Antonio Ariza | University of Oxford (original) (raw)

Papers by Antonio Ariza

Nature

The anti-cancer drug target poly(ADP-ribose) polymerase 1 (PARP1) and its close homologue, PARP2,... more The anti-cancer drug target poly(ADP-ribose) polymerase 1 (PARP1) and its close homologue, PARP2, are early responders to DNA damage in human cells1,2. After binding to genomic lesions, these enzymes use NAD+ to modify numerous proteins with mono- and poly(ADP-ribose) signals that are important for the subsequent decompaction of chromatin and the recruitment of repair factors3,4. These post-translational modifications are predominantly serine-linked and require the accessory factor HPF1, which is specific for the DNA damage response and switches the amino acid specificity of PARP1 and PARP2 from aspartate or glutamate to serine residues5,6,7,8,9,10. Here we report a co-structure of HPF1 bound to the catalytic domain of PARP2 that, in combination with NMR and biochemical data, reveals a composite active site formed by residues from HPF1 and PARP1 or PARP2 . The assembly of this catalytic centre is essential for the addition of ADP-ribose moieties after DNA damage in human cells. In response to DNA damage and occupancy of the NAD+-binding site, the interaction of HPF1 with PARP1 or PARP2 is enhanced by allosteric networks that operate within the PARP proteins, providing an additional level of regulation in the induction of the DNA damage response. As HPF1 forms a joint active site with PARP1 or PARP2, our data implicate HPF1 as an important determinant of the response to clinical PARP inhibitors.

International Journal of Molecular Sciences, 2019

Amylases are probably the best studied glycoside hydrolases and have a huge biotechnological valu... more Amylases are probably the best studied glycoside hydrolases and have a huge biotechnological value for industrial processes on starch. Multiple amylases from fungi and microbes are currently in use. Whereas bacterial amylases are well suited for many industrial processes due to their high stability, fungal amylases are recognized as safe and are preferred in the food industry, although they lack the pH tolerance and stability of their bacterial counterparts. Here, we describe three amylases, two of which have a broad pH spectrum extending to pH 8 and higher stability well suited for a broad set of industrial applications. These enzymes have the characteristic GH13 α-amylase fold with a central (β/α) 8-domain, an insertion domain with the canonical calcium binding site and a C-terminal β-sandwich domain. The active site was identified based on the binding of the inhibitor acarbose in form of a transglycosylation product, in the amylases from Thamnidium elegans and Cordyceps farinosa. The three amylases have shortened loops flanking the nonreducing end of the substrate binding cleft, creating a more open crevice. Moreover, a potential novel binding site in the C-terminal domain of the Cordyceps enzyme was identified, which might be part of a starch interaction site. In addition, Cordyceps farinosa amylase presented a successful example of using the microseed matrix screening technique to significantly speed-up crystallization.

Cell Chemical Biology, 2018

Protein ADP-ribosylation is a highly dynamic post-translational modification. The rapid turnover ... more Protein ADP-ribosylation is a highly dynamic post-translational modification. The rapid turnover is achieved, among others, by ADP-(ribosyl)hydrolases (ARHs), an ancient family of enzymes that reverses this modification. Recently ARHs came into focus due to their role as regulators of cellular stresses and tumor suppressors. Here we present a comprehensive structural analysis of the enzymatically active family members ARH1 and ARH3. These two enzymes have very distinct substrate requirements. Our data show that binding of the adenosine ribose moiety is highly diverged between the two enzymes, whereas the active sites harboring the distal ribose closely resemble each other. Despite this apparent similarity, we elucidate the structural basis for the selective inhibition of ARH3 by the ADP-ribose analogues ADP-HPD and arginine-ADP-ribose. Together, our biochemical and structural work provides important insights into the mode of enzyme-ligand interaction, helps to understand differences in their catalytic behavior, and provides useful tools for targeted drug design.

Strategies to resolve replication blocks are critical for the maintenance of genome stability. Am... more Strategies to resolve replication blocks are critical for the maintenance of genome stability. Among the factors implicated in the replication stress response is the ATP-dependent endonuclease ZRANB3. Here, we present the structure of the ZRANB3 HNH (His-Asn-His) endonuclease domain and provide a detailed analysis of its activity. We further define PCNA as a key regulator of ZRANB3 function, which recruits ZRANB3 to stalled replication forks and stimulates its endonuclease activity. Finally, we present the co-crystal structures of PCNA with two specific motifs in ZRANB3: the PIP box and the APIM motif. Our data provide important structural insights into the PCNA-APIM interaction, and reveal unexpected similarities between the PIP box and the APIM motif. We propose that PCNA and ATP-dependency serve as a multi-layered regulatory mechanism that modulates ZRANB3 activity at replication forks. Importantly, our findings allow us to interpret the functional significance of cancer associated ZRANB3 mutations.

The discovery and study of toxin-antitoxin (TA) systems helps us advance our understanding of the... more The discovery and study of toxin-antitoxin (TA) systems helps us advance our understanding of the strategies prokaryotes employ to regulate cellular processes related to the general stress response, such as defense against phages, growth control, biofilm formation, persistence, and programmed cell death. Here we identify and characterize a TA system found in various bacteria, including the global pathogen Mycobacterium tuberculosis. The toxin of the system (DarT) is a domain of unknown function (DUF) 4433, and the antitoxin (DarG) a macrodomain protein. We demonstrate that DarT is an enzyme that specifically modifies thymidines on single-stranded DNA in a sequence-specific manner by a nucleotide-type modification called ADP-ribosylation. We also show that this modification can be removed by DarG. Our results provide an example of reversible DNA ADP-ribosylation, and we anticipate potential therapeutic benefits by targeting this enzyme-enzyme TA system in bacterial pathogens such as M. tuberculosis.

ADP-ribosylation by ADP-ribosyltransferases (ARTs) has a well-established role in DNA strand brea... more ADP-ribosylation by ADP-ribosyltransferases (ARTs) has a well-established role in DNA strand break repair by promoting enrichment of repair factors at damage sites through ADP-ribose interaction domains. Here, we exploit the simple eukaryote Dictyostelium to uncover a role for ADP-ribosylation in regulating DNA interstrand crosslink repair and redundancy of this pathway with non-homologous end-joining (NHEJ). In silico searches were used to identify a protein that contains a permutated macrodomain (which we call aprataxin/APLF-and-PNKP-like protein; APL). Structural analysis reveals that this permutated macrodomain retains features associated with ADP-ribose interactions and that APL is capable of binding poly(ADP-ribose) through this macrodomain. APL is enriched in chromatin in response to cisplatin treatment, an agent that induces DNA interstrand crosslinks (ICLs). This is dependent on the macrodomain of APL and the ART Adprt2, indicating a role for ADP-ribosylation in the cellular response to cisplatin. Although adprt2 − cells are sensitive to cisplatin, ADP-ribosylation is evident in these cells owing to redundant signalling by the double-strand break (DSB)-responsive ART Adprt1a, promoting NHEJ-mediated repair. These data implicate ADP-ribosylation in DNA ICL repair and identify that NHEJ can function to resolve this form of DNA damage in the absence of Adprt2.

The industrial conversion of cellulosic plant biomass into useful products such as biofuels is a ... more The industrial conversion of cellulosic plant biomass into useful products such as biofuels is a major societal goal. These technologies harness diverse plant degrading enzymes, classical exo-and endo-acting cellulases and, increasingly, cellulose-active lytic polysaccharide monooxygenases, to deconstruct the recalcitrant-d-linked polysaccharide. A major drawback with this process is that the exo-acting cellobiohydrolases suffer from severe inhibition from their cellobiose product.-d-Glucosidases are therefore important for liberating glucose from cellobiose and thereby relieving limiting product inhibition. Here, the three-dimensional structures of two industrially important family GH3-d-glucosidases from Aspergillus fumigatus and A. oryzae, solved by molecular replacement and refined at 1.95 A ˚ resolution, are reported. Both enzymes, which share 78% sequence identity, display a three-domain structure with the catalytic domain at the interface, as originally shown for barley-d-glucan exohydrolase, the first three-dimensional structure solved from glycoside hydrolase family GH3. Both enzymes show extensive N-glycosylation, with only a few external sites being truncated to a single GlcNAc molecule. Those glycans N-linked to the core of the structure are identified purely as high-mannose trees, and establish multiple hydrogen bonds between their sugar components and adjacent protein side chains. The extensive glycans pose special problems for crystallographic refinement, and new techniques and protocols were developed especially for this work. These protocols ensured that all of the d-pyranosides in the glycosylation trees were modelled in the preferred minimum-energy 4 C 1 chair conformation and should be of general application to refinements of other crystal structures containing O-or N-glycosylation. The Aspergillus GH3 structures, in light of other recent three-dimensional structures, provide insight into fungal-d-glucosidases and provide a platform on which to inform and inspire new generations of variant enzymes for industrial application.

Hazara virus (HAZV) is a member of the Bunyaviridae family of segmented negative stranded RNA vir... more Hazara virus (HAZV) is a member of the Bunyaviridae family of segmented negative stranded RNA viruses, and shares the same serogroup as Crimean-Congo haemorrhagic fever virus (CCHFV). CCHFV is responsible for fatal human disease with a mortality rate approaching 30 %, which has an increased recent incidence within southern Europe. There are no preventative or therapeutic treatments for CCHFV-mediated disease, and thus CCHFV is classified as a hazard group 4 pathogen. In contrast HAZV is not associated with serious human disease, although infection of interferon receptor knockout mice with either CCHFV or HAZV results in similar disease progression. To characterise further similarities between HAZV and CCHFV, and support the use of HAZV as a model for CCHFV infection, we investigated the structure of the HAZV nucleocapsid protein (N) and compared it to CCHFV N. N performs an essential role in the viral life cycle by encapsidating the viral RNA genome, and thus, N represents a potential therapeutic target. Results: We present the purification, crystallisation and crystal structure of HAZV N at 2.7 Å resolution. HAZV N was expressed as an N-terminal glutathione S-transferase (GST) fusion protein then purified using glutathione affinity chromatography followed by ion-exchange chromatography. HAZV N crystallised in the P2 1 2 1 2 1 space group with unit cell parameters a = 64.99, b = 76.10, and c = 449.28 Å. HAZV N consists of a globular domain formed mostly of alpha helices derived from both the N-and C-termini, and an arm domain comprising two long alpha helices. HAZV N has a similar overall structure to CCHFV N, with their globular domains superposing with an RMSD = 0.70 Å, over 368 alpha carbons that share 59 % sequence identity. Four HAZV N monomers crystallised in the asymmetric unit, and their head-to-tail assembly reveals a potential interaction site between monomers. Conclusions: The crystal structure of HAZV N reveals a close similarity to CCHFV N, supporting the use of HAZV as a model for CCHFV. Structural similarity between the N proteins should facilitate study of the CCHFV and HAZV replication cycles without the necessity of working under containment level 4 (CL-4) conditions.

Sirtuins are an ancient family of NAD+-dependent deacylases connected with the regulation of fund... more Sirtuins are an ancient family of NAD+-dependent deacylases connected with the regulation of fundamental cellular processes including metabolic homeostasis and genome integrity. We show the existence of a hitherto unrecognized class of sirtuins, found predominantly in microbial pathogens. In contrast to earlier described classes, these sirtuins exhibit robust protein ADP-ribosylation activity. In our model organisms, Staphylococcus aureus and Streptococcus pyogenes, the activity is dependent on prior lipoylation of the target protein and can bereversed by a sirtuin-associated macrodomain protein. Together, our data describe a sirtuin-dependent reversible protein ADP-ribosylation system and establish a crosstalk between lipoylation and mono-ADP-ribosylation. We propose that these posttranslational modifications modulate microbial virulence by regulating the response to host-derived reactive oxygen species.

Journal of the American Chemical Society, 2015

Poly(ADP-ribosyl)ation is a common post-translational modification that mediates a wide variety o... more Poly(ADP-ribosyl)ation is a common post-translational modification that mediates a wide variety of cellular processes including DNA damage repair, chromatin regulation, transcription, and apoptosis. The difficulty associated with accessing poly(ADP-ribose) (PAR) in a homogeneous form has been an impediment to understanding the interactions of PAR with poly(ADP-ribose) glycohydrolase (PARG) and other binding proteins. Here we describe the chemical synthesis of the ADP-ribose dimer, and we use this compound to obtain the first human PARG substrate-enzyme cocrystal structure. Chemical synthesis of PAR is an attractive alternative to traditional enzymatic synthesis and fractionation, allowing access to products such as dimeric ADP-ribose, which has been detected but never isolated from natural sources. Additionally, we describe the synthesis of an alkynylated dimer and demonstrate that this compound can be used to synthesize PAR probes including biotin and fluorophore-labeled compounds. The fluorescently labeled ADP-ribose dimer was then utilized in a general fluorescence polarization-based PAR–protein binding assay. Finally, we use intermediates of our synthesis to access various PAR fragments, and evaluation of these compounds as substrates for PARG reveals the minimal features for substrate recognition and enzymatic cleavage. Homogeneous PAR oligomers and unnatural variants produced from chemical synthesis will allow for further detailed structural and biochemical studies on the interaction of PAR with its many protein binding partners.

RATIONALE: Bunyaviruses have become a major threat to both humans and livestock in Europe and the... more RATIONALE: Bunyaviruses have become a major threat to both humans and livestock in Europe and the Americas. The nucleocapsid (N) protein of these viruses is key to the replication cycle and knowledge of the N oligomerisation state is central to understanding the viral lifecycle and for development of therapeutic strategies. METHODS: Bunyamwera virus and Schmallenberg virus N proteins (BUNV-N and SBV-N) were expressed recombinantly in E. coli as hexahistidine-SUMO-tagged fusions, and the tag removed subsequently. Noncovalent nano-electrospray ionisation mass spectrometry was conducted in the presence and absence of short RNA oligonucleotides. Instrumental conditions were optimised for the transmission of intact protein complexes into the gas phase. The resulting protein-protein and protein-RNA complexes were identified and their stoichiometries verified by their mass. Collision-induced dissociation tandem mass spectrometry was used in cases of ambiguity. RESULTS: Both BUNV-N and SBV-N proteins reassembled into N-RNA complexes in the presence of RNA; however, SBV-N formed a wider range of complexes with varying oligomeric states. The N:RNA oligomers observed were consistent with a model of assembly via stepwise addition of N proteins. Furthermore, upon mixing the two proteins in the presence of RNA no heteromeric complexes were observed, thus revealing insights into the specificity of oligomerisation. CONCLUSIONS: Noncovalent mass spectrometry has provided the first detailed analysis of the co-populated oligomeric species formed by these important viral proteins and revealed insights into their assembly pathways. Using this technique has also enabled comparisons to be made between the two N proteins.

The M2-1 protein of the important pathogen human respiratory syncytial virus is a zinc-binding tr... more The M2-1 protein of the important pathogen human respiratory syncytial virus is a zinc-binding transcription antiterminator that is essential for viral gene expression. We present the crystal structure of full-length M2-1 protein in its native tetrameric form at a resolution of 2.5 Å. The structure reveals that M2-1 forms a disk-like assembly with tetramerization driven by a long helix forming a four-helix bundle at its center, further stabilized by contact between the zinc-binding domain and adjacent protomers. The tetramerization helix is linked to a core domain responsible for RNA binding activity by a flexible region on which lie two functionally critical serine residues that are phosphorylated during infection. The crystal structure of a phosphomimetic M2-1 variant revealed altered charge density surrounding this flexible region although its position was unaffected. Structure-guided mutagenesis identified residues that contributed to RNA binding and antitermination activity, revealing a strong correlation between these two activities, and further defining the role of phosphorylation in M2-1 antitermination activity. The data we present here identify surfaces critical for M2-1 function that may be targeted by antiviral compounds.

The impact of disulfide bonds on protein stability goes beyond simple equilibrium thermodynamics ... more The impact of disulfide bonds on protein stability goes beyond simple equilibrium thermodynamics effects associated with the conformational entropy of the unfolded state. Indeed, disulfide crosslinks may play a role in the prevention of dysfunctional association and strongly affect the rates of irreversible enzyme inactivation, highly relevant in biotechnological applications. While these kinetic-stability effects remain poorly understood, by analogy with proposed mechanisms for processes of protein aggregation and fibrillogenesis, we propose that they may be determined by the properties of sparsely-populated, partially-unfolded intermediates. Here we report the successful design, on the basis of high temperature molecular-dynamics simulations, of six thermodynamically and kinetically stabilized variants of phytase from Citrobacter braakii (a biotechnologically important enzyme) with one, two or three engineered disulfides. Activity measurements and 3D crystal structure determination demonstrate that the engineered crosslinks do not cause dramatic alterations in the native structure. The inactivation kinetics for all the variants displays a strongly non-Arrhenius temperature dependence, with the timescale for the irreversible denaturation process reaching a minimum at a given temperature within the range of the denaturation transition. We show this striking feature to be a signature of a key role played by a partially unfolded, intermediate state/ensemble. Energetic and mutational analyses confirm that the intermediate is highly unfolded (akin to a proposed critical intermediate in the misfolding of the prion protein), a result that explains the observed kinetic stabilization. Our results provide a rationale for the kinetic-stability consequences of disulfide-crosslink engineering and an experimental methodology to arrive at energetic/structural descriptions of the sparsely populated and elusive intermediates that play key roles in irreversible protein denaturation.

All orthobunyaviruses possess three genome segments of single-stranded negative sense RNA that ar... more All orthobunyaviruses possess three genome segments of single-stranded negative sense RNA that are encapsidated with the virus-encoded nu-cleocapsid (N) protein to form a ribonucleoprotein (RNP) complex, which is uncharacterized at high resolution. We report the crystal structure of both the Bunyamwera virus (BUNV) N–RNA complex and the unbound Schmallenberg virus (SBV) N protein, at resolutions of 3.20 and 2.75 A ˚ , respectively. Both N proteins crystallized as ring-like tetramers and exhibit a high degree of structural similarity despite classification into different orthobunyavirus serogroups. The structures represent a new RNA-binding protein fold. BUNV N possesses a positively charged groove into which RNA is deeply seques-tered, with the bases facing away from the solvent. This location is highly inaccessible, implying that RNA polymerization and other critical base pairing events in the virus life cycle require RNP disassembly. Mutational analysis of N protein supports a correlation between structure and function. Comparison between these crystal structures and electron microscopy images of both soluble tetra-mers and authentic RNPs suggests the N protein does not bind RNA as a repeating monomer; thus, it represents a newly described architecture for bunyavirus RNP assembly, with implications for many other segmented negative-strand RNA viruses.

Phytases hydrolyse phytate (myo-inositol hexakisphosphate), the principal form of phosphate store... more Phytases hydrolyse phytate (myo-inositol hexakisphosphate), the principal form of phosphate stored in plant seeds to produce phosphate and lower phosphorylated myo-inositols. They are used extensively in the feed industry, and have been characterised biochemically and structurally with a number of structures in the PDB. They are divided into four distinct families: histidine acid phosphatases (HAP), b-propeller phytases, cysteine phosphatases and purple acid phosphatases and also split into three enzyme classes, the 3-, 5-and 6-phytases, depending on the position of the first phosphate in the inositol ring to be removed. We report identification, cloning, purification and 3D structures of 6-phytases from two bacteria, Hafnia alvei and Yersinia kristensenii, together with their pH optima, thermal stability, and degradation profiles for phytate. An important result is the structure of the H. alvei enzyme in complex with the substrate analogue myo-inositol hexakissulphate. In contrast to the only previous structure of a ligand-bound 6-phytase, where the 3-phosphate was unexpectedly in the catalytic site, in the H. alvei complex the expected scissile 6-phosphate (sulphate in the inhibitor) is placed in the catalytic site. Citation: Ariza A, Moroz OV, Blagova EV, Turkenburg JP, Waterman J, et al. (2013) Degradation of Phytate by the 6-Phytase from Hafnia alvei : A Combined Structural and Solution Study. PLoS ONE 8(5): e65062.

Crimean-Congo hemorrhagic fever virus (CCHFV) is an emerging tick-borne virus of the Bunyaviridae... more Crimean-Congo hemorrhagic fever virus (CCHFV) is an emerging tick-borne virus of the Bunyaviridae family that is responsible for a fatal human disease for which preventative or therapeutic measures do not exist. We solved the crystal structure of the CCHFV strain Baghdad-12 nucleocapsid protein (N), a potential therapeutic target, at a resolution of 2.1 Å. N comprises a large globular domain composed of both N-and C-terminal sequences, likely involved in RNA binding, and a protruding arm domain with a conserved DEVD caspase-3 cleavage site at its apex. Alignment of our structure with that of the recently reported N protein from strain YL04057 shows a close correspondence of all folds but significant transposition of the arm through a rotation of 180 degrees and a translation of 40 Å. These observations suggest a structural flexibility that may provide the basis for switching between alternative N protein conformations during important functions such as RNA binding and oligomerization. Our structure reveals surfaces likely involved in RNA binding and oligomerization, and functionally critical residues within these domains were identified using a minigenome system able to recapitulate CCHFV-specific RNA synthesis in cells. Caspase-3 cleaves the polypeptide chain at the exposed DEVD motif; however, the cleaved N protein remains an intact unit, likely due to the intimate association of N-and C-terminal fragments in the globular domain. Structural alignment with existing N proteins reveals that the closest CCHFV relative is not another bunyavirus but the arenavirus Lassa virus instead, suggesting that current segmented negative-strand RNA virus taxonomy may need revision. C rimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne zoonotic virus responsible for a serious human disease characterized by hemorrhagic manifestations and multiple organ failure and is associated with a fatality rate of up to 50% (13, 46). The geographic range of CCHFV is exceptionally wide and reflects the broad distribution of the tick vector, which extends throughout 30 countries within Africa, Asia, the Middle East, and Southern Europe (25). Recent outbreaks of CCHFV infection in several Balkan states, southwestern Russia, and Turkey suggest that the activity of CCHFV is increasing, particularly in Southern Europe (24). The only treatment for CCHFV infection is postexposure administration of ribavirin, and the efficacy of this prophylaxis is in doubt (45). Development of effective treatments for prevention of CCHFV-mediated disease is now a priority for both public health and biodefense agencies. CCHFV is a member of the Bunyaviridae family, and together with members of the Arenaviridae and Orthomyxoviridae families, these viruses are known as segmented negative-strand RNA viruses (sNSVs) by virtue of their multiple genome segments. The Bunyaviridae family contains over 350 named isolates classified within five genera, namely, Hantavirus, Nairovirus, Orthobunya-virus, Phlebovirus, and Tospovirus (41). CCHFV is a nairovirus, with a genome comprising three RNA segments: the small (S), medium (M), and large (L) segments. The S segment encodes the nucleocapsid protein (N), the M segment encodes the viral glyco-proteins, and the L segment encodes an RNA-dependent RNA polymerase (RdRp; the L protein). The genomes of sNSVs do not exist as naked RNAs but instead are encapsidated by the viral N protein to form ribonucleoprotein (RNP) complexes. RNPs associate with their cognate RdRp to form active templates for viral RNA synthesis, resulting in generation of encapsidated replication products and unencapsidated mRNAs. Genome encapsidation is also required for RNP packaging into progeny virus particles, and for bunyaviruses, virus assembly is mediated through direct association between the RNP and viral glycoproteins (18, 31, 39, 42, 44). RNP formation is thus essential for virus multiplication and therefore represents a potential therapeutic target. In addition to RNP formation and virus assembly, the N proteins of bunyaviruses are implicated in other important functions, many of which relate to interactions with components of the host cell, including the cytoskeleton (3, 35–37, 43), cellular RNAs (26, 28), the translation machinery (8, 27), and mediators of the innate immune response (20, 30). Specifically for CCHFV, the N protein interacts with the cellular antiviral defense factor MxA (2) and recently was shown to act as a substrate for the apoptosis mediator caspase-3 (19), although the relevance of caspase-3 cleavage to the virus life cycle is unknown. We present the 2.1-Å crystal structure of the N protein from CCHFV strain Baghdad-12. The CCHFV N structure we present displays significant differences in domain position compared to the recently reported structure of N from CCHFV strain YL04057

The enzymatic degradation of plant polysaccharides is emerging as one of the key environmental go... more The enzymatic degradation of plant polysaccharides is emerging as one of the key environmental goals of the early 21st century , impacting on many processes in the textile and detergent industries as well as biomass conversion to biofuels. One of the well known problems with the use of nonstarch (nonfood)-based substrates such as the plant cell wall is that the cellulose fibers are embedded in a network of diverse polysaccharides, including xyloglucan, that renders access difficult. There is therefore increasing interest in the " accessory enzymes, " including xylog-lucanases, that may aid biomass degradation through removal of " hemicellulose " polysaccharides. Here, we report the biochemical characterization of the endo-1,4-(xylo)glucan hydrolase from Paenibacillus polymyxa with polymeric, oligomeric, and defined chromogenic aryl-oligosaccharide substrates. The enzyme displays an unusual specificity on defined xyloglucan oli-gosaccharides, cleaving the XXXG-XXXG repeat into XXX and GXXXG. Kinetic analysis on defined oligosaccharides and on aryl-glycosides suggests that both the 4 and 1 subsites show discrimination against xylose-appended glucosides. The three-dimensional structures of PpXG44 have been solved both in apo-form and as a series of ligand complexes that map the 3 to 1 and 1 to 5 subsites of the extended ligand binding cleft. Complex structures are consistent with partial intolerance of xylosides in the 4 subsites. The atypical specificity of PpXG44 may thus find use in industrial processes involving xyloglucan degradation, such as biomass conversion, or in the emerging exciting applications of defined xyloglucans in food, pharmaceuticals , and cellulose fiber modification.

As part of a drug discovery programme to discover new treatments for human African trypanosomiasi... more As part of a drug discovery programme to discover new treatments for human African trypanosomiasis, recombinant trypanothione reductase from Trypanosoma brucei has been expressed, purified and characterized. The crystal structure was solved by molecular replacement to a resolution of 2.3 Å and found to be nearly identical to the T. cruzi enzyme (root mean square deviation 0.6 Å over 482 C atoms). Kinetically , the K m for trypanothione disulphide for the T. brucei enzyme was 4.4-fold lower than for T. cruzi measured by either direct (NADPH oxidation) or DTNB-coupled assay. The K m for NADPH for the T. brucei enzyme was found to be 0.77 M using an NADPH-regenerating system coupled to reduction of DTNB. Both enzymes were assayed for inhibition at their respective S = K m values for trypanothione disulphide using a range of chemotypes, including CNS-active drugs such as clomipramine, trifluoperazine, thiori-dazine and citalopram. The relative IC 50 values for the two enzymes were found to vary by no more than 3-fold. Thus trypanothione reductases from these species are highly similar in all aspects, indicating that they may be used interchangeably for structure-based inhibitor design and high-throughput screening.

The intracellular subtilisin proteases (ISPs) are the only known members of the important and ubi... more The intracellular subtilisin proteases (ISPs) are the only known members of the important and ubiquitous subtilisin family that function exclusively within the cell, constituting a major component of the degra-dome in many Gram-positive bacteria. The first ISP structure reported herein at a spacing of 1.56 A ˚ reveals features unique among subtilisins that has enabled potential functional and physiological roles to be assigned to sequence elements exclusive to the ISPs. Unlike all other subtilisins, ISP from B. clau-sii is dimeric, with residues from the C terminus making a major contribution to the dimer interface by crossing over to contact the partner subunit. A short N-terminal extension binds back across the active site to provide a potential novel regulatory mechanism of intrinsic proteolytic activity: a proline residue conserved throughout the ISPs introduces a kink in the polypeptide backbone that lifts the target peptide bond out of reach of the catalytic residues.

Nature

International Journal of Molecular Sciences, 2019

Cell Chemical Biology, 2018

Journal of the American Chemical Society, 2015