil phagocytic activity. PGE2 (50–1000 ng/ml) inhibited the phagocytosis of Escherichia coli by HL‐60 human neutrophils in a concentration‐dependent manner. Inhibition of neutrophil phagocytosis by PGE2 correlated with increased intracellular cyclic adenosine monophosphate (cAMP) production, and forskolin, an adenosyl cyclase agonist, confirmed the inhibitory effect of cAMP stimulation on neutrophil phagocytosis. The expression of EP2 receptors by HL‐60 cells was confirmed by western blot analysis, and selective agonism of EP2 receptors mimicked the inhibition of phagocytosis by PGE2. The EP2 receptor antagonist AH‐6089 partially blocked the inhibition of neutrophil phagocytosis PGE2. Specific inhibition of phosphatase and tensin homolog (PTEN) enzyme attenuated the inhibition of neutrophil phagocytosis by PGE2, and both PGE2 and increased intracellular cAMP increased neutrophil PTEN activity, which was associated with decreased PTEN phosphorylation. The results support negative regulation of the antimicrobial activity of neutrophils (i.e., phagocytosis), which has important implications for the future management of bacterial infections.

Crystal structure of mevalonate 3,5-bisphosphate decarboxylase reveals insight into the evolution of decarboxylases in the mevalonate metabolic pathways

Mevalonate 3,5-bisphosphate decarboxylase is involved in the recently discovered Thermoplasma-type mevalonate pathway. The enzyme catalyzes the elimination of the 3-phosphate group from mevalonate 3,5-bisphosphate as well as concomitant decarboxylation of the substrate. This entire reaction of the enzyme resembles the latter half-reactions of its homologs, diphosphomevalonate decarboxylase and phosphomevalonate decarboxylase, which also catalyze ATP-dependent phosphorylation of the 3-hydroxyl group of their substrates. However, the crystal structure of mevalonate 3,5-bisphosphate decarboxylase and the structural reasons of the difference between reactions catalyzed by the enzyme and its homologs are unknown. In this study, we determined the X-ray crystal structure of mevalonate 3,5-bisphosphate decarboxylase from Picrophilus torridus, a thermoacidophilic archaeon of the order Thermoplasmatales. Structural and mutational analysis demonstrated the importance of a conserved aspartate residue for enzyme activity. In addition, although crystallization was performed in the absence of substrate or ligands, residual electron density having the shape of a fatty acid was observed at a position overlapping the ATP-binding site of the homologous enzyme, diphosphomevalonate decarboxylase. This finding is in agreement with the expected evolutionary route from phosphomevalonate decarboxylase (ATP-dependent) to mevalonate 3,5-bisphosphate decarboxylase (ATP-independent) through the loss of kinase activity. We found that the binding of geranylgeranyl diphosphate, an intermediate of the archeal isoprenoid biosynthesis pathway, evoked significant activation of mevalonate 3,5-bisphosphate decarboxylase, and several mutations at the putative geranylgeranyl diphosphate–binding site impaired this activation, suggesting the physiological importance of ligand binding as well as a possible novel regulatory system employed by the Thermoplasma-type mevalonate pathway.

In silico selectivity modeling of pyridine and pyrimidine based CYP11B1 and CYP11B2 inhibitors: A case study

Designof selective drug candidates for highly structural similar targets is a challenging task for researchers. The main objective of this study was to explore the selectivity modeling of pyridine and pyrimidine scaffold towards the highly homologous targets CYP11B1 and CYP11B2 enzymes by in silico (Molecular docking and QSAR) approaches. In this regard, a big dataset (n = 228) of CYP11B1 and CYP11B2 inhibitors were gathered and classified based on heterocyclic ring and the exhaustive analysis was carried out for pyridine and pyrimidinescaffolds. The LibDock algorithm was used to explore the binding pattern, screening, and identify the structural feature responsible for the selectivity of the ligands towards the studied targets. Finally, QSAR analysis was done to explore the correlation between various binding parameters and structural features responsible for the inhibitory activity and selectivity of the ligands in a quantitative way. The docking and QSAR analysis clearly revealed and distinguished the importance of structural features, functional groups attached for CYP11B2 and CYP11B1 selectivity for pyridine and pyrimidine analogs. Additionally, the docking analysis highlighted the differentiating amino acids residues for selectivity for ligands for each of the enzymes. The results obtained from this research work will be helpful in designing the selective CYP11B1/CYP11B2 inhibitors.

Trial in progress: A phase I trial of dual EZH 1/2 inhibitor valemetostat tosylate (DS-3201b) in pediatric, adolescent, and young adult patients with malignant solid tumors.

TPS10059 Background: Enhancer of zeste homolog enzymes (EZH1 and EZH2) form parts of the polycomb repressive complex 2 and regulate gene expression by catalyzing the tri-methylation of lysine 27 residue of histone H3. SMARCB1/INI1 is one of the core components of the SWI/SNF chromatin remodeling complex, with the loss of SMARCB1/INI1 causing the oncogenic activation of EZH2 and EZH1. The inhibition of EZH2 or both EZH2 and EZH1 can be effective against various hematological malignancies and SMARCB1/INI1-deficient solid tumors. Valemetostat tosylate (DS-3201b; valemetostat) is a potential first-in-class dual inhibitor of EZH1 and EZH2 that targets epigenetic regulations by inhibiting both EZH1 and EZH2 enzymes. A phase 2 single-arm study showed that valemetostat demonstrated promising response rates in Japanese patients with relapsed or refractory adult T-cell leukemia/lymphoma (Yoshimitsu M et al., presented at ASH Annual Meeting, 2021). Tumors characterized by SMARCB1/INI1 deficiency (a SWI/SNF mutation), such as malignant rhabdoid tumors, epithelioid sarcoma, or synovial sarcoma are quite frequently observed during childhood and adolescence, among whom valemetostat is expected to show antitumor effects. Methods: This open-label multi-center phase I trial evaluates the safety and efficacy of valemetostat in pediatric, adolescent, and young adult patients with refractory/relapsed solid tumors. The inclusion criteria are relapsed, refractory, or progressive metastatic disease; >3 and <19 years of age during the dose escalation cohort and < 29 years of age in the expanded cohort; performance status of >50 (assessed by Karnofsky Performance score in patients >16 years old, and Lansky Performance score in patients <15 years old); and adequate organ function. Valemetostat is administered orally once a day without interruption. Three dose levels (150, 200, and 250 mg/1.7 m2) are assessed using a 3+3 design during the dose escalation cohort. After determining the recommended phase 2 dose (RP2D) during dose escalation cohort, up to 30 patients will be further enrolled, and the safety and efficacy data of valemetostat are determined in the expanded cohort. The primary endpoint is the incidence of dose limiting toxicity, whereas the secondary endpoints include safety, pharmacokinetics, overall response rate, progression-free survival. The overall response rate of the tumors with SMARCB1/INI1 deficiency or SWI/SNF mutation is also evaluated as a secondary endpoint. Exploratory endpoint includes overall survival. Enrollment into this trial began in March 2020, and enrollment into the dose escalation cohort was completed. Enrollment into the expanded cohort began in November 2021. Clinical Trial Information: jRCT2031190268.

Adaptation of thermophilic enzymes from an ancestral reductive TCA cycle for carbon fixation in plants

The reductive TCA cycle (rTCA) is one of 5 carbon‐fixing cycles in prokaryotes other than the Calvin‐Benson cycle ubiquitous in plants. Adaptation of these cycles to biofuel crops is one strategy for reducing the greenhouse gas CO2 and simultaneously increasing crop yields. A minimal 5‐enzyme synthetic rTCA cycle was designed at NCSU utilizing 2 unique enzymes to carboxylate and reduce 2‐oxoglutarate to isocitrate. The enzymes are derived from an evolutionarily primitive configuration of the rTCA cycle that persists in the order Aquificales in hot springs and deep ocean thermal vents. Two strategies are being pursued to engineer mesophilic forms of these enzymes: 1) reducing the temperature of activity of the Aquificales enzymes, and 2) modifying the substrate specificity of homologous mesophilic enzymes. The first enzyme, 2‐oxoglutarate carboxylase (OGC), is a member of the biotin‐dependent carboxylase family homologous to pyruvate carboxylase. Biotin‐dependent carboxylases are multi‐subunit complexes. So far, we have identified the temperature‐dependent steps of the biotin carboxylase domain of OGC and predicted mutations to reduce the temperature of activity based on the crystal structure. Phylogenetic analysis suggests that OGC and pyruvate carboxylase diverged prior to Aquificales evolution. An ancestral promiscuous enzyme has been proposed to carboxylate both pyruvate and 2‐oxoglutarate. Ancestral enzymes are being reconstructed from phylogenetic analyses to switch the substrate specificity of mesophilic pyruvate carboxylase to carboxylate 2‐oxoglutarate. The second enzyme, oxalosuccinate reductase (OSR), is homologous with isocitrate dehydrogenase. In fact, isocitrate dehydrogenase has replaced OGC/OSR in most organisms utilizing the rTCA cycle. Isocitrate dehydrogenase favors the oxidative decarboxylase of isocitrate in the TCA cycle, while OGC/OSR favors the reductive cycle. OSR is an ancestral, non‐decarboxylating isocitrate dehydrogenase. We propose to engineer a mesophilic OSR by mutating isocitrate dehydrogenase to inhibit decarboxylation, based on phylogenic and structural studies of OSR. Ultimately engineering a mesophilic OGC/OSR will be utilized to increase carbon capture in seed oil crops like Camelina sativa that are carbon limited. Oil from Camelina seeds can be converted to jet fuels.

Temporal perturbation of histone deacetylase activity reveals a requirement for HDAC1-3 in mesendoderm cell differentiation.

Histone deacetylases (HDACs) are a class of enzymes that control chromatin state and influence cell fate. We evaluated the chromatin accessibility and transcriptome dynamics of zinc-containing HDACs during cell differentiation invitro coupled with chemical perturbation to identify the role of HDACs in mesendoderm cell fate specification. Single-cell RNA sequencing analyses of HDAC expression during human pluripotent stem cell (hPSC) differentiation invitro and mouse gastrulation invivo reveal a unique association of HDAC1 and -3 with mesendoderm gene programs during exit from pluripotency. Functional perturbation with small molecules reveals that inhibition of HDAC1 and -3, but not HDAC2, induces mesoderm while impeding endoderm and early cardiac progenitor specification. These data identify unique biological functions of the structurally homologous enzymes HDAC1-3 in influencing hPSC differentiation from pluripotency toward mesendodermal and cardiac progenitor populations.

Can ERAP1 and ERAP2 Form Functional Heterodimers? A Structural Dynamics Investigation.

Endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and ERAP2) play important roles in the generation of antigenic peptides presented by Major Histocompatibility Class I (MHCI) molecules and indirectly regulate adaptive immune responses. Although the discrete function of these enzymes has been extensively characterized, recent reports have suggested that they can also form heterodimers with functional consequences. However, lack of structural characterization of a putative ERAP1/ERAP2 dimer has limited our understanding of its biological role and significance. To address this, we employed computational molecular dynamics calculations to explore the topology of interactions between these two, based on experimentally determined homo-dimerization interfaces observed in crystal structures of ERAP2 or homologous enzymes. Our analysis of 8 possible dimerization models, suggested that the most likely ERAP1/ERAP2 heterodimerization topology involves the exon 10 loop, a non-conserved loop previously implicated in interactions between ERAP1 and the disulfide-bond shuffling chaperone ERp44. This dimerization topology allows access to the active site of both enzymes and is consistent with a previously reported construct in which ERAP1 and ERAP2 were linked by Fos/Jun zipper tags. The proposed model constitutes a tentative structural template to help understand the physiological role and significance of ERAP1/ERAP2 molecular interactions.

Evaluation of Porcine and Aspergillus oryzae α-Amylases as Possible Model for the Human Enzyme

α-amylases are ubiquitous enzymes belonging to the glycosyl hydrolase (GH13) family, whose members share a high degree of sequence identity, even between distant organisms. To understand the determinants of catalytic activity of α-amylases throughout evolution, and to investigate the use of homologous enzymes as a model for the human one, we compared human salivary α-amylase, Aspergillus oryzae α-amylase and pancreatic porcine α-amylase, using a combination of in vitro and in silico approaches. Enzyme sequences were aligned, and structures superposed, whereas kinetics were spectroscopically studied by using commercial synthetic substrates. These three enzymes show strikingly different activities, specifically mediated by different ions, despite relevant structural homology. Our study confirms that the function of α-amylases throughout evolution has considerably diverged, although key structural determinants, such as the catalytic triad and the calcium-binding pocket, have been retained. These functional differences need to be carefully considered when α-amylases, from different organisms, are used as a model for the human enzymes. In this frame, particular focus is needed for the setup of proper experimental conditions.

Insights into Mechanisms of Damage Recognition and Catalysis by APE1-like Enzymes.

Apurinic/apyrimidinic (AP) endonucleases are the key DNA repair enzymes in the base excision repair (BER) pathway, and are responsible for hydrolyzing phosphodiester bonds on the 5′ side of an AP site. The enzymes can recognize not only AP sites but also some types of damaged bases, such as 1,N6-ethenoadenosine, α-adenosine, and 5,6-dihydrouridine. Here, to elucidate the mechanism underlying such a broad substrate specificity as that of AP endonucleases, we performed a computational study of four homologous APE1-like endonucleases: insect (Drosophila melanogaster) Rrp1, amphibian (Xenopus laevis) APE1 (xAPE1), fish (Danio rerio) APE1 (zAPE1), and human APE1 (hAPE1). The contact between the amino acid residues of the active site of each homologous APE1-like enzyme and the set of damaged DNA substrates was analyzed. A comparison of molecular dynamic simulation data with the known catalytic efficiency of these enzymes allowed us to gain a deep insight into the differences in the efficiency of the cleavage of various damaged nucleotides. The obtained data support that the amino acid residues within the “damage recognition” loop containing residues Asn222–Ala230 significantly affect the catalytic-complex formation. Moreover, every damaged nucleotide has its unique position and a specific set of interactions with the amino acid residues of the active site.