Publications by authors named "Kurt Krause"

42 Publications

Synthesis and Biological Evaluation of (-) and (+)-Spiroleucettadine and Analogues.

ChemMedChem 2021 Apr 26;16(8):1308-1315. Epub 2021 Jan 26.

Department of Chemistry, University of Otago, Dunedin, 9054, New Zealand.

A second-generation enantiospecific synthesis of spiroleucettadine is described. The original reported antibacterial activity was not observed when the experiment was repeated on the synthetic samples; however, significant anti-proliferative activity was uncovered for both enantiomers of spiroleucettadine. Comparison of the optical rotational data and ORD-CD spectra of both enantiomers and the reported spectrum from the natural source have not provided a definitive answer regarding the absolute stereochemistry of naturally occurring spiroleucettadine. Efforts then focussed on alteration at the C-4 and C-5 positions of the slightly more active (-)-spiroleucettadine. Ten analogues were synthesised, with three analogues found to possess similar anti-proliferative profiles to spiroleucettadine against the H522 lung cancer cell line.
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http://dx.doi.org/10.1002/cmdc.202000954DOI Listing
April 2021

Cryo-electron microscopy structure of the 70S ribosome from Enterococcus faecalis.

Sci Rep 2020 10 1;10(1):16301. Epub 2020 Oct 1.

Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI, 02912, USA.

Enterococcus faecalis is a gram-positive organism responsible for serious infections in humans, but as with many bacterial pathogens, resistance has rendered a number of commonly used antibiotics ineffective. Here, we report the cryo-EM structure of the E. faecalis 70S ribosome to a global resolution of 2.8 Å. Structural differences are clustered in peripheral and solvent exposed regions when compared with Escherichia coli, whereas functional centres, including antibiotic binding sites, are similar to other bacterial ribosomes. Comparison of intersubunit conformations among five classes obtained after three-dimensional classification identifies several rotated states. Large ribosomal subunit protein bL31, which forms intersubunit bridges to the small ribosomal subunit, assumes different conformations in the five classes, revealing how contacts to the small subunit are maintained throughout intersubunit rotation. A tRNA observed in one of the five classes is positioned in a chimeric pe/E position in a rotated ribosomal state. The 70S ribosome structure of E. faecalis now extends our knowledge of bacterial ribosome structures and may serve as a basis for the development of novel antibiotic compounds effective against this pathogen.
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http://dx.doi.org/10.1038/s41598-020-73199-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7530986PMC
October 2020

The post-lockdown period should be used to acquire effective therapies for future resurgence in SARS-Cov-2 infections.

N Z Med J 2020 04 24;133(1513):107-111. Epub 2020 Apr 24.

Department of Microbiology and Immunology, University of Otago.

COVID-19 will be with us through the remainder of 2020 and almost certainly beyond. New Zealand needs a viable strategy to protect its populace until a vaccine is developed and in wide use. Until that time, it makes sense to protect the population by putting in place treatments that will be safe and effective, such as the use of convalescent sera and the use of direct-acting anti-virals. These treatments should be sourced externally or made locally, but steps in this direction must now begin as the lockdown ends. New Zealand has the scientists, the facilities and the will to make this happen, but the support of the government and the population will be needed if this plan is to succeed.
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April 2020

Deriving Immune Modulating Drugs from Viruses-A New Class of Biologics.

J Clin Med 2020 Mar 31;9(4). Epub 2020 Mar 31.

Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, AZ 85281, USA.

Viruses are widely used as a platform for the production of therapeutics. Vaccines containing live, dead and components of viruses, gene therapy vectors and oncolytic viruses are key examples of clinically-approved therapeutic uses for viruses. Despite this, the use of virus-derived proteins as natural sources for immune modulators remains in the early stages of development. Viruses have evolved complex, highly effective approaches for immune evasion. Originally developed for protection against host immune responses, viral immune-modulating proteins are extraordinarily potent, often functioning at picomolar concentrations. These complex viral intracellular parasites have "performed the R&D", developing highly effective immune evasive strategies over millions of years. These proteins provide a new and natural source for immune-modulating therapeutics, similar in many ways to penicillin being developed from mold or streptokinase from bacteria. Virus-derived serine proteinase inhibitors (serpins), chemokine modulating proteins, complement control, inflammasome inhibition, growth factors (e.g., viral vascular endothelial growth factor) and cytokine mimics (e.g., viral interleukin 10) and/or inhibitors (e.g., tumor necrosis factor) have now been identified that target central immunological response pathways. We review here current development of virus-derived immune-modulating biologics with efficacy demonstrated in pre-clinical or clinical studies, focusing on pox and herpesviruses-derived immune-modulating therapeutics.
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http://dx.doi.org/10.3390/jcm9040972DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7230489PMC
March 2020

Differential Inhibition of APOBEC3 DNA-Mutator Isozymes by Fluoro- and Non-Fluoro-Substituted 2'-Deoxyzebularine Embedded in Single-Stranded DNA.

Chembiochem 2020 04 19;21(7):1028-1035. Epub 2019 Dec 19.

School of Fundamental Sciences, Massey University, Private Bag 11 222, Palmerston North, 4442, New Zealand.

The APOBEC3 (APOBEC3A-H) enzyme family is part of the human innate immune system that restricts pathogens by scrambling pathogenic single-stranded (ss) DNA by deamination of cytosines to produce uracil residues. However, APOBEC3-mediated mutagenesis of viral and cancer DNA promotes its evolution, thus enabling disease progression and the development of drug resistance. Therefore, APOBEC3 inhibition offers a new strategy to complement existing antiviral and anticancer therapies by making such therapies effective for longer periods of time, thereby preventing the emergence of drug resistance. Here, we have synthesised 2'-deoxynucleoside forms of several known inhibitors of cytidine deaminase (CDA), incorporated them into oligodeoxynucleotides (oligos) in place of 2'-deoxycytidine in the preferred substrates of APOBEC3A, APOBEC3B, and APOBEC3G, and evaluated their inhibitory potential against these enzymes. An oligo containing a 5-fluoro-2'-deoxyzebularine (5FdZ) motif exhibited an inhibition constant against APOBEC3B 3.5 times better than that of the comparable 2'-deoxyzebularine-containing (dZ-containing) oligo. A similar inhibition trend was observed for wild-type APOBEC3A. In contrast, use of the 5FdZ motif in an oligo designed for APOBEC3G inhibition resulted in an inhibitor that was less potent than the dZ-containing oligo both in the case of APOBEC3G and in that of full-length wild-type APOBEC3G.
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http://dx.doi.org/10.1002/cbic.201900505DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7142307PMC
April 2020

Reconstitution of CRISPR adaptation in vitro and its detection by PCR.

Methods Enzymol 2019 12;616:411-433. Epub 2019 Jan 12.

Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand. Electronic address:

CRISPR adaptation is the initial step in CRISPR-Cas immunity and involves the acquisition of foreign invading DNA. Acquisition is facilitated by the almost universally conserved proteins Cas1 and Cas2, which form an adaptation complex. The Cas1-Cas2 complex binds fragments of invading DNA, completes final processing, and catalyzes integration into specific host loci called CRISPR arrays. Structural and biochemical studies from reconstituted complexes have provided mechanistic insight into how CRISPR adaptation occurs; however, these studies have been limited to a narrow subset of CRISPR-Cas types and may not be representative of the other types. Here we describe methods for the purification of the type I-F CRISPR adaptation complex (Cas1:Cas2-3) from Pectobacterium atrosepticum, purification of the DNA architectural protein integration host factor (IHF), and a sensitive PCR-based in vitro integration assay. This assay could easily be used to investigate mechanisms of CRISPR adaptation in other CRISPR-Cas systems, including the roles of accessory proteins.
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http://dx.doi.org/10.1016/bs.mie.2018.10.024DOI Listing
November 2019

Detection of D-glutamate production from the dual Function enzyme, 4-amino-4-deoxychorismate Lyase/D-amino Acid Transaminase, in .

Bio Protoc 2019 Jan 5;9(1):e3135. Epub 2019 Jan 5.

Department of Biochemistry, University of Otago, Dunedin, New Zealand.

D-amino acid transaminase (D-AAT) is able to synthesize both D-glutamate and D-alanine, according to the following reaction: D-alanine + α-ketoglutarate ⇌ D-glutamate + pyruvate. These two D-amino acids are essential components of the peptidoglycan layer of bacteria. In our recently published work, MSMEG_5795 from was identified as having D-amino acid transaminase (D-AAT) activity, although it has primarily been annotated as 4-amino-4-deoxychorismate lyase (ADCL). To unequivocally demonstrate D-AAT activity from MSMEG_5795 protein two coupled enzyme assays were performed in series. First, D-alanine and α-ketoglutarate were converted to D-glutamate and pyruvate by MSMEG_5795 using the D-AAT assay. Next, the products of this reaction, following removal of all protein, were used as input into an assay for glutamate racemase in which D-glutamate is converted to L-glutamate by glutamate racemase (Gallo and Knowles, 1993; Poen , 2016 ). As the only source of D-glutamate in this assay would be from the reaction of D-alanine with MSMEG_5795, positive results from this assay would confirm the D-AAT activity of MSMEG_5795 and of any enzyme tested in this manner.
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http://dx.doi.org/10.21769/BioProtoc.3135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854199PMC
January 2019

New Zealand glowworm (Arachnocampa luminosa) bioluminescence is produced by a firefly-like luciferase but an entirely new luciferin.

Sci Rep 2018 02 19;8(1):3278. Epub 2018 Feb 19.

Department of Biochemistry, University of Otago, Dunedin, New Zealand.

The New Zealand glowworm, Arachnocampa luminosa, is well-known for displays of blue-green bioluminescence, but details of its bioluminescent chemistry have been elusive. The glowworm is evolutionarily distant from other bioluminescent creatures studied in detail, including the firefly. We have isolated and characterised the molecular components of the glowworm luciferase-luciferin system using chromatography, mass spectrometry and H NMR spectroscopy. The purified luciferase enzyme is in the same protein family as firefly luciferase (31% sequence identity). However, the luciferin substrate of this enzyme is produced from xanthurenic acid and tyrosine, and is entirely different to that of the firefly and known luciferins of other glowing creatures. A candidate luciferin structure is proposed, which needs to be confirmed by chemical synthesis and bioluminescence assays. These findings show that luciferases can evolve independently from the same family of enzymes to produce light using structurally different luciferins.
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http://dx.doi.org/10.1038/s41598-018-21298-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5818473PMC
February 2018

Overexpression of a newly identified d-amino acid transaminase in Mycobacterium smegmatis complements glutamate racemase deletion.

Mol Microbiol 2018 01 7;107(2):198-213. Epub 2017 Dec 7.

Department of Biochemistry, University of Otago, Otago, New Zealand.

Glutamate racemase (MurI) has been proposed as a target for anti-tuberculosis drug development based on the inability of ΔmurI mutants of Mycobacterium smegmatis to grow in the absence of d-glutamate. In this communication, we identify ΔmurI suppressor mutants that are detected during prolonged incubation. Whole genome sequencing of these ΔmurI suppressor mutants identified the presence of a SNP, located in the promoter region of MSMEG_5795. RT-qPCR and transcriptional fusion analyses revealed that the ΔmurI suppressor mutant overexpressed MSMEG_5795 14-fold compared to the isogenic wild-type. MSMEG_5795, which is annotated as 4-amino-4-deoxychorismate lyase (ADCL) but which also has homology to d-amino acid transaminase (d-AAT), was expressed, purified and found to have d-AAT activity and to be capable of producing d-glutamate from d-alanine. Consistent with its d-amino acid transaminase function, overexpressed MSMEG_5795 is able to complement both ΔmurI deletion mutants and alanine racemase (Δalr) deletion mutants, thus confirming a multifunctional role for this enzyme in M. smegmatis.
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http://dx.doi.org/10.1111/mmi.13877DOI Listing
January 2018

Role of Alanine Racemase Mutations in Mycobacterium tuberculosis d-Cycloserine Resistance.

Antimicrob Agents Chemother 2017 12 22;61(12). Epub 2017 Nov 22.

Department of Genetics, University of Cambridge, Cambridge, United Kingdom

A screening of more than 1,500 drug-resistant strains of revealed evolutionary patterns characteristic of positive selection for three alanine racemase (Alr) mutations. We investigated these mutations using molecular modeling, MIC testing, as well as direct measurements of enzymatic activity, which demonstrated that these mutations likely confer resistance to d-cycloserine.
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http://dx.doi.org/10.1128/AAC.01575-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5700341PMC
December 2017

Spacer capture and integration by a type I-F Cas1-Cas2-3 CRISPR adaptation complex.

Proc Natl Acad Sci U S A 2017 06 13;114(26):E5122-E5128. Epub 2017 Jun 13.

Department of Microbiology and Immunology, University of Otago, Dunedin 9054, New Zealand;

CRISPR-Cas adaptive immune systems capture DNA fragments from invading bacteriophages and plasmids and integrate them as spacers into bacterial CRISPR arrays. In type I-E and II-A CRISPR-Cas systems, this adaptation process is driven by Cas1-Cas2 complexes. Type I-F systems, however, contain a unique fusion of Cas2, with the type I effector helicase and nuclease for invader destruction, Cas3. By using biochemical, structural, and biophysical methods, we present a structural model of the 400-kDa Cas1-Cas2-3 complex from with bound protospacer substrate DNA. Two Cas1 dimers assemble on a Cas2 domain dimeric core, which is flanked by two Cas3 domains forming a groove where the protospacer binds to Cas1-Cas2. We developed a sensitive in vitro assay and demonstrated that Cas1-Cas2-3 catalyzed spacer integration into CRISPR arrays. The integrase domain of Cas1 was necessary, whereas integration was independent of the helicase or nuclease activities of Cas3. Integration required at least partially duplex protospacers with free 3'-OH groups, and leader-proximal integration was stimulated by integration host factor. In a coupled capture and integration assay, Cas1-Cas2-3 processed and integrated protospacers independent of Cas3 activity. These results provide insight into the structure of protospacer-bound type I Cas1-Cas2-3 adaptation complexes and their integration mechanism.
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http://dx.doi.org/10.1073/pnas.1618421114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5495228PMC
June 2017

Resolving the cofactor-binding site in the proline biosynthetic enzyme human pyrroline-5-carboxylate reductase 1.

J Biol Chem 2017 04 3;292(17):7233-7243. Epub 2017 Mar 3.

From the Departments of Chemistry and

Pyrroline-5-carboxylate reductase (PYCR) is the final enzyme in proline biosynthesis, catalyzing the NAD(P)H-dependent reduction of Δ-pyrroline-5-carboxylate (P5C) to proline. Mutations in the gene alter mitochondrial function and cause the connective tissue disorder cutis laxa. Furthermore, PYCR1 is overexpressed in multiple cancers, and the knock-out suppresses tumorigenic growth, suggesting that PYCR1 is a potential cancer target. However, inhibitor development has been stymied by limited mechanistic details for the enzyme, particularly in light of a previous crystallographic study that placed the cofactor-binding site in the C-terminal domain rather than the anticipated Rossmann fold of the N-terminal domain. To fill this gap, we report crystallographic, sedimentation-velocity, and kinetics data for human PYCR1. Structures of binary complexes of PYCR1 with NADPH or proline determined at 1.9 Å resolution provide insight into cofactor and substrate recognition. We see NADPH bound to the Rossmann fold, over 25 Å from the previously proposed site. The 1.85 Å resolution structure of a ternary complex containing NADPH and a P5C/proline analog provides a model of the Michaelis complex formed during hydride transfer. Sedimentation velocity shows that PYCR1 forms a concentration-dependent decamer in solution, consistent with the pentamer-of-dimers assembly seen crystallographically. Kinetic and mutational analysis confirmed several features seen in the crystal structure, including the importance of a hydrogen bond between Thr-238 and the substrate as well as limited cofactor discrimination.
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http://dx.doi.org/10.1074/jbc.M117.780288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5409489PMC
April 2017

A Broad-Spectrum Chemokine-Binding Protein of Bovine Papular Stomatitis Virus Inhibits Neutrophil and Monocyte Infiltration in Inflammatory and Wound Models of Mouse Skin.

PLoS One 2016 9;11(12):e0168007. Epub 2016 Dec 9.

Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand.

Bovine papular stomatitis virus (BPSV) is a Parapoxvirus that induces acute pustular skin lesions in cattle and is transmissible to humans. Previous studies have shown that BPSV encodes a distinctive chemokine-binding protein (CBP). Chemokines are critically involved in the trafficking of immune cells to sites of inflammation and infected tissue, suggesting that the CBP plays a role in immune evasion by preventing immune cells reaching sites of infection. We hypothesised that the BPSV-CBP binds a wide range of inflammatory chemokines particularly those involved in BPSV skin infection, and inhibits the recruitment of immune cells from the blood into inflamed skin. Molecular analysis of the purified protein revealed that the BPSV-CBP is a homodimeric polypeptide with a MW of 82.4 kDa whilst a comprehensive screen of inflammatory chemokines by surface plasmon resonance showed high-affinity binding to a range of chemokines within the CXC, CC and XC subfamilies. Structural analysis of BPSV-CBP, based on the crystal structure of orf virus CBP, provided a probable explanation for these chemokine specificities at a molecular level. Functional analysis of the BPSV-CBP using transwell migration assays demonstrated that it potently inhibited chemotaxis of murine neutrophils and monocytes in response to CXCL1, CXCL2 as well as CCL2, CCL3 and CCL5 chemokines. In order to examine the effects of CBP in vivo, we used murine skin models to determine its impact on inflammatory cell recruitment such as that observed during BPSV infection. Intradermal injection of BPSV-CBP blocked the influx of neutrophils and monocytes in murine skin in which inflammation was induced with lipopolysaccharide. Furthermore, intradermal injection of BPSV-CBP into injured skin, which more closely mimics BPSV lesions, delayed the influx of neutrophils and reduced the recruitment of MHC-II+ immune cells to the wound bed. Our findings suggest that the CBP could be important in pathogenesis of BPSV infections.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0168007PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5148066PMC
July 2017

Exploring the structure of glutamate racemase from Mycobacterium tuberculosis as a template for anti-mycobacterial drug discovery.

Biochem J 2016 05 10;473(9):1267-80. Epub 2016 Mar 10.

Department of Biochemistry, University of Otago, PO Box 56, Dunedin 9054, New Zealand

Glutamate racemase (MurI) is responsible for providing D-glutamate for peptidoglycan biosynthesis in bacteria and has been a favoured target in pharmaceutical drug design efforts. It has recently been proven to be essential in Mycobacterium tuberculosis, the causative organism of tuberculosis, a disease for which new medications are urgently needed. In the present study, we have determined the protein crystal structures of MurI from both M. tuberculosis and Mycobacterium smegmatis in complex with D-glutamate to 2.3 Å and 1.8 Å resolution respectively. These structures are conserved, but reveal differences in their active site architecture compared with that of other MurI structures. Furthermore, compounds designed to target other glutamate racemases have been screened but do not inhibit mycobacterial MurI, suggesting that a new drug design effort will be needed to develop inhibitors. A new type of MurI dimer arrangement has been observed in both structures, and this arrangement becomes the third biological dimer geometry for MurI found to date. The mycobacterial MurI dimer is tightly associated, with a KD in the nanomolar range. The enzyme binds D- and L-glutamate specifically, but is inactive in solution unless the dimer interface is mutated. We created triple mutants of this interface in the M. smegmatis glutamate racemase (D26R/R105A/G194R or E) that have appreciable activity (kcat=0.056-0.160 min(-1) and KM=0.26-0.51 mM) and can be utilized to screen proposed antimicrobial candidates for inhibition.
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http://dx.doi.org/10.1042/BCJ20160186DOI Listing
May 2016

Structural plasticity and in vivo activity of Cas1 from the type I-F CRISPR-Cas system.

Biochem J 2016 Apr 29;473(8):1063-72. Epub 2016 Feb 29.

Department of Biochemistry, University of Otago, PO Box 56, Dunedin 9054, New Zealand

CRISPR-Cas systems are adaptive immune systems in prokaryotes that provide protection against viruses and other foreign DNA. In the adaptation stage, foreign DNA is integrated into CRISPR (clustered regularly interspaced short palindromic repeat) arrays as new spacers. These spacers are used in the interference stage to guide effector CRISPR associated (Cas) protein(s) to target complementary foreign invading DNA. Cas1 is the integrase enzyme that is central to the catalysis of spacer integration. There are many diverse types of CRISPR-Cas systems, including type I-F systems, which are typified by a unique Cas1-Cas2-3 adaptation complex. In the present study we characterize the Cas1 protein of the potato phytopathogen Pectobacterium atrosepticum, an important model organism for understanding spacer acquisition in type I-F CRISPR-Cas systems. We demonstrate by mutagenesis that Cas1 is essential for adaptation in vivo and requires a conserved aspartic acid residue. By X-ray crystallography, we show that although P. atrosepticum Cas1 adopts a fold conserved among other Cas1 proteins, it possesses remarkable asymmetry as a result of structural plasticity. In particular, we resolve for the first time a flexible, asymmetric loop that may be unique to type I-F Cas1 proteins, and we discuss the implications of these structural features for DNA binding and enzymatic activity.
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http://dx.doi.org/10.1042/BCJ20160078DOI Listing
April 2016

Smartphone Microscopy of Parasite Eggs Accumulated into a Single Field of View.

Am J Trop Med Hyg 2016 Jan 16;94(1):227-230. Epub 2015 Nov 16.

A Nokia Lumia 1020 cellular phone (Microsoft Corp., Auckland, New Zealand) was configured to image the ova of Ascaris lumbricoides converged into a single field of view but on different focal planes. The phone was programmed to acquire images at different distances and, using public domain computer software, composite images were created that brought all the eggs into sharp focus. This proof of concept informs a framework for field-deployable, point of care monitoring of soil-transmitted helminths.
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http://dx.doi.org/10.4269/ajtmh.15-0427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4710435PMC
January 2016

Comparative RNA seq analysis of the New Zealand glowworm Arachnocampa luminosa reveals bioluminescence-related genes.

BMC Genomics 2015 Oct 21;16:825. Epub 2015 Oct 21.

Department of Biochemistry, University of Otago, Dunedin, New Zealand.

Background: The New Zealand glowworm is the larva of a carnivorous fungus gnat that produces bioluminescence to attract prey. The bioluminescent system of the glowworm is evolutionarily distinct from other well-characterised systems, especially that of the fireflies, and the molecules involved have not yet been identified. We have used high throughput sequencing technology to produce a transcriptome for the glowworm and identify transcripts encoding proteins that are likely to be involved in glowworm bioluminescence.

Results: Here we report the sequencing and annotation of the first transcriptome of the glowworm, and a differential analysis of expression from the glowworm light organ compared with non-light organ tissue. The analysis identified six transcripts encoding proteins that are potentially involved in glowworm bioluminescence. Three of these proteins are members of the ANL superfamily of adenylating enzymes, with similar amino acid sequences to that of the luciferase enzyme found in fireflies (31 to 37 % identical), and are candidate luciferases for the glowworm bioluminescent system. The remaining three transcripts encode putative aminoacylase, phosphatidylethanolamine-binding and glutathione S-transferase proteins.

Conclusions: This research provides a basis for further biochemical studies into how the glowworm produces light, and a source of genetic information to aid future ecological and evolutionary studies of the glowworm.
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http://dx.doi.org/10.1186/s12864-015-2006-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4617951PMC
October 2015

Structures of Orf Virus Chemokine Binding Protein in Complex with Host Chemokines Reveal Clues to Broad Binding Specificity.

Structure 2015 Jul 18;23(7):1199-213. Epub 2015 Jun 18.

Department of Biochemistry, University of Otago, Dunedin 9054, New Zealand. Electronic address:

The chemokine binding protein (CKBP) from orf virus (ORFV) binds with high affinity to chemokines from three classes, C, CC, and CXC, making it unique among poxvirus CKBPs described to date. We present its crystal structure alone and in complex with three CC chemokines, CCL2, CCL3, and CCL7. ORFV CKBP possesses a β-sandwich fold that is electrostatically and sterically complementary to its binding partners. Chemokines bind primarily through interactions involving the N-terminal loop and a hydrophobic recess on the ORFV CKBP β-sheet II surface, and largely polar interactions between the chemokine 20s loop and a negatively charged surface groove located at one end of the CKBP β-sheet II surface. ORFV CKBP interacts with leukocyte receptor and glycosaminoglycan binding sites found on the surface of bound chemokines. SEC-MALLS and chromatographic evidence is presented supporting that ORFV CKBP is a dimer in solution over a broad range of protein concentrations.
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http://dx.doi.org/10.1016/j.str.2015.04.023DOI Listing
July 2015

Investigation of the essentiality of glutamate racemase in Mycobacterium smegmatis.

J Bacteriol 2014 Dec 22;196(24):4239-44. Epub 2014 Sep 22.

Department of Biochemistry, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand

The mycobacterial cell wall frequently has been used as a target for drug development, and d-glutamate, synthesized by glutamate racemase (MurI), is an important component of peptidoglycan. While the essentiality of the murI gene has been shown in several bacterial species, including Escherichia coli, Bacillus anthracis, and Streptococcus pneumoniae, studies in mycobacteria have not yet provided definitive results. This study aimed to determine whether murI is indeed essential and can serve as a possible target for structure-aided drug design. We have achieved this goal by creating a ΔmurI strain of Mycobacterium smegmatis, a close relative of Mycobacterium tuberculosis. The deletion of the murI gene in M. smegmatis could be achieved only in minimal medium supplemented with D-glutamate, demonstrating that MurI is essential for growth and that glutamate racemase is the only source of D-glutamate for peptidoglycan synthesis in M. smegmatis.
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http://dx.doi.org/10.1128/JB.02090-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4248854PMC
December 2014

The structure of alanine racemase from Acinetobacter baumannii.

Acta Crystallogr F Struct Biol Commun 2014 Sep 29;70(Pt 9):1199-205. Epub 2014 Aug 29.

Department of Biochemistry, University of Otago, Dunedin, New Zealand.

Acinetobacter baumannii is an opportunistic Gram-negative bacterium which is a common cause of hospital-acquired infections. Numerous antibiotic-resistant strains exist, emphasizing the need for the development of new antimicrobials. Alanine racemase (Alr) is a pyridoxal 5'-phosphate dependent enzyme that is responsible for racemization between enantiomers of alanine. As D-alanine is an essential component of the bacterial cell wall, its inhibition is lethal to prokaryotes, making it an excellent antibiotic drug target. The crystal structure of A. baumannii alanine racemase (AlrAba) from the highly antibiotic-resistant NCTC13302 strain has been solved to 1.9 Å resolution. Comparison of AlrAba with alanine racemases from closely related bacteria demonstrates a conserved overall fold. The substrate entryway and active site of the enzymes were shown to be highly conserved. The structure of AlrAba will provide the template required for future structure-based drug-design studies.
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http://dx.doi.org/10.1107/S2053230X14017725DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4157418PMC
September 2014

Inherent structural disorder and dimerisation of murine norovirus NS1-2 protein.

PLoS One 2012 7;7(2):e30534. Epub 2012 Feb 7.

Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand.

Human noroviruses are highly infectious viruses that cause the majority of acute, non-bacterial epidemic gastroenteritis cases worldwide. The first open reading frame of the norovirus RNA genome encodes for a polyprotein that is cleaved by the viral protease into six non-structural proteins. The first non-structural protein, NS1-2, lacks any significant sequence similarity to other viral or cellular proteins and limited information is available about the function and biophysical characteristics of this protein. Bioinformatic analyses identified an inherently disordered region (residues 1-142) in the highly divergent N-terminal region of the norovirus NS1-2 protein. Expression and purification of the NS1-2 protein of Murine norovirus confirmed these predictions by identifying several features typical of an inherently disordered protein. These were a biased amino acid composition with enrichment in the disorder promoting residues serine and proline, a lack of predicted secondary structure, a hydrophilic nature, an aberrant electrophoretic migration, an increased Stokes radius similar to that predicted for a protein from the pre-molten globule family, a high sensitivity to thermolysin proteolysis and a circular dichroism spectrum typical of an inherently disordered protein. The purification of the NS1-2 protein also identified the presence of an NS1-2 dimer in Escherichia coli and transfected HEK293T cells. Inherent disorder provides significant advantages including structural flexibility and the ability to bind to numerous targets allowing a single protein to have multiple functions. These advantages combined with the potential functional advantages of multimerisation suggest a multi-functional role for the NS1-2 protein.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0030534PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3274520PMC
July 2012

Structural features and kinetic characterization of alanine racemase from Staphylococcus aureus (Mu50).

Acta Crystallogr D Biol Crystallogr 2012 Jan 9;68(Pt 1):82-92. Epub 2011 Dec 9.

Department of Biochemistry, University of Otago, Dunedin, New Zealand.

Staphylococcus aureus is an opportunistic Gram-positive bacterium which causes a wide variety of diseases ranging from minor skin infections to potentially fatal conditions such as pneumonia, meningitis and septicaemia. The pathogen is a leading cause of nosocomial acquired infections, a problem that is exacerbated by the existence of methicillin- and glycopeptide antibiotic-resistant strains which can be challenging to treat. Alanine racemase (Alr) is a pyridoxal-5'-phosphate-dependent enzyme which catalyzes reversible racemization between enantiomers of alanine. As D-alanine is an essential component of the bacterial cell-wall peptidoglycan, inhibition of Alr is lethal to prokaryotes. Additionally, while ubiquitous amongst bacteria, this enzyme is absent in humans and most eukaryotes, making it an excellent antibiotic drug target. The crystal structure of S. aureus alanine racemase (Alr(Sas)), the sequence of which corresponds to that from the highly antibiotic-resistant Mu50 strain, has been solved to 2.15 Å resolution. Comparison of the Alr(Sas) structure with those of various alanine racemases demonstrates a conserved overall fold, with the enzyme sharing most similarity to those from other Gram-positive bacteria. Structural examination indicates that the active-site binding pocket, dimer interface and active-site entryway of the enzyme are potential targets for structure-aided inhibitor design. Kinetic constants were calculated in this study and are reported here. The potential for a disulfide bond in this structure is noted. This structural and biochemical information provides a template for future structure-based drug-development efforts targeting Alr(Sas).
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http://dx.doi.org/10.1107/S0907444911050682DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3245724PMC
January 2012

Structural features and kinetic characterization of alanine racemase from Staphylococcus aureus (Mu50).

Acta Crystallogr D Biol Crystallogr 2012 Jan 9;68(Pt 1):82-92. Epub 2011 Dec 9.

Department of Biochemistry, University of Otago, Dunedin, New Zealand.

Staphylococcus aureus is an opportunistic Gram-positive bacterium which causes a wide variety of diseases ranging from minor skin infections to potentially fatal conditions such as pneumonia, meningitis and septicaemia. The pathogen is a leading cause of nosocomial acquired infections, a problem that is exacerbated by the existence of methicillin- and glycopeptide antibiotic-resistant strains which can be challenging to treat. Alanine racemase (Alr) is a pyridoxal-5'-phosphate-dependent enzyme which catalyzes reversible racemization between enantiomers of alanine. As D-alanine is an essential component of the bacterial cell-wall peptidoglycan, inhibition of Alr is lethal to prokaryotes. Additionally, while ubiquitous amongst bacteria, this enzyme is absent in humans and most eukaryotes, making it an excellent antibiotic drug target. The crystal structure of S. aureus alanine racemase (Alr(Sas)), the sequence of which corresponds to that from the highly antibiotic-resistant Mu50 strain, has been solved to 2.15 Å resolution. Comparison of the Alr(Sas) structure with those of various alanine racemases demonstrates a conserved overall fold, with the enzyme sharing most similarity to those from other Gram-positive bacteria. Structural examination indicates that the active-site binding pocket, dimer interface and active-site entryway of the enzyme are potential targets for structure-aided inhibitor design. Kinetic constants were calculated in this study and are reported here. The potential for a disulfide bond in this structure is noted. This structural and biochemical information provides a template for future structure-based drug-development efforts targeting Alr(Sas).
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http://dx.doi.org/10.1107/S0907444911050682DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3245724PMC
January 2012

The crystal structure of alanine racemase from Streptococcus pneumoniae, a target for structure-based drug design.

BMC Microbiol 2011 May 25;11:116. Epub 2011 May 25.

Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul, Korea.

Background: Streptococcus pneumoniae is a globally important pathogen. The Gram-positive diplococcus is a leading cause of pneumonia, otitis media, bacteremia, and meningitis, and antibiotic resistant strains have become increasingly common over recent years. Alanine racemase is a ubiquitous enzyme among bacteria and provides the essential cell wall precursor, D-alanine. Since it is absent in humans, this enzyme is an attractive target for the development of drugs against S. pneumoniae and other bacterial pathogens.

Results: Here we report the crystal structure of alanine racemase from S. pneumoniae (AlrSP). Crystals diffracted to a resolution of 2.0 Å and belong to the space group P3121 with the unit cell parameters a = b = 119.97 Å, c = 118.10 Å, α = β = 90° and γ = 120°. Structural comparisons show that AlrSP shares both an overall fold and key active site residues with other bacterial alanine racemases. The active site cavity is similar to other Gram positive alanine racemases, featuring a restricted but conserved entryway.

Conclusions: We have solved the structure of AlrSP, an essential step towards the development of an accurate pharmacophore model of the enzyme, and an important contribution towards our on-going alanine racemase structure-based drug design project. We have identified three regions on the enzyme that could be targeted for inhibitor design, the active site, the dimer interface, and the active site entryway.
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http://dx.doi.org/10.1186/1471-2180-11-116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3146814PMC
May 2011

Crystallization and preliminary X-ray analysis of the chemokine-binding protein from orf virus (Poxviridae).

Acta Crystallogr Sect F Struct Biol Cryst Commun 2010 Jul 24;66(Pt 7):819-23. Epub 2010 Jun 24.

Department of Biochemistry, University of Otago, New Zealand.

The parapoxvirus orf virus (ORFV) encodes a chemokine-binding protein (CBP) that functions to downregulate the host's immune response at the site of infection by blocking the chemokine-induced recruitment of immune cells. In order to shed light on the structural determinants of CBP-chemokine binding, ORFV CBP was crystallized as part of an ongoing structure-function study on this protein. ORFV CBP crystals were obtained by the sitting-drop vapour-diffusion technique using ammonium citrate as a precipitant. The crystal quality was greatly improved through the addition of small-molecule additives to the crystallization mother liquor. ORFV CBP crystals diffracted X-rays to 2.50 A resolution and belonged to the hexagonal space group P6(1)22 or its enantiomorph P6(5)22, with unit-cell parameters a = b = 75.62, c = 282.49 A, alpha = 90, beta = 90, gamma = 120 degrees.
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http://dx.doi.org/10.1107/S1744309110018166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2898470PMC
July 2010

Biochemical and structural characterization of alanine racemase from Bacillus anthracis (Ames).

BMC Struct Biol 2009 Aug 20;9:53. Epub 2009 Aug 20.

Department of Biochemistry, University of Otago, Dunedin, New Zealand.

Background: Bacillus anthracis is the causative agent of anthrax and a potential bioterrorism threat. Here we report the biochemical and structural characterization of B. anthracis (Ames) alanine racemase (AlrBax), an essential enzyme in prokaryotes and a target for antimicrobial drug development. We also compare the native AlrBax structure to a recently reported structure of the same enzyme obtained through reductive lysine methylation.

Results: B. anthracis has two open reading frames encoding for putative alanine racemases. We show that only one, dal1, is able to complement a D-alanine auxotrophic strain of E. coli. Purified Dal1, which we term AlrBax, is shown to be a dimer in solution by dynamic light scattering and has a Vmax for racemization (L- to D-alanine) of 101 U/mg. The crystal structure of unmodified AlrBax is reported here to 1.95 A resolution. Despite the overall similarity of the fold to other alanine racemases, AlrBax makes use of a chloride ion to position key active site residues for catalysis, a feature not yet observed for this enzyme in other species. Crystal contacts are more extensive in the methylated structure compared to the unmethylated structure.

Conclusion: The chloride ion in AlrBax is functioning effectively as a carbamylated lysine making it an integral and unique part of this structure. Despite differences in space group and crystal form, the two AlrBax structures are very similar, supporting the case that reductive methylation is a valid rescue strategy for proteins recalcitrant to crystallization, and does not, in this case, result in artifacts in the tertiary structure.
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http://dx.doi.org/10.1186/1472-6807-9-53DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2743695PMC
August 2009

Advantage of being a dimer for Serratia marcescens endonuclease.

J Phys Chem B 2009 Jan;113(2):511-21

Department of Chemistry, University of Houston, Houston, Texas 77204-5003, USA.

The monomer and dimer of the bacterium Serratia marcescens endonuclease (SMnase) are each catalytically active, and the two subunits of the dimer function independently of each other. Nature, however, chooses the dimer form instead of the monomer. In order to explain this, we performed molecular dynamics (MD) simulations of both model-built complexes of a subunit of SMnase and the dimer with DNA in aqueous solution. We estimated the electrostatic binding energy, analyzed the distribution and dynamics of water around the complexes, identified water clusters in the protein, and related the dynamics of water to the protein's function. We find that the dimer form has an electrostatic advantage over the monomer to associate with DNA. Although Mg(2+) remains hexa-coordinated during the simulation, the binding pathway of DNA to Mg(2+) changes from inner-sphere binding in the monomer to outer-sphere in the dimer, which may be more energetically favorable. In addition, two water clusters in the active site of each monomer and in the dimer complex were identified and localized in two regions, named the "stabilizing" and "working" regions. Water in the "working" region in the dimer complex has larger fluctuations than that in the monomer.
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http://dx.doi.org/10.1021/jp8057838DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2645866PMC
January 2009

The alanine racemase of Mycobacterium smegmatis is essential for growth in the absence of D-alanine.

J Bacteriol 2007 Nov 7;189(22):8381-6. Epub 2007 Sep 7.

Department of Biochemistry, Otago School of Medical Sciences, University of Otago, P.O. Box 56, Dunedin, New Zealand.

Alanine racemase, encoded by the gene alr, is an important enzyme in the synthesis of d-alanine for peptidoglycan biosynthesis. Strains of Mycobacterium smegmatis with a deletion mutation of the alr gene were found to require d-alanine for growth in both rich and minimal media. This indicates that alanine racemase is the only source of d-alanine for cell wall biosynthesis in M. smegmatis and confirms alanine racemase as a viable target gene for antimycobacterial drug development.
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http://dx.doi.org/10.1128/JB.01201-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2168708PMC
November 2007

Purification and preliminary crystallization of alanine racemase from Streptococcus pneumoniae.

BMC Microbiol 2007 May 17;7:40. Epub 2007 May 17.

University of Houston, Department of Biology and Biochemistry, Houston, TX 77204-5001, USA.

Background: Over the past fifteen years, antibiotic resistance in the Gram-positive opportunistic human pathogen Streptococcus pneumoniae has significantly increased. Clinical isolates from patients with community-acquired pneumonia or otitis media often display resistance to two or more antibiotics. Given the need for new therapeutics, we intend to investigate enzymes of cell wall biosynthesis as novel drug targets. Alanine racemase, a ubiquitous enzyme among bacteria and absent in humans, provides the essential cell wall precursor, D-alanine, which forms part of the tetrapeptide crosslinking the peptidoglycan layer.

Results: The alanine racemases gene from S. pneumoniae (alrSP) was amplified by PCR and cloned and expressed in Escherichia coli. The 367 amino acid, 39854 Da dimeric enzyme was purified to electrophoretic homogeneity and preliminary crystals were obtained. Racemic activity was demonstrated through complementation of an alr auxotroph of E. coli growing on L-alanine. In an alanine racemases photometric assay, specific activities of 87.0 and 84.8 U mg-1 were determined for the conversion of D- to L-alanine and L- to D-alanine, respectively.

Conclusion: We have isolated and characterized the alanine racemase gene from the opportunistic human pathogen S. pneumoniae. The enzyme shows sufficient homology with other alanine racemases to allow its integration into our ongoing structure-based drug design project.
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http://dx.doi.org/10.1186/1471-2180-7-40DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1885262PMC
May 2007