Additional file 1 of Molecular bases for strong phenotypic effects of single synonymous codon substitutions in the E. coli ccdB toxin gene

Additional file 1

Chemoselective Cyclopropanation over Carbene Y–H Insertion Catalyzed by an Engineered Carbene Transferase

Hemoproteins have recently emerged as promising biocatalysts for promoting a variety of carbene transfer reactions including cyclopropanation and Y–H insertion (Y = N, S, Si, B). For these and synthetic carbene transfer catalysts alike, achieving high chemoselectivity toward cyclopropanation in olefin substrates bearing unprotected Y–H groups has proven remarkably challenging due to competition from the more facile carbene Y–H insertion reaction. In this report, we describe the development of a novel artificial metalloenzyme based on an engineered myoglobin incorporating a serine-ligated Co-porphyrin cofactor that is capable of offering high selectivity toward olefin cyclopropanation over N–H and Si–H insertion. Intramolecular competition experiments revealed a distinct and dramatically altered chemoselectivity of the Mb­(H64V,V68A,H93S)­[Co­(ppIX)] variant in carbene transfer reactions compared to myoglobin-based variants containing the native histidine-ligated heme cofactor or other metal/proximal ligand substitutions. These studies highlight the functional plasticity of myoglobin as a “carbene transferase” and illustrate how modulation of the cofactor environment within this metalloprotein scaffold represents a valuable strategy for accessing carbene transfer reactivity not exhibited by naturally occurring hemoproteins or transition metal catalysts

Mechanisms of Electron Transfer Rate Modulations in Cytochrome P450 BM3

Bacterial cytochromes P450 BM3 (CYP450 BM3) catalyze reactions of industrial importance. Despite many successful biotransformations, robust (re)­design for novel applications remains challenging. Rational design and evolutionary approaches are not always successful, highlighting a lack of complete understanding of the mechanisms of electron transfer (ET) modulations. Thus, the full potential of CYP450 reactions remains under-exploited. In this work, we report the first molecular dynamics (MD)-based explicit prediction of BM3 ET parameters (reorganization energies; λ and ET free energies; ΔG°), and log ET rates (log kET) using the Marcus theory. Overall, the calculated ET rates for the BM3 wild-type (WT), mutants (F393 and L86), ligand-bound state, and ion concentrations agree well with experimental data. In ligand-free (LF) BM3, mutations modulate kET via ET ΔG°. Simulations show that the experimental ET rate enhancement is due to increased driving force (more negative ΔG°) upon ligation. This increase is related to the protein reorganization required to accommodate the ligand in the binding pocket rather than binding interactions with the ligand. Our methodology (CYPWare 1.0) automates all the stages of the MD simulation step-up, energy calculations, and estimation of ET parameters. CYPWare 1.0 and this work thus represent an important advancement in the CYP450 ET rate predictions, which has the potential to guide the redesign of ET enzymes. This program and a Web tool are available on GitHub for academic research

Sequence alignment of PON1 sequences from other organisms with common homology to hPON1.

Alignment was performed using online ClustalW software (www.ebi.ac.uk/Tools/msa/clustalow). Abbreviations and NCBI accession codes are in parenthesis: h-PON1192Q isoform (h-PON1192Q; Accession code NP_000437.3), Oryctolagus cuniculus/rabbit (rPON1; NP_001075766.1), Sus scrofa/Pig (pPON1; NP_001090984.2), Macaca mulatta/monkey (moPON1; XP 001095992.1) Xenopus Silurana tropicalis/ Xenopus (xPON1; NP 001006848.1), Mus musculus/mouse (mouPON1; NP 035264.2), Cricetulus griseus/ Chinese hamster (CgPON1; XP_003506695) and Heterocephalus glaber/ Naked mole rat (nPON1; EHA98623.1). PON sequences from lower organisms such as Caenorhabditis elegans (cPON1; NP_491306), Maribactersp HTCC2170 (mPON1; YP_003862768) and Leptospira interrogans (lPON1; NP_710580) are aligned with human PON1 sequence. Only partial sequences of these proteins are shown. The important residues are highlighted manually using Jalview software. Sequences spanning the highly conserved residues (E53, D54, H115, H134, N168, D183 and H184 of h-PON1) shown are highlighted with different colors and marked with red asterisks. The amino acids of other sequences aligned with position 192 of h-PON1 are shown in black box and are marked with black asterisks.</p

Synthesis and Biological Evaluation of Novel Bisbenzimidazoles as Escherichia coli Topoisomerase IA Inhibitors and Potential Antibacterial Agents

Novel bisbenzimidazole inhibitors of bacterial type IA topoisomerase are of interest for the development of new antibacterial agents that are impacted by target-mediated cross resistance with fluoroquinolones. The present study demonstrates the successful synthesis and evaluation of bisbenzimidazole analogues as Escherichia coli topoisomerase IA inhibitors. 5-(4-Propylpiperazin-1-yl)-2-[2′-(4-ethoxyphenyl)-5′-benzimidazolyl]­benzimidazole (12b) showed significant relaxation inhibition activity against EcTopo 1A (IC50 = 2 ± 0.005 μM) and a tendency to chelate metal ion. Interestingly, these compounds did not show significant inhibition of E. coli DNA gyrase and hTop 1 even up to 100 μM. Compound 12b has shown lowest MIC against E. coli strains among 24 compounds evaluated. The binding affinity constant and binding free energy of 12b with EcTopo 1A was observed 6.8 × 106 M–1 and −10.84 kcal mol–1 from isothermal titration calorimetry (ITC), respectively. In vivo mouse systemic infection and neutropenic thigh model experimental results confirmed the therapeutic efficacy of 12b, suggesting further development of this class of compounds as antibacterial agents

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

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

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

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