22 results for Arcus, Vickery L.

  • Whole genome sequencing of Mycobacterium tuberculosis reveals slow growth and low mutation rates during latent infections in humans

    Colangeli, Roberto; Arcus, Vickery L.; Cursons, Raymond T.; Ruthe, Ali; Karalus, Noel; Coley, Kathy; Manning, Shannon D.; Kim, Soyeon; Marchiano, Emily; Alland, David (2014)

    Journal article
    University of Waikato

    Very little is known about the growth and mutation rates of Mycobacterium tuberculosis during latent infection in humans. However, studies in rhesus macaques have suggested that latent infections have mutation rates that are higher than that observed during active tuberculosis disease. Elevated mutation rates are presumed risk factors for the development of drug resistance. Therefore, the investigation of mutation rates during human latency is of high importance. We performed whole genome mutation analysis of M. tuberculosis isolates from a multi-decade tuberculosis outbreak of the New Zealand Rangipo strain. We used epidemiological and phylogenetic analysis to identify four cases of tuberculosis acquired from the same index case. Two of the tuberculosis cases occurred within two years of exposure and were classified as recently transmitted tuberculosis. Two other cases occurred more than 20 years after exposure and were classified as reactivation of latent M. tuberculosis infections. Mutation rates were compared between the two recently transmitted pairs versus the two latent pairs. Mean mutation rates assuming 20 hour generation times were 5.5X10⁻¹⁰ mutations/bp/generation for recently transmitted tuberculosis and 7.3X10⁻¹¹ mutations/bp/generation for latent tuberculosis. Generation time versus mutation rate curves were also significantly higher for recently transmitted tuberculosis across all replication rates (p = 0.006). Assuming identical replication and mutation rates among all isolates in the final two years before disease reactivation, the u20hr mutation rate attributable to the remaining latent period was 1.6×10⁻¹¹ mutations/bp/generation, or approximately 30 fold less than that calculated during the two years immediately before disease. Mutations attributable to oxidative stress as might be caused by bacterial exposure to the host immune system were not increased in latent infections. In conclusion, we did not find any evidence to suggest elevated mutation rates during tuberculosis latency in humans, unlike the situation in rhesus macaques.

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  • Structure and function of an acetyl xylan esterase (est2a) from the rumen bacterium Butyrivibrio proteoclasticus

    Till, Marisa; Goldstone, David C.; Attwood, Graeme T.; Moon, Christina D.; Kelly, Willam J.; Arcus, Vickery L. (2013)

    Journal article
    University of Waikato

    Butyrivibrio proteoclasticus is a significant component of the microbial population of the rumen of dairy cattle. It is a xylan-degrading organism whose genome encodes a large number of open reading frames annotated as fibre-degrading enzymes. We have determined the three-dimensional structure of Est2A, an acetyl xylan esterase from B. proteoclasticus, at 2.1 Å resolution, along with the structure of an inactive mutant (H351A) at 2.0 Å resolution. The structure reveals two domains - a C-terminal SGNH domain and an N-terminal jelly-roll domain typical of CE2 family structures. The structures are accompanied by experimentally determined enzymatic parameters against two model substrates, para-nitrophenyl acetate and para-nitrophenyl butyrate. The suite of fibre-degrading enzymes produced by B. proteoclasticus provides a rich source of new enzymes of potential use in industrial settings.

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  • Ribonucleases in bacterial toxin–antitoxin systems

    Cook, Gregory M.; Robson, Jennifer R.; Frampton, Rebekah A.; McKenzie, Joanna Leigh; Przybilski, Rita; Fineran, Peter C.; Arcus, Vickery L. (2013)

    Journal article
    University of Waikato

    Toxin-antitoxin (TA) systems are widespread in bacteria and archaea and play important roles in a diverse range of cellular activities. TA systems have been broadly classified into 5 types and the targets of the toxins are diverse, but the most frequently used cellular target is mRNA. Toxins that target mRNA to inhibit translation can be classified as ribosome-dependent or ribosome-independent RNA interferases. These RNA interferases are sequence-specific endoribonucleases that cleave RNA at specific sequences. Despite limited sequence similarity, ribosome-independent RNA interferases belong to a limited number of structural classes. The MazF structural family includes MazF, Kid, ParE and CcdB toxins. MazF members cleave mRNA at 3-, 5- or 7-base recognition sequences in different bacteria and have been implicated in controlling cell death (programmed) and cell growth, and cellular responses to nutrient starvation, antibiotics, heat and oxidative stress. VapC endoribonucleases belong to the PIN-domain family and inhibit translation by either cleaving tRNAfMet in the anticodon stem loop, cleaving mRNA at -AUA(U/A)-hairpin-G- sequences or by sequence-specific RNA binding. VapC has been implicated in controlling bacterial growth in the intracellular environment and in microbial adaptation to nutrient limitation (nitrogen, carbon) and heat shock. ToxN shows structural homology to MazF and is also a sequence-specific endoribonuclease. ToxN confers phage resistance by causing cell death upon phage infection by cleaving cellular and phage RNAs, thereby interfering with bacterial and phage growth. Notwithstanding our recent progress in understanding ribonuclease action and function in TA systems, the environmental triggers that cause release of the toxin from its cognate antitoxin and the precise cellular function of these systems in many bacteria remain to be discovered. This article is part of a Special Issue entitled: RNA Decay mechanisms.

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  • The Structure of the Oligomerization Domain of Lsr2 from Mycobacterium tuberculosis Reveals a Mechanism for Chromosome Organization and Protection

    Summers, Emma Louise; Meindl, Kathrin; Usón, Isabel; Mitra, Alok K.; Radjainia, Mazdak; Colangeli, Roberto; Alland, David; Arcus, Vickery L. (2012)

    Journal article
    University of Waikato

    Lsr2 is a small DNA-binding protein present in mycobacteria and related actinobacteria that regulates gene expression and influences the organization of bacterial chromatin. Lsr2 is a dimer that binds to AT-rich regions of chromosomal DNA and physically protects DNA from damage by reactive oxygen intermediates (ROI). A recent structure of the C-terminal DNA-binding domain of Lsr2 provides a rationale for its interaction with the minor groove of DNA, its preference for AT-rich tracts, and its similarity to other bacterial nucleoid-associated DNA-binding domains. In contrast, the details of Lsr2 dimerization (and oligomerization) via its N-terminal domain, and the mechanism of Lsr2-mediated chromosomal cross-linking and protection is unknown. We have solved the structure of the N-terminal domain of Lsr2 (N-Lsr2) at 1.73 Å resolution using crystallographic ab initio approaches. The structure shows an intimate dimer of two ß-ß-a motifs with no close homologues in the structural databases. The organization of individual N-Lsr2 dimers in the crystal also reveals a mechanism for oligomerization. Proteolytic removal of three N-terminal residues from Lsr2 results in the formation of an anti-parallel β-sheet between neighboring molecules and the formation of linear chains of N-Lsr2. Oligomerization can be artificially induced using low concentrations of trypsin and the arrangement of N-Lsr2 into long chains is observed in both monoclinic and hexagonal crystallographic space groups. In solution, oligomerization of N-Lsr2 is also observed following treatment with trypsin. A change in chromosomal topology after the addition of trypsin to full-length Lsr2-DNA complexes and protection of DNA towards DNAse digestion can be observed using electron microscopy and electrophoresis. These results suggest a mechanism for oligomerization of Lsr2 via protease-activation leading to chromosome compaction and protection, and concomitant down-regulation of large numbers of genes. This mechanism is likely to be relevant under conditions of stress where cellular proteases are known to be upregulated.

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  • Superantigen architecture: Functional decoration on a conserved scaffold

    Arcus, Vickery L.; Baker, Edward N. (2007)

    Book item
    University of Waikato

    A defining and consistent feature of the bacterial superantigens from Staphylococcus aureus and Streptococcus pyogenes is their strongly conserved three-dimensional structure. Structural studies to date show that the array of more than 280 amino acid sequences known for superantigens (SAgs) and staphylococcal superantigen-like (SSL) proteins all have the same fold-a structure in which the same three-dimensional arrangement of α-helices and β-sheets is traced by each amino acid sequence, with the same topology (for recent reviews, see references 29 and 43). A typical SAg structure comprises two domains-an N-terminal β -barrel domain called an OB-fold (4, 25) and a C-terminal β-grasp domain in which a long α-helix packs on to a mixed parallel and antiparallel β-sheet. These two domains are traversed by an α-helix that lies at the N terminus of the protein and packs against the β-grasp domain, thus linking the N- and C-terminal domains.

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  • Toxin-antitoxin systems of mycobacterium smegmatis are essential for cell survival

    Frampton, Rebekah A.; Aggio, Rachael B.M.; Villas-Bôas, Silas G; Arcus, Vickery L.; Cook, Gregory M. (2011)

    Journal article
    University of Waikato

    The role of chromosomal toxin-antitoxin (TA) modules in bacterial physiology remains enigmatic despite their abundance in the genomes of many bacteria. Mycobacterium smegmatis contains three putative TA systems, VapBC, MazEF, and Phd/Doc, and previous work from our group has shown VapBC to be a bona fide TA system. In this study, we show that MazEF and Phd/Doc are also TA systems that are constitutively expressed, transcribed as leaderless transcripts, and subject to autoregulation, and expression of the toxin component leads to growth inhibition that can be rescued by the cognate antitoxin. No phenotype was identified for deletions of the individual TA systems, but a triple deletion strain (ΔvapBC, mazEF, phd/doc), designated ΔTAtriple, exhibited a survival defect in complex growth medium demonstrating an essential role for these TA modules in mycobacterial survival. Transcriptomic analysis revealed no significant differences in gene expression between wild type and the ΔTAtriple mutant under these conditions suggesting that the growth defect was not at a transcriptional level. Metabolomic analysis demonstrated that in response to starvation in complex medium, both the wild type and ΔTAtriple mutant consumed a wide range of amino acids from the external milieu. Analysis of intracellular metabolites revealed a significant difference in the levels of branched-chain amino acids between the wild type and ΔTAtriple mutant, which are proposed to play essential roles in monitoring the nutritional supply and physiological state of the cell and linking catabolic with anabolic reactions. Disruption of this balance in the ΔTAtriple mutant may explain the survival defect in complex growth medium.

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  • Infinite stacking of alternating polyfluoroaryl rings and bromide anions

    Arcus, Vickery L.; Bernstein, Daniel R.; Crombie, Cameron W.; Saunders, Graham C. (2013)

    Journal article
    University of Waikato

    The crystal structure of 1-(4-bromo-2,3,5,6-tetrafluorophenyl)-3-benzylimidazolium bromide comprises columns of parallel bromotetrafluorophenyl rings with an interplanar distance of 6.936(6) Å separated by bromide anions.

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  • Structural analysis of the GH43 enzyme Xsa43E from Butyrivibrio proteoclasticus

    Till, Marisa; Goldstone, D.; Card, G.; Attwood, Graeme T.; Moon, Christina D.; Arcus, Vickery L. (2014-08-29)

    Journal article
    University of Waikato

    The rumen of dairy cattle can be thought of as a large, stable fermentation vat and as such it houses a large and diverse community of microorganisms. The bacterium Butyrivibrio proteoclasticus is a representative of a significant component of this microbial community. It is a xylan-degrading organism whose genome encodes a large number of open reading frames annotated as fibre-degrading enzymes. This suite of enzymes is essential for the organism to utilize the plant material within the rumen as a fuel source, facilitating its survival in this competitive environment. Xsa43E, a GH43 enzyme from B. proteoclasticus, has been structurally and functionally characterized. Here, the structure of selenomethionine-derived Xsa43E determined to 1.3 Å resolution using single-wavelength anomalous diffraction is reported. Xsa43E possesses the characteristic five-bladed β-propeller domain seen in all GH43 enzymes. GH43 enzymes can have a range of functions, and the functional characterization of Xsa43E shows it to be an arabinofuranosidase capable of cleaving arabinose side chains from short segments of xylan. Full functional and structural characterization of xylan-degrading enzymes will aid in creating an enzyme cocktail that can be used to completely degrade plant material into simple sugars. These molecules have a range of applications as starting materials for many industrial processes, including renewable alternatives to fossil fuels.

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  • Structure of a eukaryotic nonribosomal peptide synthetase adenylation domain that activates a large hydroxamate amino acid in siderophore biosynthesis

    Lee, T. Verne; Johnson, Linda J.; Johnson, Richard D.; Koulman, Albert; Lane, Geoffrey A.; Lott, J. Shaun; Arcus, Vickery L. (2010)

    Journal article
    University of Waikato

    Nonribosomal peptide synthetases (NRPSs) are large, multidomain proteins that are involved in the biosynthesis of an array of secondary metabolites. We report the structure of the third adenylation domain from the siderophore-synthesizing NRPS, SidN, from the endophytic fungus Neotyphodium lolii. This is the first structure of a eukaryotic NRPS domain, and it reveals a large binding pocket required to accommodate the unusual amino acid substrate, N-delta-cis-anhydromevalonyl-N-delta-hydroxy-L-ornithine (cis-AMHO). The specific activation of cis-AMHO was confirmed biochemically, and an AMHO moiety was unambiguously identified as a component of the fungal siderophore using mass spectroscopy. The protein structure shows that the substrate binding pocket is defined by 17 amino acid residues, in contrast to both prokaryotic adenylation domains and to previous predictions based on modeling. Existing substrate prediction methods for NRPS adenylation domains fail for domains from eukaryotes due to the divergence of their signature sequences from those of prokaryotes. Thus, this new structure will provide a basis for improving prediction methods for eukaryotic NRPS enzymes that play important and diverse roles in the biology of fungi.

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  • VapC Toxins from Mycobacterium tuberculosis are ribonucleases that differentially inhibit growth and are neutralized by cognate VapB antitoxins

    Ahidjo, Bintou Ahmadou; Kuhnert, Diane; McKenzie, Joanna Leigh; Machowski, Edith E.; Gordhan, Bhavna G.; Arcus, Vickery L.; Abrahams, Garth L.; Mizrahi, Valerie (2011)

    Journal article
    University of Waikato

    The chromosome of Mycobacterium tuberculosis (Mtb) encodes forty seven toxin-antitoxin modules belonging to the VapBC family. The role of these modules in the physiology of Mtb and the function(s) served by their expansion are unknown. We investigated ten vapBC modules from Mtb and the single vapBC from M. smegmatis. Of the Mtb vapCs assessed, only Rv0549c, Rv0595c, Rv2549c and Rv2829c were toxic when expressed from a tetracycline-regulated promoter in M. smegmatis. The same genes displayed toxicity when conditionally expressed in Mtb. Toxicity of Rv2549c in M. smegmatis correlated with the level of protein expressed, suggesting that the VapC level must exceed a threshold for toxicity to be observed. In addition, the level of Rv2456 protein induced in M. smegmatis was markedly lower than Rv2549c, which may account for the lack of toxicity of this and other VapCs scored as ‘non-toxic’. The growth inhibitory effects of toxic VapCs were neutralized by expression of the cognate VapB as part of a vapBC operon or from a different chromosomal locus, while that of non-cognate antitoxins did not. These results demonstrated a specificity of interaction between VapCs and their cognate VapBs, a finding corroborated by yeast two-hybrid analyses. Deletion of selected vapC or vapBC genes did not affect mycobacterial growth in vitro, but rendered the organisms more susceptible to growth inhibition following toxic VapC expression. However, toxicity of ‘non-toxic’ VapCs was not unveiled in deletion mutant strains, even when the mutation eliminated the corresponding cognate VapB, presumably due to insufficient levels of VapC protein. Together with the ribonuclease (RNase) activity demonstrated for Rv0065 and Rv0617 – VapC proteins with similarity to Rv0549c and Rv3320c, respectively – these results suggest that the VapBC family potentially provides an abundant source of RNase activity in Mtb, which may profoundly impact the physiology of the organism.

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  • Change in heat capacity for enzyme catalysis determines temperature dependence of enzyme catalyzed ratesnzyme Catalyzed Rates

    Hobbs, Joanne K.; Jiao, Wanting; Easter, Ashley Davys; Parker, Emily J.; Schipper, Louis A.; Arcus, Vickery L. (2013)

    Journal article
    University of Waikato

    The increase in enzymatic rates with temperature up to an optimum temperature (Topt) is widely attributed to classical Arrhenius behavior, with the decrease in enzymatic rates above Topt ascribed to protein denaturation and/or aggregation. This account persists despite many investigators noting that denaturation is insufficient to explain the decline in enzymatic rates above Topt. Here we show that it is the change in heat capacity associated with enzyme catalysis (ΔC‡p) and its effect on the temperature dependence of ΔG‡ that determines the temperature dependence of enzyme activity. Through mutagenesis, we demonstrate that the Topt of an enzyme is correlated with ΔC‡p and that changes to ΔC‡p are sufficient to change Topt without affecting the catalytic rate. Furthermore, using X-ray crystallography and molecular dynamics simulations we reveal the molecular details underpinning these changes in ΔC‡p. The influence of ΔC‡p on enzymatic rates has implications for the temperature dependence of biological rates from enzymes to ecosystems.

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  • Identification of extracellular siderophores and a related peptide from the endophytic fungus Epichloë festucae in culture and endophyte-infected Lolium perenne

    Koulman, Albert; Lee, T. Verne; Fraser, Karl; Johnson, Linda J.; Arcus, Vickery L.; Lott, J. Shaun; Rasmussen, Carl Edward; Lane, Geoffrey A. (2012)

    Journal article
    University of Waikato

    A number of genes encoding non-ribosomal peptide synthetases (NRPSs) have been identified in fungi of Epichloë/Neotyphodium species, endophytes of Pooid grasses, including sidN, putatively encoding a ferrichrome siderophore-synthesizing NRPS. Targeted gene replacement and complementation of sidN in Epichloë festucae has established that extracellular siderophore epichloënin A is the major product of the SidN enzyme complex (Johnson et al., 2007a). We report here high resolution mass spectrometric fragmentation experiments and NMR analysis of an isolated fraction establishing that epichloënin A is a siderophore of the ferrichrome family, comprising a cyclic sequence of four glycines, a glutamine and three Nδ-trans-anhydromevalonyl–Nδ-hydroxyornithine (AMHO) moieties. Epichloënin A is unusual among ferrichrome siderophores in comprising an octapeptide rather than hexapeptide sequence, and in incorporating a glutamine residue. During this investigation we have established that desferrichrome siderophores with pendant trans-AMHO groups can be distinguished from those with pendant cis-AMHO groups by the characteristic neutral loss of an hydroxyornithine moiety in the MS/MS spectrum. A minor component, epichloënin B, has been characterized as the triglycine variant by mass spectrometry. A peptide characterized by mass spectrometry as the putative deoxygenation product, epichloëamide has been detected together with ferriepichloënin A in guttation fluid from ryegrass (Lolium perenne) plants infected with wild-type E. festucae, but not in plants infected with the ΔsidN mutant strain, and also detected at trace levels in wild-type E. festucae fungal culture.

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  • The Structure of the Oligomerization Domain of Lsr2 from Mycobacterium tuberculosis Reveals a Mechanism for Chromosome Organization and Protection

    Summers, Emma Louise; Meindl, Kathrin; Usón, Isabel; Mitra, Alok K.; Radjainia, Mazdak; Colangeli, Roberto; Alland, David; Arcus, Vickery L. (2012-06-13)

    Journal article
    University of Waikato

    Lsr2 is a small DNA-binding protein present in mycobacteria and related actinobacteria that regulates gene expression and influences the organization of bacterial chromatin. Lsr2 is a dimer that binds to AT-rich regions of chromosomal DNA and physically protects DNA from damage by reactive oxygen intermediates (ROI). A recent structure of the C-terminal DNAbinding domain of Lsr2 provides a rationale for its interaction with the minor groove of DNA, its preference for AT-rich tracts, and its similarity to other bacterial nucleoid-associated DNA-binding domains. In contrast, the details of Lsr2 dimerization (and oligomerization) via its N-terminal domain, and the mechanism of Lsr2-mediated chromosomal crosslinking and protection is unknown. We have solved the structure of the N-terminal domain of Lsr2 (N-Lsr2) at 1.73 A˚ resolution using crystallographic ab initio approaches. The structure shows an intimate dimer of two ß–ß–a motifs with no close homologues in the structural databases. The organization of individual N-Lsr2 dimers in the crystal also reveals a mechanism for oligomerization. Proteolytic removal of three N-terminal residues from Lsr2 results in the formation of an anti-parallel b-sheet between neighboring molecules and the formation of linear chains of N-Lsr2. Oligomerization can be artificially induced using low concentrations of trypsin and the arrangement of N-Lsr2 into long chains is observed in both monoclinic and hexagonal crystallographic space groups. In solution, oligomerization of N-Lsr2 is also observed following treatment with trypsin. A change in chromosomal topology after the addition of trypsin to full-length Lsr2-DNA complexes and protection of DNA towards DNAse digestion can be observed using electron microscopy and electrophoresis. These results suggest a mechanism for oligomerization of Lsr2 via protease-activation leading to chromosome compaction and protection, and concomitant down-regulation of large numbers of genes. This mechanism is likely to be relevant under conditions of stress where cellular proteases are known to be upregulated.

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  • Determination of ribonuclease sequence-specificity using Pentaprobes and mass spectrometry

    McKenzie, Joanna Leigh; Duyvestyn, Johanna Maria; Smith, Tony C.; Bendak, Katerina; Mackay, Joel P.; Cursons, Raymond T.; Cook, Gregory M.; Arcus, Vickery L. (2012)

    Journal article
    University of Waikato

    The VapBC toxin-antitoxin (TA) family is the largest of nine identified TA families. The toxin, VapC, is a metal-dependent ribonuclease that is inhibited by its cognate antitoxin, VapB. Although the VapBCs are the largest TA family, little is known about their biological roles. Here we describe a new general method for the overexpression and purification of toxic VapC proteins and subsequent determination of their RNase sequence-specificity. Functional VapC was isolated by expression of the nontoxic VapBC complex, followed by removal of the labile antitoxin (VapB) using limited trypsin digestion. We have then developed a sensitive and robust method for determining VapC ribonuclease sequence-specificity. This technique employs the use of Pentaprobes as substrates for VapC. These are RNA sequences encoding every combination of five bases. We combine the RNase reaction with MALDI-TOF MS to detect and analyze the cleavage products and thus determine the RNA cut sites. Successful MALDI-TOF MS analysis of RNA fragments is acutely dependent on sample preparation methods. The sequencespecificity of four VapC proteins from two different organisms (VapCPAE0151 and VapCPAE2754 from Pyrobaculum aerophilum, and VapCRv0065 and VapCRv0617 from Mycobacterium tuberculosis) was successfully determined using the described strategy. This rapid and sensitive method can be applied to determine the sequence-specificity of VapC ribonucleases along with other RNA interferases (such as MazF) from a range of organisms. Published by Cold Spring Harbor Laboratory Press.

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  • On the origin and evolution of thermophily: Reconstruction of functional precambrian enzymes from ancestors of Bacillus

    Hobbs, Joanne K.; Shepherd, Caris; Saul, David; Demetras, Nicholas J.; Haaning, Svend; Monk, Colin R.; Daniel, Roy M.; Arcus, Vickery L. (2011)

    Journal article
    University of Waikato

    Thermophily is thought to be a primitive trait, characteristic of early forms of life on Earth, that has been gradually lost over evolutionary time. The genus Bacillus provides an ideal model for studying the evolution of thermophily as it is an ancient taxon and its contemporary species inhabit a range of thermal environments. The thermostability of reconstructed ancestral proteins has been used as a proxy for ancient thermal adaptation. The reconstruction of ancestral “enzymes” has the added advantages of demonstrable activity, which acts as an internal control for accurate inference, and providing insights into the evolution of enzymatic catalysis. Here, we report the reconstruction of the structurally complex core metabolic enzyme LeuB (3-isopropylmalate dehydrogenase, E. C. 1.1.1.85) from the last common ancestor (LCA) of Bacillus using both maximum likelihood (ML) and Bayesian inference. ML LeuB from the LCA of Bacillus shares only 76% sequence identity with its closest contemporary homolog, yet it is fully functional, thermophilic, and exhibits high values for kcat, kcat/KM, and ΔG‡ for unfolding. The Bayesian version of this enzyme is also thermophilic but exhibits anomalous catalytic kinetics. We have determined the 3D structure of the ML enzyme and found that it is more closely aligned with LeuB from deeply branching bacteria, such as Thermotoga maritima, than contemporary Bacillus species. To investigate the evolution of thermophily, three descendents of LeuB from the LCA of Bacillus were also reconstructed. They reveal a fluctuating trend in thermal evolution, with a temporal adaptation toward mesophily followed by a more recent return to thermophily. Structural analysis suggests that the determinants of thermophily in LeuB from the LCA of Bacillus and the most recent ancestor are distinct and that thermophily has arisen in this genus at least twice via independent evolutionary paths. Our results add significant fluctuations to the broad trend in thermal adaptation previously proposed and demonstrate that thermophily is not exclusively a primitive trait, as it can be readily gained as well as lost. Our findings also demonstrate that reconstruction of complex functional Precambrian enzymes is possible and can provide empirical access to the evolution of ancient phenotypes and metabolisms

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  • The PIN-domain ribonucleases and the prokaryotic VapBC toxin–antitoxin array

    Arcus, Vickery L.; McKenzie, Joanna Leigh; Robson, Jennifer R.; Cook, Gregory M. (2010)

    Journal article
    University of Waikato

    The PIN-domains are small proteins of ∼130 amino acids that are found in bacteria, archaea and eukaryotes and are defined by a group of three strictly conserved acidic amino acids. The conserved three-dimensional structures of the PIN-domains cluster these acidic residues in an enzymatic active site. PIN-domains cleave single-stranded RNA in a sequence-specific, Mg²⁺- or Mg²⁺- dependent manner. These ribonucleases are toxic to the cells which express them and to offset this toxicity, they are co-expressed with tight binding protein inhibitors. The genes encoding these two proteins are adjacent in the genome of all prokaryotic organisms where they are found. This sequential arrangement of inhibitor-RNAse genes conforms to that of the so-called toxin–antitoxin (TA) modules and the PIN-domain TAs have been named VapBC TAs (virulence associated proteins, VapB is the inhibitor which contains a transcription factor domain and VapC is the PIN-domain ribonuclease). The presence of large numbers of vapBC loci in disparate prokaryotes has motivated many researchers to investigate their biochemical and biological functions. For example, the devastating human pathogen Mycobacterium tuberculosis has 45 vapBC loci encoded in its genome whereas its non-pathogenic relative, Mycobacterium smegmatis has just one vapBC operon. On another branch of the prokaryotic tree, the nitrogen-fixing symbiont of legumes, Sinorhizobium meliloti has 21 vapBC loci and at least one of these loci have been implicated in the regulation of growth in the plant nodule. A range of biological functions has been suggested for these operons and this review sets out to survey the PIN-domains and summarise the current knowledge about the vapBC TA systems and their roles in diverse bacteria.

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  • Structural and functional characterization of a promiscuous feruloyl esterase (Est1E) from the rumen bacterium Butyrivibrio proteoclasticus

    Goldston, David C.; Villas-Bôas, Silas G; Till, Marisa; Kelly, William J.; Attwood, Graeme T.; Arcus, Vickery L. (2010)

    Journal article
    University of Waikato

    The release of polysaccharide from the plant cell wall is a key process to release the stored energy from plant biomass. Within the ruminant digestive system, a host of commensal microorganisms speed the breakdown of plant cell matter releasing fermentable sugars. The presence of phenolic compounds, most notably ferulic acid (FA), esterified within the cell wall is thought to pose a significant impediment to the degradation of the plant cell wall. The structure of a FA esterase from the ruminant bacterium Butyrivibrio proteoclasticus has been determined in two different space groups, in both the apo-form, and the ligand bound form with FA located in the active site. The structure reveals a new lid domain that has no structural homologues in the PDB. The flexibility of the lid domain is evident by the presence of three different conformations adopted by different molecules in the crystals. In the FA-bound structures, these conformations show sequential binding and closing of the lid domain over the substrate. Enzymatic activity assays demonstrate a broad activity against plant-derived hemicellulose, releasing at least four aromatic compounds including FA, coumaric acid, coumarin-3-carboxylic acid, and cinnamic acid. The rumen is a complex ecosystem that efficiently degrades plant biomass and the genome of B. proteoclasticus contains greater than 130 enzymes, which are potentially involved in this process of which Est1E is the first to be well characterized.

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  • Expression and purification of an adenylation domain from a eukaryotic nonribosomal peptide synthetase: Using structural genomics tools for a challenging target

    Lee, T. Verne; Lott, J. Shaun; Johnson, Richard D.; Arcus, Vickery L. (2010)

    Journal article
    University of Waikato

    Nonribosomal peptide synthetases (NRPSs) are large multimodular and multidomain enzymes that are involved in synthesising an array of molecules that are important in human and animal health. NRPSs are found in both bacteria and fungi but most of the research to date has focused on the bacterial enzymes. This is largely due to the technical challenges in producing active fungal NRPSs, which stem from their large size and multidomain nature. In order to target fungal NRPS domains for biochemical and structural characterisation, we tackled this challenge by using the cloning and expression tools of structural genomics to screen the many variables that can influence the expression and purification of proteins. Using these tools we have screened 32 constructs containing 16 different fungal NRPS domains or domain combinations for expression and solubility. Two of these yielded soluble protein with one, the third adenylation domain of the SidN NRPS (SidNA3) from the grass endophyte Neotyphodium lolii, being tractable for purification using Ni-affinity resin. The initial purified protein exhibited poor solution behaviour but optimisation of the expression construct and the buffer conditions used for purification, resulted in stable recombinant protein suitable for biochemical characterisation, crystallisation and structure determination.

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  • Editorial: Biotechnological Uses of Archaeal Proteins

    Pecorari, Frédéric; Arcus, Vickery L.; Wiegel, Juergen (2015-01-01)

    Journal article
    University of Waikato

    Many industrial/biotechnological processes take place under extreme conditions of temperature, pH, salinity, or pressure which are not suitable for activities of proteins from model eukaryotic or common neutrophilic, mesophilic, and prokaryotic microorganisms. In contrast, Archaea offer a large panel of extremophile organisms that express proteins that are able to remain properly folded and functional under the harshest biophysical conditions.

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  • A structural basis for the reduced toxicity of dinophysistoxin-2

    Huhn, Jason; Jeffrey, Philip D.; Larsen, Kristofer; Rundberget, Thomas; Rise, Frode; Cox, Neil R.; Arcus, Vickery L.; Shi, Yigong; Miles, Christopher O. (2009)

    Journal article
    University of Waikato

    Okadaic acid (OA), dinophysistoxin-1 (DTX-1), and dinophysistoxin-2 (DTX-2) are algal toxins that can accumulate in shellfish and cause diarrhetic shellfish poisoning. Recent studies indicate that DTX-2 is about half as toxic and has about half the affinity for protein phosphatase 2A (PP2A) as OA. NMR structural studies showed that DTX-1 possessed an equatorial 35-methyl group but that DTX-2 had an axial 35-methyl group. Molecular modeling studies indicated that an axial 35-methyl could exhibit unfavorable interactions in the PP2A binding site, and this has been proposed as the reason for the reduced toxicity of DTX-2. Statistical analyses of published data indicate that the affinity of PP2A for DTX-1 is 1.6-fold higher, and for DTX-2 is 2-fold lower, than for OA. We obtained X-ray crystal structures of DTX-1 and DTX-2 bound to PP2A. The crystal structures independently confirm the C-35 stereochemistries determined in the earlier NMR study. The structure for the DTX-1 complex was virtually identical to that of the OA-PP2A complex, except for the presence of the equatorial 35-methyl on the ligand. The favorable placement of the equatorial 35-methyl group of DTX-1 against the aromatic π-bonds of His191 may account for the increased affinity of PP2A toward DTX-1. In contrast, the axial 35-methyl of DTX-2 caused the side chain of His191 to rotate 140° so that it pointed toward the solvent, thereby opening one end of the hydrophobic binding cage. This rearrangement to accommodate the unfavorable interaction from the axial 35-methyl of DTX-2 reduces the binding energy and appears to be responsible for the reduced affinity of PP2A for DTX-2. These results highlight the potential of molecular modeling studies for understanding the relative toxicity of analogues once the binding site at the molecular target has been properly characterized.

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