Experimental validation of in silico model-predicted isocitrate dehydrogenase and phosphomannose isomerase from Dehalococcoides mccartyi

Gene sequences annotated as proteins of unknown or non-specific function and hypothetical proteins account for a large fraction of most genomes. In the strictly anaerobic and organohalide respiring Dehalococcoides mccartyi, this lack of annotation plagues almost half the genome. Using a combination of bioinformatics analyses and genome-wide metabolic modelling, new or more specific annotations were proposed for about 80 of these poorly annotated genes in previous investigations of D. mccartyi metabolism. Herein, we report the experimental validation of the proposed reannotations for two such genes (KB1_0495 and KB1_0553) from D. mccartyi strains in the KB-1 community. KB1_0495 or DmIDH was originally annotated as an NAD+-dependent isocitrate dehydrogenase, but biochemical assays revealed its activity primarily with NADP+ as a cofactor. KB1_0553, also denoted as DmPMI, was originally annotated as a hypothetical protein/sugar isomerase domain protein. We previously proposed that it was a bifunctional phosphoglucose isomerase/phosphomannose isomerase, but only phosphomannose isomerase activity was identified and confirmed experimentally. Further bioinformatics analyses of these two protein sequences suggest their affiliation to potentially novel enzyme families within their respective larger enzyme super families

Structural and Biochemical Characterization of <i>Acinetobacter</i> spp. Aminoglycoside Acetyltransferases Highlights Functional and Evolutionary Variation among Antibiotic Resistance Enzymes

Modification of aminoglycosides by <i>N-</i>acetyltransferases (AACs) is one of the major mechanisms of resistance to these antibiotics in human bacterial pathogens. More than 50 enzymes belonging to the AAC(6′) subfamily have been identified in Gram-negative and Gram-positive clinical isolates. Our understanding of the molecular function and evolutionary origin of these resistance enzymes remains incomplete. Here we report the structural and enzymatic characterization of AAC(6′)-Ig and AAC(6′)-Ih from <i>Acinetobacter</i> spp. The crystal structure of AAC(6′)-Ig in complex with tobramycin revealed a large substrate-binding cleft remaining partially unoccupied by the substrate, which is in stark contrast with the previously characterized AAC(6′)-Ib enzyme. Enzymatic analysis indicated that AAC(6′)-Ig and -Ih possess a broad specificity against aminoglycosides but with significantly lower turnover rates as compared to other AAC(6′) enzymes. Structure- and function-informed phylogenetic analysis of AAC(6′) enzymes led to identification of at least three distinct subfamilies varying in oligomeric state, active site composition, and drug recognition mode. Our data support the concept of AAC(6′) functionality originating through convergent evolution from diverse Gcn5-related-<i>N</i>-acetyltransferase (GNAT) ancestral enzymes, with AAC(6′)-Ig and -Ih representing enzymes that may still retain ancestral nonresistance functions in the cell as provided by their particular active site properties

Type III Effector NleH2 from <i>Escherichia coli</i> O157:H7 str. Sakai Features an Atypical Protein Kinase Domain

The crystal structure of a C-terminal domain of enterohemorrhagic <i>Escherichia coli</i> type III effector NleH2 has been determined to 2.6 Å resolution. The structure resembles those of protein kinases featuring the catalytic, activation, and glycine-rich loop motifs and ATP-binding site. The position of helix αC and the lack of a conserved arginine within an equivalent HRD motif suggested that the NleH2 kinase domain’s active conformation might not require phosphorylation. The activation segment markedly contributed to the dimerization interface of NleH2, which can also accommodate the NleH1–NleH2 heterodimer. The C-terminal PDZ-binding motif of NleH2 provided bases for interaction with host proteins

Substrate Recognition by a Colistin Resistance Enzyme from <i>Moraxella catarrhalis</i>

Lipid A phosphoethanolamine (PEtN) transferases render bacteria resistant to the last resort antibiotic colistin. The recent discoveries of pathogenic bacteria harboring plasmid-borne PEtN transferase (<i>mcr</i>) genes have illustrated the serious potential for wide dissemination of these resistance elements. The origin of <i>mcr-1</i> is traced to <i>Moraxella</i> species co-occupying environmental niches with Enterobacteriaceae. Here, we describe the crystal structure of the catalytic domain of the chromosomally encoded colistin resistance PEtN transferase, ICR^<i>Mc</i> (for intrinsic colistin resistance) of <i>Moraxella catarrhalis</i>. The ICR^<i>Mc</i> structure in complex with PEtN reveals key molecular details including specific residues involved in catalysis and PEtN binding. It also demonstrates that ICR^<i>Mc</i> catalytic domain dimerization is required for substrate binding. Our structure-guided phylogenetic analysis provides sequence signatures defining potentially colistin-active representatives in this enzyme family. Combined, these results advance the molecular and mechanistic understanding of PEtN transferases and illuminate their origins

Structural and Functional Survey of Environmental Aminoglycoside Acetyltransferases Reveals Functionality of Resistance Enzymes

Aminoglycoside <i>N</i>-acetyltransferases (AACs) confer resistance against the clinical use of aminoglycoside antibiotics. The origin of AACs can be traced to environmental microbial species representing a vast reservoir for new and emerging resistance enzymes, which are currently undercharacterized. Here, we performed detailed structural characterization and functional analyses of four metagenomic AAC (meta-AACs) enzymes recently identified in a survey of agricultural and grassland soil microbiomes (Forsberg et al. Nature 2014, 509, 612). These enzymes are new members of the Gcn5-Related-<i>N</i>-Acetyltransferase superfamily and confer resistance to the aminoglycosides gentamicin C, sisomicin, and tobramycin. Moreover, the meta-AAC0020 enzyme demonstrated activity comparable with an AAC(3)-I enzyme that serves as a model AAC enzyme identified in a clinical bacterial isolate. The crystal structure of meta-AAC0020 in complex with sisomicin confirmed an unexpected AAC(6′) regiospecificity of this enzyme and revealed a drug binding mechanism distinct from previously characterized AAC(6′) enzymes. Together, our data highlights the presence of highly active antibiotic-modifying enzymes in the environmental microbiome and reveals unexpected diversity in substrate specificity. These observations of additional AAC enzymes must be considered in the search for novel aminoglycosides less prone to resistance

Structural Analysis of HopPmaL Reveals the Presence of a Second Adaptor Domain Common to the HopAB Family of <i>Pseudomonas syringae</i> Type III Effectors

HopPmaL is a member of the HopAB family of type III effectors present in the phytopathogen <i>Pseudomonas syringae</i>. Using both X-ray crystallography and solution nuclear magnetic resonance, we demonstrate that HopPmaL contains two structurally homologous yet functionally distinct domains. The N-terminal domain corresponds to the previously described Pto-binding domain, while the previously uncharacterised C-terminal domain spans residues 308–385. While structurally similar, these domains do not share significant sequence similarity and most importantly demonstrate significant differences in key residues involved in host protein recognition, suggesting that each of them targets a different host protein

A Pathogen Type III Effector with a Novel E3 Ubiquitin Ligase Architecture

<div><p>Type III effectors are virulence factors of Gram-negative bacterial pathogens delivered directly into host cells by the type III secretion nanomachine where they manipulate host cell processes such as the innate immunity and gene expression. Here, we show that the novel type III effector XopL from the model plant pathogen <em>Xanthomonas campestris</em> pv. <em>vesicatoria</em> exhibits E3 ubiquitin ligase activity <em>in vitro</em> and <em>in planta</em>, induces plant cell death and subverts plant immunity. E3 ligase activity is associated with the C-terminal region of XopL, which specifically interacts with plant E2 ubiquitin conjugating enzymes and mediates formation of predominantly K11-linked polyubiquitin chains. The crystal structure of the XopL C-terminal domain revealed a single domain with a novel fold, termed XL-box, not present in any previously characterized E3 ligase. Mutation of amino acids in the central cavity of the XL-box disrupts E3 ligase activity and prevents XopL-induced plant cell death. The lack of cysteine residues in the XL-box suggests the absence of thioester-linked ubiquitin-E3 ligase intermediates and a non-catalytic mechanism for XopL-mediated ubiquitination. The crystal structure of the N-terminal region of XopL confirmed the presence of a leucine-rich repeat (LRR) domain, which may serve as a protein-protein interaction module for ubiquitination target recognition. While the E3 ligase activity is required to provoke plant cell death, suppression of PAMP responses solely depends on the N-terminal LRR domain. Taken together, the unique structural fold of the E3 ubiquitin ligase domain within the <em>Xanthomonas</em> XopL is unprecedented and highlights the variation in bacterial pathogen effectors mimicking this eukaryote-specific activity.</p> </div

Plazomicin Retains Antibiotic Activity against Most Aminoglycoside Modifying Enzymes

Plazomicin is a next-generation, semisynthetic aminoglycoside antibiotic currently under development for the treatment of infections due to multidrug-resistant <i>Enterobacteriaceae</i>. The compound was designed by chemical modification of the natural product sisomicin to provide protection from common aminoglycoside modifying enzymes that chemically alter these drugs via <i>N</i>-acetylation, <i>O</i>-adenylylation, or <i>O</i>-phosphorylation. In this study, plazomicin was profiled against a panel of isogenic strains of <i>Escherichia coli</i> individually expressing twenty-one aminoglycoside resistance enzymes. Plazomicin retained antibacterial activity against 15 of the 17 modifying enzyme-expressing strains tested. Expression of only two of the modifying enzymes, <i>aac­(2′)-Ia</i> and <i>aph­(2″)-IVa,</i> decreased plazomicin potency. On the other hand, expression of 16S rRNA ribosomal methyltransferases results in a complete lack of plazomicin potency. <i>In vitro</i> enzymatic assessment confirmed that AAC(2′)-Ia and APH(2′′)-IVa (aminoglycoside acetyltransferase, AAC; aminoglycoside phosphotransferase, APH) were able to utilize plazomicin as a substrate. AAC(2′)-Ia and APH(2′′)-IVa are limited in their distribution to <i>Providencia stuartii</i> and Enterococci, respectively. These data demonstrate that plazomicin is not modified by a broad spectrum of common aminoglycoside modifying enzymes including those commonly found in <i>Enterobacteriaceae</i>. However, plazomicin is inactive in the presence of 16S rRNA ribosomal methyltransferases, which should be monitored in future surveillance programs

XopL displays E2 specificity <i>in vitro</i>.

<p>(<b>A</b>) <i>In vitro</i> ubiquitin ligase assay with ATP, ubiquitin, E1, human UBE2D2 (E2D2) or different <i>Arabidopsis thaliana</i> E2s (ATUBC28, 11, 13 or 19) in the presence (+) or absence (−) of His₆-XopL[aa 1–660]. The left panel shows the western blot reacted with ubiquitin antibodies (α-Ub) after 5 hours incubation, while the right panel shows the Coomassie stained gel of the reactants at the start of the reaction. Polyubiquitination is indicated by (Ub)_n. A lower-molecular weight impurity or degradation product in the full-length XopL protein purification is denoted by †. (<b>B</b>) Ubiquitin ligase assay described in (A) using His₆-XopL[aa 474–660], AtUBC28 and mutant derivatives R5A, F62A, K63A and A96D. Reaction times are indicated. The left panel shows the western blot reacted with ubiquitin antibodies (α-Ub), while the right panel shows the Coomassie-stained gel at the equivalent time points. (Ub)_n indicates polyubiquitination, and positions on the western blot or Coomassie-stained gels corresponding to ubiquitin (Ub), di-ubiquitin (Ub)2, AtUBC28, mono-ubiquitinated AtUBC28 (AtUBC28-Ub) and His₆-XopL[aa 474–660] (E3) are labeled.</p

XopL inhibits pathogen-associated molecular pattern (PTI)-induced defense gene expression.

<p><i>Arabidopsis thaliana</i> Col-0 protoplasts were co-transformed with <i>pNHL10-LUC</i> (luciferase) as reporter, the <i>p35S-</i>effector gene constructs <i>xopL</i>, <i>xopL_Q612A</i>, <i>xopL_LRR</i> and <i>xopL_CTD</i> or <i>p35S-cfp</i> and p<i>35S</i>-<i>avrPto</i> (negative and positive control, respectively), and <i>pUBQ10-GUS</i> (β-glucuronidase) for normalization. 14 h after transformation, protoplasts were treated with H₂O (<b>A</b>), 100 nM elf18 (<b>B</b>) and 100 nM flg22 (<b>C</b>), and luciferase activity was monitored for 3 h. Results are depicted as LUC/GUS ratios (with the zero timepoint, H₂O-treated sample set at a reference value of one). (<b>D</b>) Protein extracts of transformed protoplasts were taken 10 min after treatment and analyzed by immunoblotting using a pTepY-antibody (specific for activated MAP-Kinases) and HA-specific antibodies for detection of HA-tagged effector-or CFP-fusion proteins. MPK3, 4, 6, 11: mitogen activated protein kinase 3, 4, 6, 11. The experiments were performed three times with similar results.</p