Predicting substrate selectivity between UGT1A9 and UGT1A10 using molecular modelling and molecular dynamics approach

<div><p>Uridine 5′-diphospho-glucuronosyltransferase-1A9 (UGT1A9) expressed in the liver, shows good sequence identity with UGT1A10, expressed in the intestine. Both uridine 5′-diphospho-glucuronosyltransferase (UGT) isoforms show comprehensive overlapping substrate selectivity but there are differences in stereoselectivity, regiospecificity and rate of glucuronidation of the substrates. Multiple sequence alignment analyses of UGT1A9 and UGT1A10 showed that 13% of the residues in N-terminal domain (NTD) are non-identical between them. Herein, authors attempted homology modelling of UGT1A9 and UGT1A10 and validation using software tools and reported mutagenic studies. A molecular docking study of the known substrates is performed on UGT1A9 and UGT1A10 homology models. The non-identical N-terminal residues ranging from 111 to 117 in UGT1A9 and UGT1A10 were identified to play a central role in the substrate selectivity. However, substrate binding is performed by Ser111, Gly115 and Leu117 in UGT1A10 and Gly111, Asp115 and Phe117 in UGT1A9. This study reports new residues in NTD, showing interaction with uridine 5′-diphospho-glucuronic acid which binds with C-terminal domain. Further, molecular dynamics simulations were carried out to study the role of non-identical residues in substrate identification. The study demonstrates the folding of the UGT enzyme, particularly, helix-loop-helix transition and movement of Nα3-2 helix, in response to substrate and co-substrate binding.</p></div

3D-QSAR and molecular docking analysis of (4-piperidinyl)-piperazines as acetyl-CoA carboxylases inhibitors

Acetyl-CoA carboxylase (ACC) is a crucial metabolic enzyme, which plays a vital role in fatty acid metabolism and obesity induced type 2 diabetes. Herein, we have performed 3D-QSAR and molecular docking analysis on a novel series of (4-piperidinyl)-piperazines to design potent ACC inhibitors. This study correlates the ACC inhibitory activities of 68 (4-piperidinyl)-piperazine derivatives with several stereo-chemical parameters representing steric, electrostatic, hydrophobic, hydrogen bond donor and acceptor fields. The CoMFA and CoMSIA models exhibited excellent rncv2 values of 0.974 and 0.985, and rcv2 values of 0.671 and 0.693, respectively. CoMFA predicted rpred2 of 0.910 and CoMSIA predicted rpred2 of 0.963 showed that the predicted values were in good agreement with experimental values. Glide5.5 program was used to explore the binding mode of inhibitors inside the active site of ACC. We have accordingly designed novel ACC inhibitors by utilising the LeapFrog and predicted with excellent inhibitory activity in the developed models

Toward Understanding the Catalytic Mechanism of Human Paraoxonase 1: Site-Specific Mutagenesis at Position 192.

Human paraoxonase 1 (h-PON1) is a serum enzyme that can hydrolyze a variety of substrates. The enzyme exhibits anti-inflammatory, anti-oxidative, anti-atherogenic, anti-diabetic, anti-microbial and organophosphate-hydrolyzing activities. Thus, h-PON1 is a strong candidate for the development of therapeutic intervention against a variety conditions in human. However, the crystal structure of h-PON1 is not solved and the molecular details of how the enzyme hydrolyzes different substrates are not clear yet. Understanding the catalytic mechanism(s) of h-PON1 is important in developing the enzyme for therapeutic use. Literature suggests that R/Q polymorphism at position 192 in h-PON1 dramatically modulates the substrate specificity of the enzyme. In order to understand the role of the amino acid residue at position 192 of h-PON1 in its various hydrolytic activities, site-specific mutagenesis at position 192 was done in this study. The mutant enzymes were produced using Escherichia coli expression system and their hydrolytic activities were compared against a panel of substrates. Molecular dynamics simulation studies were employed on selected recombinant h-PON1 (rh-PON1) mutants to understand the effect of amino acid substitutions at position 192 on the structural features of the active site of the enzyme. Our results suggest that, depending on the type of substrate, presence of a particular amino acid residue at position 192 differentially alters the micro-environment of the active site of the enzyme resulting in the engagement of different subsets of amino acid residues in the binding and the processing of substrates. The result advances our understanding of the catalytic mechanism of h-PON1

Comparison of the distance between catalytic calcium and the ligand in the protein ligand complex.

<p>The position of the ligand substrate in the active site was monitored by plotting the distance between catalytic calcium and the centre of the mass of substrate. (A-D) depict the distance between the catalytic calcium of the rh-PON1_(wt) (<b>—</b>), rh-PON1_(H115W,R192) (<b>—</b>),rh-PON1_{(H115W,R192K)} (<b>—</b>), and rh-PON1_{(H115W,R192I)} (<b>—</b>) proteins and the bound ligands over the course of the MD simulations. The ligands used were panel (A)–Pxn; panel (B)—Pha, panel (C)—<i>δ</i>-val, and panel (D)—TBBL.</p

Molecular surface representation of the active site of rh-PON1_{(H115W,R192I)} protein containing <i>δ</i>-val (A) and TBBL (B) ligands.

<p>The molecular surface of the active site of the protein is shown in grey colour and the active site residues W115, D183, H184, I178, D269 and the catalytic calcium are indicated by red, blue, green, yellow, cyan and magenta colours, respectively. <i>δ</i>-val and TBBL are shown in stick model and colour by atom type (red—oxygen; yellow—sulphur; orange—carbon). Note that in the rh-PON1_{(H115W,R192I)} containing TBBL <b>(B)</b>, the oxygen atom of TBBL is oriented towards the carboxyl oxygen of D183 (blue).</p

H-bonding network in the active site of the TBBL-bound rh-PON1 protein complexes.

<p>The amino acid residues of the rh-PON1 proteins are shown in stick format and colour by atom type (<b>red</b>–oxygen; <b>blue</b>–nitrogen). The yellow broken lines show H-bonding interaction between the amino acid residues. Catalytic calcium is represented by yellow spheres. <b>Panels (A-D)</b> depict rh-PON1_(wt), rh-PON1_(H115W,R192), rh-PON1_{(H115W,R192K)}, and rh-PON1_{(H115W,R192I)} proteins. Differential H-bonding network around position 192 was observed in these proteins.</p

Lactone-hydrolyzing activity of the rh-PON1 enzymes.

<p>(A) and (B) compare the TBBL- and <i>δ</i>-val-hydrolyzing activity of the rh-PON1 enzymes, respectively. The TBBL-hydrolyzing activity was determined using Ellman-based colorimetric assay. Equal amounts of the rh-PON1 enzymes were separately incubated with 0.5 mM TBBL in the activity buffer containing 0.3 mM DTNB and the hydrolysis of TBBL was monitored at 412 nm. The <i>δ</i>-val-hydrolyzing activity of the rh-PON1 enzymes was determined by pH-indicator assay. The enzymes were incubated with 1 mM (in 50 mM bicine buffer pH 8.3, 1 mM CaCl₂) and the hydrolysis of <i>δ</i>-val was monitored at 577 nm using <i>m</i>-cresol purple as the indicator. The hydrolytic activity of rh-PON1_(wt) was taken 100% and the percentage activities of all the rh-PON1 mutants were calculated. Enzymatic assays were performed in duplicate. Various mutants were named with single letter code representing the particular amino acid at position 192. <b>Legends:</b> same as in the legends of Fig 3.</p

OP-hydrolyzing activity of the rh-PON1 enzymes.

<p>(A) compares the Pxn-hydrolyzing activity of the recombinant enzymes. The Pxn-hydrolyzing activity was determined using direct assay. Equal amounts of the rh-PON1 enzymes were incubated with 1 mM paraoxon in the activity buffer (50 mM Tris-HCl, pH 8.0 and containing 1 mM CaCl₂) and the hydrolysis of Pxn was recorded at 405 nm. (B) and (C) depict the CPO- and DFP- hydrolyzing activity of the enzymes, determined by using an indirect AChE-inhibition assay, as described in the Experimental procedure. The concentration of CPO and DFP used were 75 μM and 200 μM (final concentration), respectively. The hydrolytic activity of rh-PON1_(wt) was taken 100% and the percentage activities of the rh-PON1 mutants were calculated. Enzymatic assays were performed in duplicate. Various mutants were named with single letter code representing the particular amino acid at position 192. <b>Legends:</b> wt, rh-PON1_(wt); K, rh-PON1_{(H115W,R192K)}; R, rh-PON1_(H115W,R192); Q, rh-PON1_{(H115W,R192Q);} N, rh-PON1_{(H115W,R192N)}; D, rh-PON1_{(H115W,R192D)}; E, rh-PON1_{(H115W,R192E);} S, rh-PON1_{(H115W,R192S);} T, rh-PON1_{(H115W,R192T)}; W, rh-PON1_{(H115W,R192W)}; Y, rh-PON1_{(H115W,R192Y);} F, rh-PON1_{(H115W,R192F);} L, rh-PON1_{(H115W,R192L)}; I, rh-PON1_{(H115W,R192I)}; V, rh-PON1_{(H115W,R192V);} P, rh-PON1_{(H115W,R192P);} G, rh-PON1_{(H115W,R192G)}; A, rh-PON1_{(H115W,R192A)}.</p

Kinetic parameters for hydrolysis of Pxn by rh-PON1 mutants.

<p>a,b,c- Data derived from Bajaj et al. (2014) Biochimie, 22: 210–214.</p