Additional file 3: Online Resource 4. of Arabidopsis protein disulfide isomerase-8 is a type I endoplasmic reticulum transmembrane protein with thiol-disulfide oxidase activity

Validation of antibody specificity in tissues for microscopy. a Section of a root apical cell from the pdi8 antisense line, AS1, stained with anti-PDI8 antiserum. b Section of a shoot apical cell from AS1, stained with anti-PDI8 antiserum. c Section of a WT root apical cell, labeled with rabbit pre-immune serum. d Section of a root apical cell from the line OE1 labeled with anti-PDI8 antiserum. (JPG 9403 kb

Additional file 1: Online Resource 2. of Arabidopsis protein disulfide isomerase-8 is a type I endoplasmic reticulum transmembrane protein with thiol-disulfide oxidase activity

Relative levels of PDI8 transcripts across various plant organs of Arabidopsis. An electronic fluorescent pictograph depicting the relative expression level of PDI8 across different Arabidopsis tissues based on publicly available microarray data. (JPG 587 kb

Additional file 4: Online Resource 1. of Arabidopsis protein disulfide isomerase-8 is a type I endoplasmic reticulum transmembrane protein with thiol-disulfide oxidase activity

Sequences of PDI-B subfamily proteins identified by database searches. A compilation of the deduced products of PDI-B genes found in available sequenced plant genomes. (DOCX 134 kb

Structural and Functional Basis for Targeting <i>Campylobacter jejuni</i> Agmatine Deiminase To Overcome Antibiotic Resistance

<i>Campylobacter jejuni</i> is the most common bacterial cause of gastroenteritis and a major contributor to infant mortality in the developing world. The increasing incidence of antibiotic-resistant <i>C. jejuni</i> only adds to the urgency to develop effective therapies. Because of the essential role that polyamines play, particularly in protection from oxidative stress, enzymes involved in the biosynthesis of these metabolites are emerging as promising antibiotic targets. The recent description of an alternative pathway for polyamine synthesis, distinct from that in human cells, in <i>C. jejuni</i> suggests this pathway could be a target for novel therapies. To that end, we determined X-ray crystal structures of <i>C. jejuni</i> agmatine deiminase (CjADI) and demonstrated that loss of CjADI function contributes to antibiotic sensitivity, likely because of polyamine starvation. The structures provide details of key molecular features of the active site of this protein. Comparison of the unliganded structure (2.1 Å resolution) to that of the CjADI–agmatine complex (2.5 Å) reveals significant structural rearrangements that occur upon substrate binding. The shift of two helical regions of the protein and a large conformational change in a loop near the active site generate a narrow binding pocket around the bound substrate. This change optimally positions the substrate for catalysis. In addition, kinetic analysis of this enzyme demonstrates that CjADI is an iminohydrolase that effectively deiminates agmatine. Our data suggest that <i>C. jejuni</i> agmatine deiminase is a potentially important target for combatting antibiotic resistance, and these results provide a valuable framework for guiding future drug development

Mechanism of MenE Inhibition by Acyl-Adenylate Analogues and Discovery of Novel Antibacterial Agents

MenE is an <i>o</i>-succinylbenzoyl-CoA (OSB-CoA) synthetase in the bacterial menaquinone biosynthesis pathway and is a promising target for the development of novel antibacterial agents. The enzyme catalyzes CoA ligation via an acyl-adenylate intermediate, and we have previously reported tight-binding inhibitors of MenE based on stable acyl-sulfonyladenosine analogues of this intermediate, including OSB-AMS (<b>1</b>), which has an IC₅₀ value of ≤25 nM for <i>Escherichia coli</i> MenE. Herein, we show that OSB-AMS reduces menaquinone levels in <i>Staphylococcus aureus</i>, consistent with its proposed mechanism of action, despite the observation that the antibacterial activity of OSB-AMS is ∼1000-fold lower than the IC₅₀ for enzyme inhibition. To inform the synthesis of MenE inhibitors with improved antibacterial activity, we have undertaken a structure–activity relationship (SAR) study stimulated by the knowledge that OSB-AMS can adopt two isomeric forms in which the OSB side chain exists either as an open-chain keto acid or a cyclic lactol. These studies revealed that negatively charged analogues of the keto acid form bind, while neutral analogues do not, consistent with the hypothesis that the negatively charged keto acid form of OSB-AMS is the active isomer. X-ray crystallography and site-directed mutagenesis confirm the importance of a conserved arginine for binding the OSB carboxylate. Although most lactol isomers tested were inactive, a novel difluoroindanediol inhibitor (<b>11</b>) with improved antibacterial activity was discovered, providing a pathway toward the development of optimized MenE inhibitors in the future