Structure-based redesign of the bacterial prolidase active-site pocket for efficient enhancement of methyl-parathion hydrolysis

2021 ◽  
Author(s):  
Jian Yang ◽  
Yunzhu Xiao ◽  
Yu Liu ◽  
Ru Li ◽  
Lijuan Long
Keyword(s):  
Active Site ◽  
Methyl Parathion ◽  
Rapid Evolution ◽  
Active Site Pocket ◽  
Molecular Determinants

The recruitment and rapid evolution of promiscuous catalysis provide insights into the precise mechanisms underlying enzyme repurposing and help understand molecular determinants of the remarkable adaptability of proteins. Starting from...

scholarly journals Specificity and mechanism of carbohydrate demethylation by cytochrome P450 monooxygenases

2018 ◽  
Vol 475 (23) ◽  
pp. 3875-3886 ◽  
Author(s):  
Craig S. Robb ◽  
Lukas Reisky ◽  
Uwe T. Bornscheuer ◽  
Jan-Hendrik Hehemann
Keyword(s):  
Cytochrome P450 ◽  
Active Site ◽  
Substrate Recognition ◽  
High Energy ◽  
Cytochrome P450 Monooxygenases ◽  
P450 Monooxygenases ◽  
High Energy Barrier ◽  
Molecular Determinants ◽  
Cytochrome P450 Family ◽  
Carbohydrate Ligand

Degradation of carbohydrates by bacteria represents a key step in energy metabolism that can be inhibited by methylated sugars. Removal of methyl groups, which is critical for further processing, poses a biocatalytic challenge because enzymes need to overcome a high energy barrier. Our structural and computational analysis revealed how a member of the cytochrome P450 family evolved to oxidize a carbohydrate ligand. Using structural biology, we ascertained the molecular determinants of substrate specificity and revealed a highly specialized active site complementary to the substrate chemistry. Invariance of the residues involved in substrate recognition across the subfamily suggests that they are critical for enzyme function and when mutated, the enzyme lost substrate recognition. The structure of a carbohydrate-active P450 adds mechanistic insight into monooxygenase action on a methylated monosaccharide and reveals the broad conservation of the active site machinery across the subfamily.

P4-286 The C-terminal PAL motif in presenilins constitutes part of the gamma-secretase active site pocket

2004 ◽  
Vol 25 ◽  
pp. S556-S557
Author(s):  
Jun Wang ◽  
Dirk Beher ◽  
Mark S. Shearman ◽  
Alison Goate
Keyword(s):  
Active Site ◽  
Gamma Secretase ◽  
Active Site Pocket

scholarly journals Engineered Factor Xa Variants Retain Procoagulant Activity Independent of Direct Factor Xa-Inhibitors

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 126-126
Author(s):  
Daniël Verhoef ◽  
Mark Schreuder ◽  
Ka Lei Cheung ◽  
Pieter H. Reitsma ◽  
Mettine H.A. Bos
Keyword(s):  
Active Site ◽  
Thrombin Generation ◽  
Activated Protein C ◽  
Factor Xa ◽  
Hydrophobic Interaction Chromatography ◽  
Size Exclusion ◽  
Bleeding Events ◽  
Surgical Interventions ◽  
Anionic Phospholipids ◽  
Active Site Pocket

Abstract The venom of the Australian Elapid snake Pseudonaja textilis contains a prothrombin-activating complex that consists of factor Xa (FXa) and factor Va (FVa) homologs which are evolutionary adapted to derail the hemostatic system of its prey, leading to runaway coagulation. These adaptations include functional resistance to inactivation by the main inhibitors of coagulation, antithrombin and activated protein C. Further studies revealed that venom FXa, unlike other FXa species, is also resistant to inhibition by direct oral FXa-inhibitors such as rivaroxaban and apixaban (Ki >1000 nM for venom FXa vs. 1 nM for human FXa). Accordingly, venom FXa is able to support thrombin generation (TG) in FX-depleted plasma spiked with pharmacological concentrations (0.4-2 μM) of these FXa-inhibitors. While human FXa-initiated TG resulted in a 8-fold prolonged lag time and a 70% reduced thrombin peak, those parameters were within normal range in venom FXa-triggered TG. Venom FX homologs produced by Elapid snakes comprise a heterogeneous insertion between His91-Tyr99 (chymotrypsin numbering) in the serine protease domain. A recent crystal structure of one of these homologs shows that this insertion is in close proximity of the active site pocket. In contrast, P. textilis liver-derived plasma FX, which, when activated, is fully inhibited by the FXa-inhibitors (Ki10 nM), lacks this structural feature. We investigated whether the His91-Tyr99 insertion is at the basis of the reduced sensitivity of venom FXa towards FXa-inhibitors. To do so, we constructed and stably expressed human-snake FX chimeras (FX-A, -B, -C) that incorporate His91-Tyr99 insertions from three venom FXa homologs. The chimeric FX variants were purified by successive ion-exchange and hydrophobic interaction chromatography steps, and FXa was generated following RVV-X-activation and size-exclusion chromatography. Evaluation of the kinetic parameters of prothrombin conversion in the presence of saturating amounts of FVa and anionic phospholipids revealed that the chimeric FXa variants exhibit an up to ~4-fold enhanced affinity for prothrombin (Km 0.11-0.29 μM) as compared to recombinant human FXa (rhFXa; Km 0.41 μM). The rate of prothrombin activation was 3-10-fold reduced (kcat 118-370 min-1 vs. 1243 min-1 for rhFXa), which may be indicative of a modified active site conformation. Consistent with this, the rate of chimeric FXa inhibition by antithrombin was impaired (kapp 0.12-0.95 x 103 M-1 s-1 vs. 4.07 x 103 M-1 s-1 for rhFXa). Furthermore, the variant that was most poorly inhibited by antithrombin (variant A) also exhibited the lowest catalytic rate of prothrombin conversion and vice versa (variant C). Conversely, apixaban or edoxaban inhibition of the FXa variants assembled into prothrombinase led to the highest Ki for chimeric variant C (2.3 or 0.3 µM), followed by variants B (1.4 or 0.2 µM), and A (0.2 or 0.006 µM) compared to rhFXa (0.004 or 0.0005 µM). Evaluation of the inhibition of uncomplexed FXa variants employing peptidyl substrate conversion revealed a similar decrease in sensitivity to the FXa-inhibitors (≤500-fold). These data suggest that insertion of the snake venom His91-Tyr99 regions indeed results in impaired engagement of the FXa active site pocket. We next assessed whether chimeric variant C, which is most resistant to inhibition by the direct FXa-inhibitors, is able to restore thrombin generation in a plasma system in the presence of apixaban or edoxaban. While rhFXa-triggered (5 nM) thrombin formation in FX-depleted plasma was inhibited by 2 μM apixaban, initiation with FXa-C (5 nM) resulted in normal thrombin generation parameters (peak thrombin 98%). In addition, the zymogen form of variant C also supported tissue factor-initiated (2 pM) thrombin generation in FX-depleted plasma with inhibitor concentrations up to 6 μM (apixaban) or 2 μM (edoxaban). Under these conditions, little if any thrombin was formed with rhFX present (peak thrombin 5%). We obtained similar results when performing these experiments in normal pooled plasma. Taken together, these results show that chimeric FX is able to restore hemostasis in plasma inhibited by the direct FXa-inhibitors, both in the zymogen as well as protease state. As such, these variants have the potential to serve as rescue therapeutic agents to overcome the effect of FXa-inhibitors in case of potential life-threatening bleeding events or emergency surgical interventions. Disclosures Bos: Bayer Hemophilia Awards: Research Funding.

scholarly journals Characterization of a factor IX variant with a glycine207 to glutamic acid mutation

Blood ◽  
1994 ◽  
Vol 84 (6) ◽  
pp. 1866-1873 ◽  
Author(s):  
SW Lin ◽  
CN Lin ◽  
N Hamaguchi ◽  
KJ Smith ◽  
MC Shen
Keyword(s):  
Glutamic Acid ◽  
Heavy Chain ◽  
Active Site ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Intrinsic Fluorescence ◽  
Factor Ix ◽  
Active Site Pocket ◽  
Factor Ixa ◽  
Factor Xia

Factor IXTaipei9 is a factor IX variant from a hemophilia B patient with reduced levels of circulating protein molecules (cross-reacting material reduced, CRM). This variant contained a glycine (Gly) to glutamic acid (Glu) substitution at the 207th codon of mature factor IX. The functional consequences of the Gly-->Glu mutation in factor IXTaipei9 (IXG207E) were characterized in this study. Plasma-derived IXG207E exhibited a mobility similar to that of normal factor IX on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its specific activity was estimated to be 3.5% that of the purified normal factor IX in a one-stage partial thromboplastin time assay (aPTT). Cleavage of factor IXG207E by factor XIa or factor VIIa-tissue factor complex appeared to be normal. When the calcium-dependent conformational change was examined by monitoring quenching of intrinsic fluorescence, both normal factor IX and IXG207E exhibited equivalent intrinsic fluorescence quenching. Activated factor IXG207E (IXaG207E) also binds antithrombin III equally as well as normal factor IXa. However, aberrant binding of the active site probe p-aminobenzamidine was observed for factor XIa-activated factor IXG207E, indicating that the active site pocket of the heavy chain of factor IXaG207E was abnormal. Moreover, the rate of activation of factor X by factor IXaG207E, as measured in a purified system using chromogenic substrates, was estimated to be 1/40 of that of normal factor IXa. A computer-modeled heavy-chain structure of factor IXa predicts a hydrophobic environment surrounding Gly-207 and this Gly forms a hydrogen bound to the active site serine-365. The molecular mechanism of the Gly-->Glu mutation in factor IXTaipei9 might result in the alteration of the microenvironment of the active site pocket which renders the active site serine-365 inaccessible to its substrate.

scholarly journals Characterization of a factor IX variant with a glycine207 to glutamic acid mutation

Blood ◽  
1994 ◽  
Vol 84 (6) ◽  
pp. 1866-1873 ◽  
Author(s):  
SW Lin ◽  
CN Lin ◽  
N Hamaguchi ◽  
KJ Smith ◽  
MC Shen
Keyword(s):  
Glutamic Acid ◽  
Heavy Chain ◽  
Active Site ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Intrinsic Fluorescence ◽  
Factor Ix ◽  
Active Site Pocket ◽  
Factor Ixa ◽  
Factor Xia

Abstract Factor IXTaipei9 is a factor IX variant from a hemophilia B patient with reduced levels of circulating protein molecules (cross-reacting material reduced, CRM). This variant contained a glycine (Gly) to glutamic acid (Glu) substitution at the 207th codon of mature factor IX. The functional consequences of the Gly-->Glu mutation in factor IXTaipei9 (IXG207E) were characterized in this study. Plasma-derived IXG207E exhibited a mobility similar to that of normal factor IX on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its specific activity was estimated to be 3.5% that of the purified normal factor IX in a one-stage partial thromboplastin time assay (aPTT). Cleavage of factor IXG207E by factor XIa or factor VIIa-tissue factor complex appeared to be normal. When the calcium-dependent conformational change was examined by monitoring quenching of intrinsic fluorescence, both normal factor IX and IXG207E exhibited equivalent intrinsic fluorescence quenching. Activated factor IXG207E (IXaG207E) also binds antithrombin III equally as well as normal factor IXa. However, aberrant binding of the active site probe p-aminobenzamidine was observed for factor XIa-activated factor IXG207E, indicating that the active site pocket of the heavy chain of factor IXaG207E was abnormal. Moreover, the rate of activation of factor X by factor IXaG207E, as measured in a purified system using chromogenic substrates, was estimated to be 1/40 of that of normal factor IXa. A computer-modeled heavy-chain structure of factor IXa predicts a hydrophobic environment surrounding Gly-207 and this Gly forms a hydrogen bound to the active site serine-365. The molecular mechanism of the Gly-->Glu mutation in factor IXTaipei9 might result in the alteration of the microenvironment of the active site pocket which renders the active site serine-365 inaccessible to its substrate.

scholarly journals DOCKING ANTIOXIDANT ACTIVITY ON HYDROXY (DIPHENYL) ACETICACID AND ITS DERIVATIVES

2017 ◽  
Vol 10 (7) ◽  
pp. 263 ◽  
Author(s):  
Sudha Rajendran ◽  
Nithya G ◽  
Brindha Devi P ◽  
Charles C Kanakam
Keyword(s):  
Antioxidant Activity ◽  
Active Site ◽  
Data Bank ◽  
Docking Study ◽  
Protein Crystal ◽  
Molecular Docking Study ◽  
Ligand Interaction ◽  
Active Site Pocket ◽  
Study Results

Objectives: The antioxidant activity of the synthesized compounds along with the standard compound for comparison is reported. There is comparison of binding analysis and the ligand interaction of the compound. Methods: The protein crystal structure complexed with 4-methyl-6-[2-(5-morpholin-4-ylpyridin-3-yl)ethyl]pyridin-2-amine inhibitor was selected from Protein Data Bank (5FVP) for our study. Results: The docking studies and structure-activity relationship reveals that the compound 2’-chloro-4-methoxy-3nitro benzilic acid after three different docking strategies reveals that the score was found to be higher compared with others.Conclusion: Based on the in vitro antioxidant results, the compounds synthesized were investigated for the molecular docking study to identify the amino acid interactions in the active site pocket of nitric oxide synthase enzyme. Based on the docking score results, all the compounds were oriented toward the active site pocket occupied by the cocrystallized ligand

scholarly journals Isolation and sequence analysis of serine protease cDNAs from mouse cytolytic T lymphocytes.

1988 ◽  
Vol 168 (5) ◽  
pp. 1839-1854 ◽  
Author(s):  
B S Kwon ◽  
D Kestler ◽  
E Lee ◽  
M Wakulchik ◽  
J D Young
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
T Lymphocytes ◽  
Serine Protease ◽  
Active Site ◽  
Serine Proteases ◽  
Cdna Clones ◽  
Lacz Gene ◽  
Cytolytic T Lymphocytes ◽  
Active Site Pocket

Three new cDNA clones (designated MCSP-1, MCSP-2, and MCSP-3) encoding mouse serine proteases were isolated from cloned cytolytic T lymphocytes (CTL) by a modified differential screening procedure. The putative mature proteins of MCSP-2 and MCSP-3 are each composed of 228 amino acids with molecular weights of 25,477 and 25,360, respectively. NH2-terminal amino acids of MCSP-2- and MCSP-3-predicted proteins were identical to those reported for granzyme E and F, respectively. The third species, MCSP-1, was closely related to the two other cDNA species but approximately 30 amino acids equivalents of the NH2-terminal portion of the cDNA were not cloned. The amino acids forming the active sites of serine proteases were well conserved among the three predicted proteins. The active site pocket residue positioned six residues before the active-site Ser184 is alanine in MCSP-1, threonine in MCSP-2, and serine in MCSP-3, indicating that both MCSP-2 and MCSP-3 may have chymotrypsin-like specificity. There are three potential asparagine-linked glycosylation sites in MCSP-1 and MCSP-3, and four in MCSP-2-deduced amino acid sequences. Amino acid comparison of MCSP-1 with four other reported serine proteases whose active site pocket residue is alanine revealed that MCSP-1 was substantially different from the other molecules, indicating that MCSP-1 may be a new member of mouse T cell serine protease family. Antibodies made against a MCSP-1 lacZ gene fusion protein stain granules of CTL and react on immunoblots with two distinct granule protein bands of 29 and 35-40 kD. Only the 35-kD species labels with [3H]DFP. Since a protease cascade may play a key role in cytolytic lymphocyte activation, our isolation of cDNAs representative of unique serine esterases should help to investigate such a cascade process.

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