The biosynthesis of the lincosamide antibiotics lincomycin A and celesticetin involves the pyridoxal-5'-phosphate (PLP)-dependent enzymes LmbF and CcbF, which are responsible for bifurcation of the biosynthetic pathways. Despite recognizing the same S-glycosyl-L-cysteine structure of the substrates, LmbF catalyses thiol formation through β-elimination, whereas CcbF produces S-acetaldehyde through decarboxylation-coupled oxidative deamination. The structural basis for the diversification mechanism remains largely unexplored. Here we conduct structure-function analyses of LmbF and CcbF. X-ray crystal structures, docking and molecular dynamics simulations reveal that active-site aromatic residues play important roles in controlling the substrate binding mode and the reaction outcome. Furthermore, the reaction selectivity and oxygen-utilization of LmbF and CcbF were rationally engineered through structure- and calculation-based mutagenesis. Thus, the catalytic function of CcbF was switched to that of LmbF, and, remarkably, both LmbF and CcbF variants gained the oxidative-amidation activity to produce an unnatural S-acetamide derivative of lincosamide.
This study investigates the factors modulating the reactivity of 5'-deoxyadenosyl (5'dAdo ̇) radical, a potent hydrogen atom abstractor that forms in the active sites of radical SAM enzymes and that otherwise undergoes a rapid self-decay in aqueous solution. Here, we compare hydrogen atom abstraction (HAA) reactions between native substrates of radical SAM enzymes and 5'dAdo ̇ in aqueous solution and in two enzymatic microenvironments. With that we reveal that HAA efficiency of 5'dAdo ̇ is due to (i) the in situ formation of 5'dAdo ̇ in a pre-ordered complex with a substrate, which attenuates the unfavorable effect of substrate:5'dAdo ̇ complex formation, and (ii) the prevention of the conformational changes associated with self-decay by a tight active-site cavity. The enzymatic cavity, however, does not have a strong effect on the HAA activity of 5'dAdo ̇. Thus, we performed an analysis of in-water HAA performed by 5'dAdo ̇ based on a three-component thermodynamic model incorporating the diagonal effect of the free energy of reaction, and the off-diagonal effect of asynchronicity and frustration. To this aim, we took advantage of the straightforward relationship between the off-diagonal thermodynamic effects and the electronic-structure descriptor - the redistribution of charge between the reactants during the reaction. It allows to access HAA-competent redox and acidobasic properties of 5'dAdo ̇ that are otherwise unavailable due to its instability upon one-electron reduction and protonation. The results show that all reactions feature a favourable thermodynamic driving force and tunneling, the latter of which lowers systematically barriers by ∼2 kcal mol-1. In addition, most of the reactions experience a favourable off-diagonal thermodynamic contribution. In HAA reactions, 5'dAdo ̇ acts as a weak oxidant as well as a base, also 5'dAdo ̇-promoted HAA reactions proceed with a quite low degree of asynchronicity of proton and electron transfer. Finally, the study elucidates the crucial and dual role of asynchronicity. It directly lowers the barrier as a part of the off-diagonal thermodynamic contribution, but also indirectly increases the non-thermodynamic part of the barrier by presumably controlling the adiabatic coupling between proton and electron transfer. The latter signals that the reaction proceeds as a hydrogen atom transfer rather than a proton-coupled electron transfer.
YKL-40, also known as human cartilage glycoprotein-39 (HC-gp39) or CHI3L1, shares structural similarities with chitotriosidase (CHIT1), an active chitinase, but lacks chitinase activity. Despite being a biomarker for inflammatory disorders and cancer, the reasons for YKL-40's inert chitinase function have remained elusive. This study reveals that the loss of chitinase activity in YKL-40 has risen from multiple sequence modifications influencing its chitin affinity. Contrary to the common belief associating the lack of chitinase activity with amino acid substitutions in the catalytic motif, attempts to activate YKL-40 by creating two amino acid mutations in the catalytic motif (MT-YKL-40) proved ineffective. Subsequent exploration that included creating chimeras of MT-YKL-40 and CHIT1 catalytic domains (CatDs) identified key exons responsible for YKL-40 inactivation. Introducing YKL-40 exons 3, 6, or 8 into CHIT1 CatD resulted in chitinase inactivation. Conversely, incorporating CHIT1 exons 3, 6, and 8 into MT-YKL-40 led to its activation. Our recombinant proteins exhibited properly formed disulfide bonds, affirming a defined structure in active molecules. Biochemical and evolutionary analysis indicated that the reduced chitinase activity of MT-YKL-40 correlates with specific amino acids in exon 3. M61I and T69W substitutions in CHIT1 CatD diminished chitinase activity and increased chitin binding. Conversely, substituting I61 with M and W69 with T in MT-YKL-40 triggered chitinase activity while reducing the chitin-binding activity. Thus, W69 plays a crucial role in a unique subsite within YKL-40. These findings emphasize that YKL-40, though retaining the structural framework of a mammalian chitinase, has evolved to recognize chitin while surrendering chitinase activity.
- MeSH
- chitin * metabolismus chemie MeSH
- chitinasy metabolismus genetika chemie MeSH
- exony MeSH
- hexosaminidasy metabolismus chemie genetika MeSH
- katalytická doména MeSH
- lidé MeSH
- molekulární evoluce MeSH
- protein CHI3L1 * metabolismus genetika chemie MeSH
- sekvence aminokyselin MeSH
- substituce aminokyselin MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Inhibition of hypoxanthine-guanine-xanthine phosphoribosyltransferase activity decreases the pool of 6-oxo and 6-amino purine nucleoside monophosphates required for DNA and RNA synthesis, resulting in a reduction in cell growth. Therefore, inhibitors of this enzyme have potential to control infections, caused by Plasmodium falciparum and Plasmodium vivax, Trypanosoma brucei, Mycobacterium tuberculosis, and Helicobacter pylori. Five compounds synthesized here that contain a purine base covalently linked by a prolinol group to one or two phosphonate groups have Ki values ranging from 3 nM to >10 μM, depending on the structure of the inhibitor and the biological origin of the enzyme. X-ray crystal structures show that, on binding, these prolinol-containing inhibitors stimulated the movement of active site loops in the enzyme. Against TBr in cell culture, a prodrug exhibited an EC50 of 10 μM. Thus, these compounds are excellent candidates for further development as drug leads against infectious diseases as well as being potential anticancer agents.
- MeSH
- inhibitory enzymů * farmakologie chemie chemická syntéza MeSH
- katalytická doména MeSH
- krystalografie rentgenová MeSH
- lidé MeSH
- molekulární modely MeSH
- molekulární struktura MeSH
- pentosyltransferasy * antagonisté a inhibitory metabolismus MeSH
- racionální návrh léčiv * MeSH
- Trypanosoma brucei brucei účinky léků enzymologie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
HDAC11 is a class IV histone deacylase with no crystal structure reported so far. The catalytic domain of HDAC11 shares low sequence identity with other HDAC isoforms, which makes conventional homology modeling less reliable. AlphaFold is a machine learning approach that can predict the 3D structure of proteins with high accuracy even in absence of similar structures. However, the fact that AlphaFold models are predicted in the absence of small molecules and ions/cofactors complicates their utilization for drug design. Previously, we optimized an HDAC11 AlphaFold model by adding the catalytic zinc ion and minimization in the presence of reported HDAC11 inhibitors. In the current study, we implement a comparative structure-based virtual screening approach utilizing the previously optimized HDAC11 AlphaFold model to identify novel and selective HDAC11 inhibitors. The stepwise virtual screening approach was successful in identifying a hit that was subsequently tested using an in vitro enzymatic assay. The hit compound showed an IC50 value of 3.5 μM for HDAC11 and could selectively inhibit HDAC11 over other HDAC subtypes at 10 μM concentration. In addition, we carried out molecular dynamics simulations to further confirm the binding hypothesis obtained by the docking study. These results reinforce the previously presented AlphaFold optimization approach and confirm the applicability of AlphaFold models in the search for novel inhibitors for drug discovery.
The protozoan parasite Trichomonas vaginalis (Tv) causes trichomoniasis, the most common non-viral sexually transmitted infection in the world. Although Tv has been linked to significant health complications, only two closely related 5-nitroimidazole drugs are approved for its treatment. The emergence of resistance to these drugs and lack of alternative treatment options poses an increasing threat to public health, making development of novel anti-Trichomonas compounds an urgent need. The proteasome, a critical enzyme complex found in all eukaryotes has three catalytic subunits, β1, β2, and β5 and has been validated as a drug target to treat trichomoniasis. With the goal of developing tools to study the Tv proteasome, we isolated the enzyme complex and identified inhibitors that preferentially inactivate either one or two of the three catalytic subunits. Using a mass spectrometry-based peptide digestion assay, these inhibitors were used to define the substrate preferences of the β1, β2 and β5 subunits. Subsequently, three model fluorogenic substrates were designed, each specific for one of the catalytic subunits. This novel substrate profiling methodology will allow for individual subunit characterization of other proteasomes of interest. Using the new substrates, we screened a library of 284 peptide epoxyketone inhibitors against Tv and determined the subunits targeted by the most active compounds. The data show that inhibition of the Tv β5 subunit alone is toxic to the parasite. Taken together, the optimized proteasome subunit substrates will be instrumental for understanding the molecular determinants of proteasome specificity and for accelerating drug development against trichomoniasis.
- MeSH
- inhibitory proteasomu farmakologie chemie MeSH
- katalytická doména * MeSH
- proteasomový endopeptidasový komplex * metabolismus chemie MeSH
- protozoální proteiny chemie metabolismus antagonisté a inhibitory genetika MeSH
- substrátová specifita MeSH
- Trichomonas vaginalis * enzymologie MeSH
- Publikační typ
- časopisecké články MeSH
Complex II (CII) activity controls phenomena that require crosstalk between metabolism and signaling, including neurodegeneration, cancer metabolism, immune activation, and ischemia-reperfusion injury. CII activity can be regulated at the level of assembly, a process that leverages metastable assembly intermediates. The nature of these intermediates and how CII subunits transfer between metastable complexes remains unclear. In this work, we identify metastable species containing the SDHA subunit and its assembly factors, and we assign a preferred temporal sequence of appearance of these species during CII assembly. Structures of two species show that the assembly factors undergo disordered-to-ordered transitions without the appearance of significant secondary structure. The findings identify that intrinsically disordered regions are critical in regulating CII assembly, an observation that has implications for the control of assembly in other biomolecular complexes.
- MeSH
- katalytická doména * MeSH
- sekundární struktura proteinů MeSH
- Publikační typ
- časopisecké články MeSH
Our knowledge on the genetic diversity of the human genome is exponentially growing. However, our capacity to establish genotype-phenotype correlations on a large scale requires a combination of detailed experimental and computational work. This is a remarkable task in human proteins which are typically multifunctional and structurally complex. In addition, mutations often prevent the determination of mutant high-resolution structures by X-ray crystallography. We have characterized here the effects of five mutations in the active site of the disease-associated NQO1 protein, which are found either in cancer cell lines or in massive exome sequencing analysis in human population. Using a combination of H/D exchange, rapid-flow enzyme kinetics, binding energetics and conformational stability, we show that mutations in both sets may cause counterintuitive functional effects that are explained well by their effects on local stability regarding different functional features. Importantly, mutations predicted to be highly deleterious (even those affecting the same protein residue) may cause mild to catastrophic effects on protein function. These functional effects are not well explained by current predictive bioinformatic tools and evolutionary models that account for site conservation and physicochemical changes upon mutation. Our study also reinforces the notion that naturally occurring mutations not identified as disease-associated can be highly deleterious. Our approach, combining protein biophysics and structural biology tools, is readily accessible to broadly increase our understanding of genotype-phenotype correlations and to improve predictive computational tools aimed at distinguishing disease-prone against neutral missense variants in the human genome.
- MeSH
- katalytická doména genetika MeSH
- lidé MeSH
- missense mutace * MeSH
- molekulární biologie MeSH
- mutace MeSH
- NAD(P)H dehydrogenasa (chinon) genetika metabolismus MeSH
- proteiny * chemie MeSH
- výpočetní biologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: S1-like nucleases are widespread enzymes commonly used in biotechnology and molecular biology. Although it is commonly believed that they are mainly Zn2+-dependent acidic enzymes, we have found that numerous members of this family deviate from this rule. Therefore, in this work, we decided to check how broad is the range of non‐zinc-dependent S1-like nucleases and what is the molecular basis of their activities. METHODS: S1-like nucleases chosen for analysis were achieved through heterologous expression in appropriate eukaryotic hosts. To characterize nucleases' active-site properties, point mutations were introduced in selected positions. The enzymatic activities of wild-type and mutant nucleases were tested by in-gel nuclease activity assay. RESULTS: We discovered that S1-like nucleases encoded by non-vascular plants and single-celled protozoa, like their higher plant homologues, exhibit a large variety of catalytic properties. We have shown that these individual properties are determined by specific non-conserved active site residues. CONCLUSIONS: Our findings demonstrate that mutations that occur during evolution can significantly alter the catalytic properties of S1-like nucleases. As a result, different ions can compete for particular S1-type nucleases' active sites. This phenomenon undermines the existing classification of S1-like nucleases. GENERAL SIGNIFICANCE: Our findings have numerous implications for applications and understanding the S1-like nucleases' biological functions. For example, new biotechnological applications should take into account their unexpected catalytic properties. Moreover, these results demonstrate that the trinuclear zinc-based model commonly used to characterize the catalytic activities of S1-like nucleases is insufficient to explain the actions of non‐zinc-dependent members of this family.
Alisertib (MLN8237), a novel Aurora A kinase inhibitor, is currently being clinically tested in late-phase trials for the therapy of various malignancies. In the present work, we describe alisertib's potential to perpetrate pharmacokinetic drug-drug interactions (DDIs) and/or to act as an antagonist of multidrug resistance (MDR). In accumulation assays, alisertib potently inhibited ABCC1 transporter, but not ABCB1 or ABCG2. The results of molecular modeling suggested a bifunctional mechanism for interaction on ABCC1. In addition, alisertib was characterized as a low- to moderate-affinity inhibitor of recombinant CYP3A4, CYP2C8, CYP2C9, CYP2C19, and CYP2D6 isoenzymes, but without potential clinical relevance. Drug combination studies revealed the capability of alisertib to synergistically antagonize ABCC1-mediated resistance to daunorubicin. Although alisertib exhibited substrate characteristics toward ABCB1 transporter in monolayer transport assays, comparative proliferation studies showed lack of its MDR-victim behavior in cells overexpressing ABCB1 as well as ABCG2 and ABCC1. Lastly, alisertib did not affect the expression of ABCC1, ABCG2, ABCB1 transporters and CYP1A2, CYP3A4, CYP2B6 isozymes on mRNA level in various systemic and tumoral models. In conclusion, our study suggests that alisertib is a drug candidate with negligible potential for perpetrating systemic pharmacokinetic DDIs on ABCB1, ABCG2 and cytochromes P450. In addition, we introduce alisertib as an effective dual-activity chemosensitizer whose MDR-antagonistic capacities are not impaired by efflux or effect on MDR phenotype. Our in vitro findings provide important pieces of information for clinicians when introducing alisertib into the clinical area.
- MeSH
- ABC transportér z rodiny G, člen 2 genetika metabolismus MeSH
- azepiny farmakokinetika farmakologie MeSH
- buněčné linie MeSH
- katalytická doména MeSH
- konformace proteinů MeSH
- lékové interakce MeSH
- lidé MeSH
- molekulární modely MeSH
- P-glykoprotein genetika metabolismus MeSH
- proteiny spojené s mnohočetnou rezistencí k lékům antagonisté a inhibitory MeSH
- psi MeSH
- pyrimidiny farmakokinetika farmakologie MeSH
- regulace genové exprese účinky léků MeSH
- simulace molekulového dockingu MeSH
- systém (enzymů) cytochromů P-450 metabolismus MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- psi MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
