substrate docking
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Rutinosidases (α-l-rhamnopyranosyl-(1-6)-β-d-glucopyranosidases, EC 3.2.1.168, CAZy GH5) are diglycosidases that cleave the glycosidic bond between the disaccharide rutinose and the respective aglycone. Similar to many retaining glycosidases, rutinosidases can also transfer the rutinosyl moiety onto acceptors with a free -OH group (so-called transglycosylation). The recombinant rutinosidase from Aspergillus niger (AnRut) is selectively produced in Pichia pastoris. It can catalyze transglycosylation reactions as an unpurified preparation directly from cultivation. This enzyme exhibits catalytic activity towards two substrates; in addition to rutinosidase activity, it also exhibits β-d-glucopyranosidase activity. As a result, new compounds are formed by β-glucosylation or rutinosylation of acceptors such as alcohols or strong inorganic nucleophiles (NaN3). Transglycosylation products with aliphatic aglycones are resistant towards cleavage by rutinosidase, therefore, their side hydrolysis does not occur, allowing higher transglycosylation yields. Fourteen compounds were synthesized by glucosylation or rutinosylation of selected acceptors. The products were isolated and structurally characterized. Interactions between the transglycosylation products and the recombinant AnRut were analyzed by molecular modeling. We revealed the role of a substrate tunnel in the structure of AnRut, which explained the unusual catalytic properties of this glycosidase and its specific transglycosylation potential. AnRut is attractive for biosynthetic applications, especially for the use of inexpensive substrates (rutin and isoquercitrin).
- MeSH
- Aspergillus niger enzymologie MeSH
- disacharidy chemie metabolismus MeSH
- fungální proteiny chemie metabolismus MeSH
- glykosidhydrolasy chemie metabolismus MeSH
- glykosylace MeSH
- hydrolýza MeSH
- katalytická doména MeSH
- rekombinantní proteiny metabolismus MeSH
- rutin chemie metabolismus MeSH
- simulace molekulového dockingu MeSH
- substrátová specifita MeSH
- Publikační typ
- časopisecké články MeSH
Molecular dynamics simulations of complexes between Norwalk virus RNA dependent RNA polymerase and its natural CTP and 2dCTP (both containing the O5'-C5'-C4'-O4' sequence of atoms bridging the triphosphate and sugar moiety) or modified coCTP (C5'-O5'-C4'-O4'), cocCTP (C5'-O5'-C4'-C4'') substrates were produced by means of CUDA programmable graphical processing units and the ACEMD software package. It enabled us to gain microsecond MD trajectories clearly showing that similar nucleoside triphosphates can bind surprisingly differently into the active site of the Norwalk virus RNA dependent RNA polymerase. It corresponds to their different modes of action (CTP-substrate, 2dCTP-poor substrate, coCTP-chain terminator, cocCTP-inhibitor). Moreover, extremely rare events-as repetitive pervasion of Arg182 into a potentially reaction promoting arrangement-were captured.
- MeSH
- cytidintrifosfát analogy a deriváty metabolismus MeSH
- infekce viry z čeledi Caliciviridae virologie MeSH
- lidé MeSH
- Norovirus enzymologie metabolismus MeSH
- RNA-dependentní RNA-polymerasa metabolismus MeSH
- simulace molekulární dynamiky MeSH
- simulace molekulového dockingu MeSH
- substrátová specifita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Based on the significant anti-inflammatory activity of natural quinone primin (5a), series of 1,4-benzoquinones, hydroquinones, and related resorcinols were designed, synthesized, characterized and tested for their ability to inhibit the activity of cyclooxygenase (COX-1 and COX-2) and 5-lipoxygenase (5-LOX) enzymes. Structural modifications resulted in the identification of two compounds 5b (2-methoxy-6-undecyl-1,4-benzoquinone) and 6b (2-methoxy-6-undecyl-1,4-hydroquinone) as potent dual COX/5-LOX inhibitors. The IC50 values evaluated in vitro using enzymatic assay were for compound 5b IC50 = 1.07, 0.57, and 0.34 μM and for compound 6b IC50 = 1.07, 0.55, and 0.28 μM for COX-1, COX-2, and 5-LOX enzyme, respectively. In addition, compound 6d was identified as the most potent 5-LOX inhibitor (IC50 = 0.14 μM; reference inhibitor zileuton IC50 = 0.66 μM) from the tested compounds while its inhibitory potential against COX enzymes (IC50 = 2.65 and 2.71 μM for COX-1 and COX-2, respectively) was comparable with the reference inhibitor ibuprofen (IC50 = 4.50 and 2.46 μM, respectively). The most important structural modification leading to increased inhibitory activity towards both COXs and 5-LOX was the elongation of alkyl chain in position 6 from 5 to 11 carbons. Moreover, the monoacetylation in ortho position of bromo-hydroquinone 13 led to the discovery of potent (IC50 = 0.17 μM) 5-LOX inhibitor 17 (2-bromo-6-methoxy-1,4-benzoquinone) while bromination stabilized the hydroquinone form. Docking analysis revealed the interaction of compounds with Tyr355 and Arg120 in the catalytic site of COX enzymes, while the hydrophobic parts of the molecules filled the hydrophobic substrate channel leading up to Tyr385. In the allosteric catalytic site of 5-LOX, compounds bound to Tyr142 and formed aromatic interactions with Arg138. Taken together, we identified optimal alkyl chain length for dual COX/5-LOX inhibition and investigated other structural modifications influencing COX and 5-LOX inhibitory activity.
- MeSH
- benzochinony chemie MeSH
- inhibitory cyklooxygenasy chemická syntéza chemie farmakologie MeSH
- inhibitory lipoxygenas chemická syntéza chemie farmakologie MeSH
- katalytická doména MeSH
- oxidace-redukce MeSH
- počítačová simulace MeSH
- resorcinoly chemie MeSH
- simulace molekulového dockingu MeSH
- spektrální analýza metody MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
Plant aminoaldehyde dehydrogenases (AMADHs, EC 1.2.1.19) belong to the family 10 of aldehyde dehydrogenases and participate in the metabolism of compounds related to amino acids such as polyamines or osmoprotectants. Their broad specificity covers ω-aminoaldehydes, aliphatic and aromatic aldehydes as well as nitrogen-containing heterocyclic aldehydes. The substrate preference of plant AMADHs is determined by the presence of aspartic acid and aromatic residues in the substrate channel. In this work, 15 new N-acyl derivates of 3-aminopropanal (APAL) and 4-aminobutanal (ABAL) were synthesized and confirmed as substrates of two pea AMADH isoenzymes (PsAMADH 1 and 2). The compounds were designed considering the previously demonstrated conversion of N-acetyl derivatives as well as substrate channel dimensions (5-8 Å × 14 Å). The acyl chain length and its branching were found less significant for substrate properties than the length of the initial natural substrate. In general, APAL derivatives were found more efficient than the corresponding ABAL derivatives because of the prevailing higher conversion rates and lower K m values. Differences in enzymatic performance between the two isoenzymes corresponded in part to their preferences to APAL to ABAL. The higher PsAMADH2 affinity to substrates correlated with more frequent occurrence of an excess substrate inhibition. Molecular docking indicated the possible auxiliary role of Tyr163, Ser295 and Gln451 in binding of the new substrates. The only derivative carrying a free carboxyl group (N-adipoyl APAL) was surprisingly better substrate than ABAL in PsAMADH2 reaction indicating that also negatively charged aldehydes might be good substrates for ALDH10 family.
- MeSH
- aldehyddehydrogenasa chemie metabolismus MeSH
- aldehydy chemie metabolismus MeSH
- hrách setý chemie enzymologie MeSH
- kinetika MeSH
- molekulární struktura MeSH
- propylaminy chemie metabolismus MeSH
- rostlinné proteiny chemie metabolismus MeSH
- simulace molekulového dockingu MeSH
- substrátová specifita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Phenyl valerate (PV) is a substrate for measuring the PVase activity of neuropathy target esterase (NTE), a key molecular event of organophosphorus-induced delayed neuropathy. A protein with PVase activity in chicken (model for delayed neurotoxicity) was identified as butyrylcholinesterase (BChE). Purified human butyrylcholinesterase (hBChE) showed PVase activity with a similar sensitivity to inhibitors as its cholinesterase (ChE) activity. Further kinetic and theoretical molecular simulation studies were performed. The kinetics did not fit classic competition models among substrates. Partially mixed inhibition was the best-fitting model to acetylthiocholine (AtCh) interacting with PVase activity. ChE activity showed substrate activation, and non-competitive inhibition was the best-fitting model to PV interacting with the non-activated enzyme and partial non-competitive inhibition was the best fitted model for PV interacting with the activated enzyme by excess of AtCh. The kinetic results suggest that other sites could be involved in those activities. From the theoretical docking analysis, we deduced other more favorable sites for binding PV related with Asn289 residue, situated far from the catalytic site ("PV-site"). Both substrates acethylcholine (ACh) and PV presented similar docking values in both the PV-site and catalytic site pockets, which explained some of the observed substrate interactions. Molecular dynamic simulations based on the theoretical structure of crystallized hBChE were performed. Molecular modeling studies suggested that PV has a higher potential for non-competitive inhibition, being also able to inhibit the hydrolysis of ACh through interactions with the PV-site. Further theoretical studies also suggested that PV could yet be able to promote competitive inhibition. We concluded that the kinetic and theoretical studies did not fit the simple classic competition among substrates, but were compatible with the interaction with two different binding sites.
To investigate structure-function relationships of cytochromes P450 (CYP), 3-azidiamantane was employed for photoaffinity labeling of rabbit microsomal CYP2B4. Four diamantane labeled tryptic fragments were identified by mass spectrometry and sequencing: peptide I (Leu359-Lys373), peptide II (Leu30-Arg48), peptide III (Phe127-Arg140), and peptide IV (Arg434-Arg443). Their positions were projected into CYP2B4 model structures and compared with substrate binding sites, proposed by docking of diamantane. We identified novel binding regions outside the active site of CYP2B4. One of them, defined with diamantane modified Arg133, marks a possible entrance to the active site from the heme proximal face. In addition to crystal structures of CYP2B4 chimeras and molecular dynamics simulations, our data of photoaffinity labeling of the full CYP2B4 molecule provide further insight into functional and structural aspects of substrate binding.
- MeSH
- adamantan analogy a deriváty chemie MeSH
- aromatické hydroxylasy chemie ultrastruktura MeSH
- chemické modely MeSH
- financování organizované MeSH
- fluorescenční mikroskopie metody MeSH
- fotochemie metody MeSH
- konformace proteinů MeSH
- molekulární modely MeSH
- počítačová simulace MeSH
- substrátová specifita MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
Haloalkane dehalogenases catalyze the hydrolysis of halogen-carbon bonds in organic halogenated compounds and as such are of great utility as biocatalysts. The crystal structures of the haloalkane dehalogenase DhlA from the bacterium from Xanthobacter autotrophicus GJ10, specifically adapted for the conversion of the small 1,2-dichloroethane (DCE) molecule, display the smallest catalytic site (110 Å3) within this enzyme family. However, during a substrate-specificity screening, we noted that DhlA can catalyze the conversion of far bulkier substrates, such as the 4-(bromomethyl)-6,7-dimethoxy-coumarin (220 Å3). This large substrate cannot bind to DhlA without conformational alterations. These conformational changes have been previously inferred from kinetic analysis, but their structural basis has not been understood. Using molecular dynamic simulations, we demonstrate here the intrinsic flexibility of part of the cap domain that allows DhlA to accommodate bulky substrates. The simulations displayed two routes for transport of substrates to the active site, one of which requires the conformational change and is likely the route for bulky substrates. These results provide insights into the structure-dynamics function relationships in enzymes with deeply buried active sites. Moreover, understanding the structural basis for the molecular adaptation of DhlA to 1,2-dichloroethane introduced into the biosphere during the industrial revolution provides a valuable lesson in enzyme design by nature.
- MeSH
- ethylendichloridy metabolismus MeSH
- halogenace MeSH
- hydrolasy chemie metabolismus MeSH
- katalytická doména MeSH
- kinetika MeSH
- konformace proteinů MeSH
- krystalografie rentgenová MeSH
- kumariny chemie metabolismus MeSH
- metylace MeSH
- simulace molekulární dynamiky MeSH
- simulace molekulového dockingu MeSH
- substrátová specifita MeSH
- Xanthobacter chemie enzymologie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Substrate specificity is a key feature of enzymes determining their applicability in biomaterials and biotechnologies. Experimental testing of activities with novel substrates is a time-consuming and inefficient process, typically resulting in many failures. Here, we present an experimentally validated in silico method for the discovery of novel substrates of enzymes with a known reaction mechanism. The method was developed for a model system of biotechnologically relevant enzymes, haloalkane dehalogenases. On the basis of the parametrization of six different haloalkane dehalogenases with 30 halogenated substrates, mechanism-based geometric criteria for reactivity approximation were defined. These criteria were subsequently applied to the previously experimentally uncharacterized haloalkane dehalogenase DmmA. The enzyme was computationally screened against 41,366 compounds, yielding 548 structurally unique compounds as potential substrates. Eight out of 16 experimentally tested top-ranking compounds were active with DmmA, indicating a 50% success rate for the prediction of substrates. The remaining eight compounds were able to bind to the active site and inhibit enzymatic activity. These results confirmed good applicability of the method for prioritizing active compounds-true substrates and binders-for experimental testing. All validated substrates were large compounds often containing polyaromatic moieties, which have never before been considered as potential substrates for this enzyme family. Whereas four of these novel substrates were specific to DmmA, two substrates showed activity with three other tested haloalkane dehalogenases, i.e., DhaA, DbjA, and LinB. Additional validation of the developed screening strategy with the data set of over 200 known substrates of Candida antarctica lipase B confirmed its applicability for the identification of novel substrates of other biotechnologically relevant enzymes with an available tertiary structure and known reaction mechanism.
- MeSH
- chemické databáze MeSH
- fungální proteiny chemie metabolismus MeSH
- hydrolasy chemie genetika metabolismus MeSH
- lipasa chemie metabolismus MeSH
- počítačová simulace MeSH
- preklinické hodnocení léčiv metody MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- simulace molekulového dockingu MeSH
- substrátová specifita MeSH
- výpočetní biologie metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
