Biochemical Studies and Ligand-bound Structures of Biphenyl Dehydrogenase from Pandoraea pnomenusa Strain B-356 Reveal a Basis for Broad Specificity of the Enzyme

ABSTRACT: Biphenyl dehydrogenase, a member of short-chain dehydrogenase/reductase enzymes, catalyzes the second step of the biphenyl/polychlorinated biphenyls catabolic pathway in bacteria. To understand the molecular basis for the broad substrate specificity of Pandoraea pnomenusa strain B-356 biphenyl dehydrogenase (BphB(B-356)), the crystal structures of the apo-enzyme, the binary complex with NAD(+), and the ternary complexes with NAD(+)-2,3-dihydroxybiphenyl and NAD(+)-4,4'-dihydroxybiphenyl were determined at 2.2-, 2.5-, 2.4-, and 2.1-A resolutions, respectively. A crystal structure representing an intermediate state of the enzyme was also obtained in which the substrate binding loop was ordered as compared with the apo and binary forms but it was displaced significantly with respect to the ternary structures. These five structures reveal that the substrate binding loop is highly mobile and that its conformation changes during ligand binding, starting from a disorganized loop in the apo state to a well organized loop structure in the ligand-bound form. Conformational changes are induced during ligand binding; forming a well defined cavity to accommodate a wide variety of substrates. This explains the biochemical data that shows BphB(B-356) converts the dihydrodiol metabolites of 3,3'-dichlorobiphenyl, 2,4,4'-trichlorobiphenyl, and 2,6-dichlorobiphenyl to their respective dihydroxy metabolites. For the first time, a combination of structural, biochemical, and molecular docking studies of BphB(B-356) elucidate the unique ability of the enzyme to transform the cis-dihydrodiols of double meta-, para-, and ortho-substituted chlorobiphenyls

Structure-Function Studies of DNA Binding Domain of Response Regulator KdpE Reveals Equal Affinity Interactions at DNA Half-Sites

Expression of KdpFABC, a K+ pump that restores osmotic balance, is controlled by binding of the response regulator KdpE to a specific DNA sequence (kdpFABCBS) via the winged helix-turn-helix type DNA binding domain (KdpEDBD). Exploration of E. coli KdpEDBD and kdpFABCBS interaction resulted in the identification of two conserved, AT-rich 6 bp direct repeats that form half-sites. Despite binding to these half-sites, KdpEDBD was incapable of promoting gene expression in vivo. Structure-function studies guided by our 2.5 Å X-ray structure of KdpEDBD revealed the importance of residues R193 and R200 in the α-8 DNA recognition helix and T215 in the wing region for DNA binding. Mutation of these residues renders KdpE incapable of inducing expression of the kdpFABC operon. Detailed biophysical analysis of interactions using analytical ultracentrifugation revealed a 2∶1 stoichiometry of protein to DNA with dissociation constants of 200±100 and 350±100 nM at half-sites. Inactivation of one half-site does not influence binding at the other, indicating that KdpEDBD binds independently to the half-sites with approximately equal affinity and no discernable cooperativity. To our knowledge, these data are the first to describe in quantitative terms the binding at half-sites under equilibrium conditions for a member of the ubiquitous OmpR/PhoB family of proteins

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Isolation, purification, crystallization and preliminary crystallographic studies of chitinase from tamarind (Tamarindus indica) seeds

A 34 kDa chitinase from tamarind (T. indica) seeds was purified, crystallized and characterized using X-ray diffraction

Structure of KdpE_DBD.

<p><b>A.</b> A cartoon representation of a molecule showing the wHTH motif in progressive coloring; the rest is in gray. To maintain continuity with the structure of the N-terminal receiver domain of KdpE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030102#pone.0030102-ToroRoman1" target="_blank">[25]</a>, the β-strands and α-helices of KdpE_DBD are labeled starting with β-6 and α-6. The side chains shown in stick representation are residues R193 and R200 in α8 and T215 in β11 targeted for mutagenesis. N and C refer to the amino- and carboxyl- termini. <b>B.</b> Conservation of the sequence in the wHTH motif across members of the OmpR/PhoB family (upper panel) and between KdpE orthologs (lower panel) presented in logo format derived from multiple sequence alignments <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030102#pone.0030102-Crooks1" target="_blank">[61]</a>. The Y-axis represents sequence conservation in bits. The residues targeted for mutagenesis in KdpE are boxed, the triangles represent residues involved in base specific interactions in PhoB-DNA complex (PDB code: 1GXP), and the residue numbering is that of KdpE sequence. Shown below the logo representation are the sequences of the wHTH motif of KdpE and PhoB (upper panel) and that of KdpE in the lower panel. The gap in the lower panel represents a three residue insertion in few of the KdpE orthologs used in sequence alignment. The schematic of the secondary structure was derived from the structure of KdpE_DBD. <b>C.</b> Superposition of KdpE_DBD onto the structure of PhoB bound to DNA (PDB code: 1GXP). Only wHTH motifs of KdpE_DBD and chain A of PhoB in 1GXP and part of the DNA are shown. The coloring scheme: green, KdpE_DBD; purple, PhoB and yellow/orange, DNA strands. The following side chains of residues of PhoB (and in parenthesis equivalent residues in KdpE_DBD labeled in blue) are shown as sticks: T194 (Y191), V197 (I194), R201 (H198) and R219 (T217, not shown), R203 (R200) and T217 (T215) and D196 (R193). Residues T194, V197, R201 and R219 (that penetrates the minor groove is labeled in red) of PhoB have been shown to be form base specific interactions.</p

<i>Binding analysis of the half-sites of kdpFABC_BS</i>.

<p>SE analysis of binding of KdpE_DBD to S1 (<i>kdpFABC_BS—7</i>) (<b>A</b>)and S2 (<i>kdpFABC_BS</i>—<i>1</i>) (<b>B</b>) half-sites revealed a 1∶1 stoichiometry. Mixtures of KdpE_DBD and DNA were spun at 9,000 (•), 19,800 (□) and 34,000 (Δ) rpm. The <i>K_d</i>s obtained for KdpE_DBD binding at half-sites S1 was 350±100 nM and for S2 was 200±100 nM using a one site binding model (AB) in SEDPHAT. The molecular weights calculated from the SE data were 30,000±1,500 for <i>kdpFABC_BS—1</i> and 30,000±2,500 for <i>kdpFABC_BS—7</i>.</p

Biochemical and functional characterization of KdpE_DBD.

<p><b>A.</b> Sedimentation velocity analysis of the KdpE_DBD to detect self-association. The c(s) distribution of the KdpE_DBD at 21 (dots), 42 (solid line), and 84 µM (dashes) shows a single species of 1.4 S. No concentration-dependent formation of higher-order species was observed. <b>B.</b> Interaction of KdpE_DBD protein with <i>kdpFABC_BS</i> and <i>ompF_Pro</i> DNA sequences analyzed by EMSA. The triangles represent increasing molar ratios of 1∶0, 1∶1, 1∶2, and 1∶3 of DNA to purified KdpE_DBD. The lower and upper bands represent free DNA and DNA-KdpE_DBD complex, respectively. <b>C. </b><i>In vivo</i> analysis of expression of the β-galactosidase gene fused to <i>kdpFABC_Pro</i>. <i>E. coli</i> RH003 cells lacking the histidine kinase (<i>kdpD</i>) and RR (<i>kdpE</i>) were used to express full-length KdpD alone as well as KdpD combined with KdpE or KdpE_DBD. As described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030102#s2" target="_blank">methods</a>, the cells were grown in K0 (▪) and K10 (□) media prior to analysis of gene expression. Growth in K0 medium mimics stresses resulting from external K⁺ depletion. The β-galactosidase activity expressed as Miller units represents the mean of three independent experiments; error bars represent standard error.</p

Sedimentation velocity analysis of KdpE_DBD—<i>kdpFABC_BS</i> association.

<p><b>A.</b> Continuous distribution of sedimentation coefficients [c(s)] as a function of increasing concentration of protein against a fixed concentration of <i>kdpFABC_BS</i> DNA (0.5 µM). The protein concentrations used varied between 0.25 and 16 µM as shown. The largest complex with sedimentation coefficient of 4.1 S was observed at protein concentration of 4 to 16 µM. Independent experiments established the sedimentation coefficients of KdpE_DBD and <i>kdpFABC_BS</i> at 1.4 S and 2.8 S respectively (data not shown). <b>B.</b> A plot of the weight average sedimentation coefficients (S_w) against the concentration of KdpE_DBD is shown. Analysis of the isotherm indicated that DNA was saturated beginning at 8-fold molar excess of KdpE_DBD protein. <b>C.</b> SV c(s) distributions comparing binding of KdpE_DBD to the S1 and S2 sites individually and to the both sites simultaneously. Wild-type DNA with both sites intact (<i>kdpFABC_BS</i>), functional S1 (<i>kdpFABC_BS —</i>7) and S2 (<i>kdpFABC_BS —</i>1) sites were analyzed with a 16-fold molar excess of Kdp_DBD. Complexes with DNA possessing single sites have sedimentation coefficients of 3.5 S whereas when both sites were occupied a 4.1 S species was formed.</p

Identification and characterization of half-sites S1 and S2 on DNA that interacts with KdpE_DBD.

<p><b>A.</b> Sequence logo representation to highlight conserved sequences in a 24 bp stretch of <i>kdpFABC_BS</i>. In the logo, the height of the letter represents its frequency of occurrence in a multiple sequence alignment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030102#pone.0030102.s003" target="_blank">Fig. S3</a>) and the error bars indicate the sampling error at individual positions. Two 6 bp imperfect direct repeats (TTTATA and TTTACA) separated by a 5 bp sequence are shown in dashed boxes below the logo. <b>B.</b> Identification of the minimal length of DNA required for binding KdpE. For EMSA, double-stranded DNA molecules with progressive deletions (indicated by Δ) at either 5′, 3′, or both ends were used (the nomenclature for oligonucleotides: 5′Δ2, 3′Δ8 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030102#pone-0030102-g004" target="_blank">Fig. 4B</a>, lane 9) refers to deletion of 2 and 8 bp from the 5′ and 3′ ends respectively of the wild-type (30 bp) DNA molecule; oligonucleotides used are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030102#pone.0030102.s005" target="_blank">Table S2</a>). The interpretation of EMSA was qualitative: discreet band shifts as observed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030102#pone-0030102-g004" target="_blank">Fig. 4B</a>, lane 1 were considered a positive reaction (+), whereas no shift (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030102#pone-0030102-g004" target="_blank">Fig. 4B</a>, lane 3) was scored negative (−) and smeared bands as exemplified by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030102#pone-0030102-g004" target="_blank">Fig. 4B</a>, lane 2 were considered partial binding. <b>C.</b> Effects of changes in DNA sequence on the KdpE_DBD-DNA interaction. A summary of EMSA data (data not shown) using the 30 bp <i>kdpFABC_BS</i> sequence and modified oligonucleotides (only specific two or one nucleotide substitutions are noted) are presented. The scoring of EMSA analysis was as described above. The dashed boxes represent the 6 bp direct repeats that form half-sites S1 and S2.</p

Effects of mutation of residues conserved in <i>kdpE_DBD</i>.

<p><b>A.</b> Comparison of β-galactosidase activities of KdpE mutants and wild-type KdpE in the <i>kdpFABC_Pro-lacZ</i> fusion strain HAK003. Residues located in the α-8 (R193 and R200) and β-hairpin (T215) of KdpE (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030102#pone-0030102-g002" target="_blank">Fig. 2</a>) were targeted for mutagenesis to alanine. β-galactosidase (a reporter for <i>kdpFABC</i> expression) was measured in cells grown in media containing either K10 (white bar, 10 mM K⁺) or K0 (gray bar, 0 mM K⁺). <b>B.</b> EMSA showing effects of mutations in KdpE on interaction with the 30 bp DNA fragment representing its binding site. The triangles represent increasing molar ratios of 1∶0, 1∶1, 1∶2, 1∶4, and 1∶8 of DNA to purified mutants as indicated and wild-type KdpE_DBD.</p