Inverting the Enantiopreference of Nitrilase‐Catalyzed Desymmetric Hydrolysis of Prochiral Dinitriles by Reshaping the Binding Pocket with a Mirror‐Image Strategy

Tervalent cations of the lanthanide (rare-earth) elements reversibly inhibit bacterial collagenase (clostridiopeptidase A; EC 3.4.24.3). Sm3+, whose ionic radius is closest to that of Ca2+, is the most effective inhibitor, completely suppressing clostridiopeptidase activity at a concentration of 100μm in the presence of 5mm-Ca2+. Er3+ and Lu3+, which both have ionic radii smaller than either Ca2+ or Sm3+, inhibit less efficiently, and La3+, which is slightly larger than Ca2+ or Sm3+, inhibits only weakly. These findings indicate a closely fitting, stereospecific, Ca2+-binding pocket in clostridiopeptidase, which excludes ions that are only slightly larger than Ca2+ [ionic radius 0.099nm (0.99 Ȧ)]. By contrast, trypsin, an enzyme whose activity does not depend on Ca2+, requires lanthanide concentrations 50–100-fold greater for inhibition. Furthermore, the relative efficiency of inhibition of trypsin by lanthanides increases as the lanthanide ions become smaller and the charge/volume ratio increases. At a concentration of 50μm, Sm3+ lowers the apparent Km for the hydrolysis of Pz-peptide by clostridiopeptidase from 5.4mm to 0.37mm and the apparent Vmax. from 0.29 Wünsch–Heidrich unit to 0.018 unit. Thus Sm3+ enhances the affinity of this enzyme for its substrate; inhibition of hydrolysis of Pz-peptide may result from the excessive stability of the enzyme–Sm3+–substrate complex. Inhibition by Sm3+ is competitive with regard to Ca2+. The apparent dissociation constant, Kd, of Ca2+ is 0.27mm, where the Ki for Sm3+ is 12μm. Clostridiopeptidase is more thermolabile in the absence of Ca2+. With Sm3+, thermoinactivation of the enzyme at 53°C or 60°C is initially accelerated, but then becomes retarded as heating continues. Lanthanide ions bind to gelatin and collagen. In so doing, they appear to protect these substrates from lysis by clostridiopeptidase through mechanisms additional to supplanting Ca2+ at its binding site on the enzyme. Collagen and gelatin sequester sufficient lanthanide ions to gain partial protection from clostridiopeptidase in the absence of an extraneous source of these inhibitors.

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The Structure of Sucrose-Soaked Levansucrase Crystals from Erwinia tasmaniensis reveals a Binding Pocket for Levanbiose

International Journal of Molecular Sciences ◽

10.3390/ijms21010083 ◽

2019 ◽

Vol 21 (1) ◽

pp. 83 ◽

Cited By ~ 4

Author(s):

Ivan Polsinelli ◽

Rosanna Caliandro ◽

Nicola Demitri ◽

Stefano Benini

Keyword(s):

Crystal Structure ◽

Potential Role ◽

Binding Pocket ◽

Chain Elongation ◽

High Yield ◽

Negative Bacterium ◽

Yield Production ◽

Wide Range ◽

Hydrolysis Of ◽

Erwinia Tasmaniensis

Given its potential role in the synthesis of novel prebiotics and applications in the pharmaceutical industry, a strong interest has developed in the enzyme levansucrase (LSC, EC 2.4.1.10). LSC catalyzes both the hydrolysis of sucrose (or sucroselike substrates) and the transfructosylation of a wide range of acceptors. LSC from the Gram-negative bacterium Erwinia tasmaniensis (EtLSC) is an interesting biocatalyst due to its high-yield production of fructooligosaccharides (FOSs). In order to learn more about the process of chain elongation, we obtained the crystal structure of EtLSC in complex with levanbiose (LBS). LBS is an FOS intermediate formed during the synthesis of longer-chain FOSs and levan. Analysis of the LBS binding pocket revealed that its structure was conserved in several related species. The binding pocket discovered in this crystal structure is an ideal target for future mutagenesis studies in order to understand its biological relevance and to engineer LSCs into tailored products.

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Substantially improving the enantioconvergence of PvEH1, a Phaseolus vulgaris epoxide hydrolase, towards m-chlorostyrene oxide by laboratory evolution

Microbial Cell Factories ◽

10.1186/s12934-019-1252-4 ◽

2019 ◽

Vol 18 (1) ◽

Cited By ~ 2

Author(s):

Xun-Cheng Zong ◽

Chuang Li ◽

Yao-Hui Xu ◽

Die Hu ◽

Bo-Chun Hu ◽

...

Keyword(s):

Phaseolus Vulgaris ◽

Epoxide Hydrolase ◽

Substrate Binding ◽

Binding Pocket ◽

Saturation Mutagenesis ◽

Substrate Binding Pocket ◽

Laboratory Evolution ◽

E Coli ◽

Enantioconvergent Hydrolysis ◽

Hydrolysis Of

Abstract Background Epoxide hydrolase can regioselectively catalyze the oxirane ring-opening hydrolysis of rac-epoxides producing the corresponding chiral diols. In our laboratory, a gene named pveh1 encoding an EH from Phaseolus vulgaris was cloned. Although the directed modification of PvEH1 was carried out, the mutant PvEH1Y3 showed a limited degree of enantioconvergence towards racemic (rac-) m-chlorostyrene oxide (mCSO). Results PvEH1 and PvEH1Y3 were combinatively subjected to laboratory evolution to further enhance the enantioconvergence of PvEH1Y3 towards rac-mCSO. Firstly, the substrate-binding pocket of PvEH1 was identified using a CAVER 3.0 software, and divided into three zones. After all residues in zones 1 and 3 were subjected to leucine scanning, two E. coli transformants, E. coli/pveh1Y149L and /pveh1P184L, were selected, by which rac-mCSO was transformed into (R)-m-chlorophenyl-1,2-ethanediol (mCPED) having 55.1% and 27.2% eep. Secondly, two saturation mutagenesis libraries, E. coli/pveh1Y149X and /pveh1P184X (X: any one of 20 residues) were created at sites Y149 and P184 of PvEH1. Among all transformants, both E. coli/pveh1Y149L (65.8% αS and 55.1% eep) and /pveh1P184W (66.6% αS and 59.8% eep) possessed the highest enantioconvergences. Finally, the combinatorial mutagenesis was conducted by replacements of both Y149L and P184W in PvEH1Y3, constructing E. coli/pveh1Y3Z2, whose αS reached 97.5%, higher than that (75.3%) of E. coli/pveh1Y3. In addition, the enantioconvergent hydrolysis of 20 mM rac-mCSO was performed by E. coli/pveh1Y3Z2, giving (R)-mCPED with 95.2% eep and 97.2% yield. Conclusions In summary, the enantioconvergence of PvEH1Y3Z2 was successfully improved by laboratory evolution, which was based on the study of substrate-binding pocket by leucine scanning. Our present work introduced an effective strategy for the directed modification of enantioconvergence of PvEH1.

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Highly Potent and Selective Ectonucleoside Triphosphate Diphosphohydrolase (ENTPDase1, 2, 3 and 8) Inhibitors Having 2-substituted-7- trifluoromethyl-thiadiazolopyrimidones Scaffold

Medicinal Chemistry ◽

10.2174/1573406415666190614095821 ◽

2020 ◽

Vol 16 (5) ◽

pp. 689-702 ◽

Cited By ~ 1

Author(s):

Saira Afzal ◽

Sumera Zaib ◽

Behzad Jafari ◽

Peter Langer ◽

Joanna Lecka ◽

...

Keyword(s):

Inhibitory Activity ◽

Binding Pocket ◽

Docking Studies ◽

Extracellular Nucleotides ◽

Nucleoside Triphosphate ◽

Selective Inhibitors ◽

Docking Analysis ◽

Inhibitory Potential ◽

Malachite Green Assay ◽

Hydrolysis Of

Background: The ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) terminate nucleotide signaling via the hydrolysis of extracellular nucleoside-5'-triphosphate and nucleoside- 5'-diphosphate, to nucleoside-5'-monophosphate and composed of eight Ca2+/Mg2+ dependent ectonucleotidases (NTPDase1-8). Extracellular nucleotides are involved in a variety of physiological mechanisms. However, they are rapidly inactivated by ectonucleotidases that are involved in the sequential removal of phosphate group from nucleotides with the release of inorganic phosphate and their respective nucleoside. Ectonucleoside triphosphate diphosphohydrolases (NTPDases) represent the key enzymes responsible for nucleotides hydrolysis and their overexpression has been related to certain pathological conditions. Therefore, the inhibitors of NTPDases are of particular importance in order to investigate their potential to treat various diseases e.g., cancer, ischemia and other disorders of the cardiovascular and immune system. Methods: Keeping in view the importance of NTPDase inhibitors, a series of thiadiazolopyrimidones were evaluated for their potential inhibitory activity towards NTPDases by the malachite green assay. Results: The results suggested that some of the compounds were found as non-selective inhibitors of isozyme of NTPDases, however, most of the compounds act as potent and selective inhibitors. In case of substituted amino derivatives (4c-m), the compounds 4m (IC50 = 1.13 ± 0.09 μM) and 4g (IC50 = 1.72 ± 0.08 μM) were found to be the most potent inhibitors of h-NTPDase1 and 2, respectively. Whereas, compound 4d showed the best inhibitory potential for both h-NTPDase3 (IC50 = 1.25 ± 0.06 μM) and h-NTPDase8 (0.21 ± 0.02 μM). Among 5a-t derivatives, compounds 5e (IC50 = 2.52 ± 0.15 μM), 5p (IC50 = 3.17 ± 0.05 μM), 5n (IC50 = 1.22 ± 0.06 μM) and 5b (IC50 = 0.35 ± 0.001 μM) were found to be the most potent inhibitors of h-NTPDase1, 2, 3 and 8, respectively. Interestingly, the inhibitory concentration values of above-mentioned inhibitors were several folds greater than suramin, a reference control. In order to determine the binding interactions, molecular docking studies of the most potent inhibitors were conducted into the homology models of NTPDases and the putative binding analysis further confirmed that selective and potent compounds bind deep inside the active pocket of the respective enzymes. Conclusions: The docking analysis proposed that the inhibitory activity correlates with the hydrogen bonds inside the binding pocket. Thus, these derivatives are of interest and may further be investigated for their importance in medicinal chemistry.

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Elucidation of the Molecular Basis for Arabinoxylan-Debranching Activity of a Thermostable Family GH62 α-l-Arabinofuranosidase from Streptomyces thermoviolaceus

Applied and Environmental Microbiology ◽

10.1128/aem.00685-14 ◽

2014 ◽

Vol 80 (17) ◽

pp. 5317-5329 ◽

Cited By ~ 28

Author(s):

Weijun Wang ◽

Galina Mai-Gisondi ◽

Peter J. Stogios ◽

Amrit Kaur ◽

Xiaohui Xu ◽

...

Keyword(s):

Hydrophobic Interactions ◽

Complex Structure ◽

Three Dimensional ◽

Binding Pocket ◽

Content Type ◽

Wheat Arabinoxylan ◽

Debranching Enzymes ◽

The Family ◽

Multiple Binding ◽

Hydrolysis Of

ABSTRACTXylan-debranching enzymes facilitate the complete hydrolysis of xylan and can be used to alter xylan chemistry. Here, the family GH62 α-l-arabinofuranosidase fromStreptomyces thermoviolaceus(SthAbf62A) was shown to have a half-life of 60 min at 60°C and the ability to cleave α-1,3l-arabinofuranose (l-Araf) from singly substituted xylopyranosyl (Xylp) backbone residues in wheat arabinoxylan; low levels of activity on arabinan as well as 4-nitrophenyl α-l-arabinofuranoside were also detected. After selective removal of α-1,3l-Arafsubstituents from disubstituted Xylpresidues present in wheat arabinoxylan, SthAbf62A could also cleave the remaining α-1,2l-Arafsubstituents, confirming the ability of SthAbf62A to remove α-l-Arafresidues that are (1→2) and (1→3) linked to monosubstituted β-d-Xylpsugars. Three-dimensional structures of SthAbf62A and its complex with xylotetraose andl-arabinose confirmed a five-bladed β-propeller fold and revealed a molecular Velcro in blade V between the β1 and β21 strands, a disulfide bond between Cys27 and Cys297, and a calcium ion coordinated in the central channel of the fold. The enzyme-arabinose complex structure further revealed a narrow and seemingly rigidl-arabinose binding pocket situated at the center of one side of the β propeller, which stabilized the arabinofuranosyl substituent through several hydrogen-bonding and hydrophobic interactions. The predicted catalytic amino acids were oriented toward this binding pocket, and the catalytic essentiality of Asp53 and Glu213 was confirmed by site-specific mutagenesis. Complex structures with xylotetraose revealed a shallow cleft for xylan backbone binding that is open at both ends and comprises multiple binding subsites above and flanking thel-arabinose binding pocket.

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Structural insight into the rearrangement of the switch I region in GTP-bound G12A K-Ras

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798317015418 ◽

2017 ◽

Vol 73 (12) ◽

pp. 970-984 ◽

Cited By ~ 13

Author(s):

Shenyuan Xu ◽

Brian N. Long ◽

Gabriel H. Boris ◽

Anqi Chen ◽

Shuisong Ni ◽

...

Keyword(s):

Transition State ◽

Binding Pocket ◽

High Energy ◽

Side Chain ◽

Conformation Change ◽

Gtp Hydrolysis ◽

Structural Insight ◽

Hydrolysis Of ◽

Insight Into ◽

Intrinsic Gtpase Activity

K-Ras, a molecular switch that regulates cell growth, apoptosis and metabolism, is activated when it undergoes a conformation change upon binding GTP and is deactivated following the hydrolysis of GTP to GDP. Hydrolysis of GTP in water is accelerated by coordination to K-Ras, where GTP adopts a high-energy conformation approaching the transition state. The G12A mutation reduces intrinsic K-Ras GTP hydrolysis by an unexplained mechanism. Here, crystal structures of G12A K-Ras in complex with GDP, GTP, GTPγS and GppNHp, and of Q61A K-Ras in complex with GDP, are reported. In the G12A K-Ras–GTP complex, the switch I region undergoes a significant reorganization such that the Tyr32 side chain points towards the GTP-binding pocket and forms a hydrogen bond to the GTP γ-phosphate, effectively stabilizing GTP in its precatalytic state, increasing the activation energy required to reach the transition state and contributing to the reduced intrinsic GTPase activity of G12A K-Ras mutants.

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Spiegelzymes: Sequence Specific Hydrolysis of L-RNA with Mirror Image Hammerhead Ribozymes and DNAzymes

PLoS ONE ◽

10.1371/journal.pone.0054741 ◽

2013 ◽

Vol 8 (1) ◽

pp. e54741 ◽

Cited By ~ 14

Author(s):

Eliza Wyszko ◽

Maciej Szymański ◽

Heinz Zeichhardt ◽

Florian Müller ◽

Jan Barciszewski ◽

...

Keyword(s):

Mirror Image ◽

Hammerhead Ribozymes ◽

Hydrolysis Of

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Functional convergence of structurally distinct thioesterases from cyanobacteria and plants involved in phylloquinone biosynthesis

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444913015771 ◽

2013 ◽

Vol 69 (10) ◽

pp. 1876-1888 ◽

Cited By ~ 5

Author(s):

Fabienne Furt ◽

William J. Allen ◽

Joshua R. Widhalm ◽

Peter Madzelan ◽

Robert C. Rizzo ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Active Site ◽

Active Sites ◽

Binding Pocket ◽

Flowering Plant ◽

Structural Basis ◽

Photosynthetic Organisms ◽

Functional Convergence ◽

Hotdog Fold ◽

Hydrolysis Of

The synthesis of phylloquinone (vitamin K1) in photosynthetic organisms requires a thioesterase that hydrolyzes 1,4-dihydroxy-2-naphthoyl-CoA (DHNA-CoA) to release 1,4-dihydroxy-2-naphthoate (DHNA). Cyanobacteria and plants contain distantly related hotdog-fold thioesterases that catalyze this reaction, although the structural basis of these convergent enzymatic activities is unknown. To investigate this, the crystal structures of hotdog-fold DHNA-CoA thioesterases from the cyanobacteriumSynechocystis(Slr0204) and the flowering plantArabidopsis thaliana(AtDHNAT1) were determined. These enzymes form distinct homotetramers and use different active sites to catalyze hydrolysis of DHNA-CoA, similar to the 4-hydroxybenzoyl-CoA (4-HBA-CoA) thioesterases fromPseudomonasandArthrobacter. Like the 4-HBA-CoA thioesterases, the DHNA-CoA thioesterases contain either an active-site aspartate (Slr0204) or glutamate (AtDHNAT1) that are predicted to be catalytically important. Computational modeling of the substrate-bound forms of both enzymes indicates the residues that are likely to be involved in substrate binding and catalysis. Both enzymes are selective for DHNA-CoA as a substrate, but this selectivity is achieved using divergent predicted binding strategies. The Slr0204 binding pocket is predominantly hydrophobic and closely conforms to DHNA, while that of AtDHNAT1 is more polar and solvent-exposed. Considered in light of the related 4-HBA-CoA thioesterases, these structures indicate that hotdog-fold thioesterases using either an active-site aspartate or glutamate diverged into distinct clades prior to the evolution of strong substrate specificity in these enzymes.

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Novel xylan degrading enzymes from polysaccharide utilizing loci of Prevotella copri DSM18205

10.1101/2020.12.10.419226 ◽

2020 ◽

Author(s):

Javier A. Linares-Pastén ◽

Johan Sebastian Hero ◽

José Horacio Pisa ◽

Cristina Teixeira ◽

Margareta Nyman ◽

...

Keyword(s):

3D Structure ◽

Extracellular Enzyme ◽

Binding Pocket ◽

Human Gastrointestinal Tract ◽

Intracellular Location ◽

Inflammatory Conditions ◽

Hydrolysis Products ◽

Encoding Genes ◽

Hydrolysis Of

AbstractPrevotella copri DSM18205 is a bacterium, classified under Bacteroidetes that can be found in the human gastrointestinal tract (GIT). The role of P. copri in the GIT is unclear, and elevated numbers of the microbe have been reported both in dietary fiber-induced improvement in glucose metabolism but also in conjunction with certain inflammatory conditions. These findings raised our interest in investigating the possibility of P. copri to grow on xylan, and identify the enzyme systems playing a role in digestion of xylan-based dietary fibers in P. copri, which currently are unexplored. Two xylan degrading polysaccharide utilizing loci (PUL10 and 15) were found in the genome, with three and eight GH-encoding genes, respectively. Three of the eight gene products were successfully produced in Escherichia coli: One monomeric two-domain extracellular enzyme from GH43 (subfamily 12, in PUL10, 60 kDa) and two dimeric single module enzymes from PUL15, one extracellular GH10 (41 kDa), and one intracellular GH43 subfamily 1 enzyme (37 kDa). The GH43_12 enzyme was hydrolysing arabinofuranose residues from different substrates, and a model of the 3D-structure revealed a single arabinose binding pocket. The GH10 (1) and GH43_1 are cleaving the xylan backbone. Hydrolysis products of GH10 (1) were DP2-4, and seven subsites (−3 to +4) were predicted in the 3D-model of the GH10 active site. GH43_1 mainly produced xylose (in line with its intracellular location). Based on our results we propose that in PUL15, GH10 (1) is an extracellular endo-1,4-β-xylanase, that hydrolyses mainly glucuronosylated xylan polymers to xylooligosaccharides (XOS); while, GH43_1 in the same PUL, is an intracellular β-xylosidase, catalysing complete hydrolysis of the XOS to xylose. In PUL10, the characterized GH43_12 is an arabinofuranosidase, with a role in degradation of arabinoxylan, catalysing removal of arabinose-residues on xylan polymers.

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