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Papers by luke guddat

Acta crystallographica. Section A, Foundations and advances, Aug 22, 2023

Protein & Cell

The ATP-binding cassette (ABC) transporter, IrtAB, plays a vital role in the replication and viab... more The ATP-binding cassette (ABC) transporter, IrtAB, plays a vital role in the replication and viability of Mycobacterium tuberculosis (Mtb), where its function is to import iron-loaded siderophores. Unusually, it adopts the canonical type IV exporter fold. Herein, we report the structure of unliganded Mtb IrtAB and its structure in complex with ATP, ADP, or ATP analogue (AMP-PNP) at resolutions ranging from 2.8 to 3.5 Å. The structure of IrtAB bound ATP-Mg 2+ shows a “head-to-tail” dimer of nucleotide-binding domains (NBDs), a closed amphipathic cavity within the transmembrane domains (TMDs) and a metal ion liganded to three histidine residues of IrtA in the cavity. Cryo-electron microscopy (Cryo-EM) structures and ATP hydrolysis assays show that the NBD of IrtA has a higher affinity for nucleotides and increased ATPase activity compared with IrtB. Moreover, the metal ion located in the TM region of IrtA is critical for the stabilization of the conformation of IrtAB during the transp...

an evolutionary link to Ser/Thr protein phosphatases?

Antimicrobial Agents and Chemotherapy, 2021

The structural diversity in metallo-β-lactamases (MBLs), especially in the vicinity of the active... more The structural diversity in metallo-β-lactamases (MBLs), especially in the vicinity of the active site, has been a major hurdle in the development of clinically effective inhibitors. Representatives from three variants of the B3 MBL subclass, containing either the canonical HHH/DHH active-site motif (present in the majority of MBLs in this subclass) or the QHH/DHH (B3-Q) or HRH/DQK (B3-RQK) variations were reported previously.

Scientific Reports, 2021

All medically important unicellular protozoans cannot synthesize purines de novo and they entirel... more All medically important unicellular protozoans cannot synthesize purines de novo and they entirely rely on the purine salvage pathway (PSP) for their nucleotide generation. Therefore, purine derivatives have been considered as a promising source of anti-parasitic compounds since they can act as inhibitors of the PSP enzymes or as toxic products upon their activation inside of the cell. Here, we characterized a Trypanosoma brucei enzyme involved in the salvage of adenine, the adenine phosphoribosyl transferase (APRT). We showed that its two isoforms (APRT1 and APRT2) localize partly in the cytosol and partly in the glycosomes of the bloodstream form (BSF) of the parasite. RNAi silencing of both APRT enzymes showed no major effect on the growth of BSF parasites unless grown in artificial medium with adenine as sole purine source. To add into the portfolio of inhibitors for various PSP enzymes, we designed three types of acyclic nucleotide analogs as potential APRT inhibitors. Out of f...

Scientific Reports, 2020

Genes that confer antibiotic resistance can rapidly be disseminated from one microorganism to ano... more Genes that confer antibiotic resistance can rapidly be disseminated from one microorganism to another by mobile genetic elements, thus transferring resistance to previously susceptible bacterial strains. The misuse of antibiotics in health care and agriculture has provided a powerful evolutionary pressure to accelerate the spread of resistance genes, including those encoding β-lactamases. These are enzymes that are highly efficient in inactivating most of the commonly used β-lactam antibiotics. However, genes that confer antibiotic resistance are not only associated with pathogenic microorganisms, but are also found in non-pathogenic (i.e. environmental) microorganisms. Two recent examples are metal-dependent β-lactamases (MBLs) from the marine organisms Novosphingobium pentaromativorans and Simiduia agarivorans. Previous studies have demonstrated that their β-lactamase activity is comparable to those of well-known MBLs from pathogenic sources (e.g. NDM-1, AIM-1) but that they also ...

Molecular Pharmacology, 2020

Due to -lactamase mediated resistance, -lactam antibiotics were long considered ineffective dru... more Due to -lactamase mediated resistance, -lactam antibiotics were long considered ineffective drugs for tuberculosis (TB) treatment. However, some -lactams, including meropenem and faropenem, are being re-evaluated in patients infected with TB. Penicillin-binding protein 3 (PBP3, or ftsI) is an essential transpeptidase in Mycobacterium tuberculosis (Mtb) required for cell division, thus is an important drug target. Structures of apo MtbPBP3 and of complexes with five -lactams, including meropenem and faropenem, reveal how they cause inactivation via formation of hydrolytically stable acyl-enzyme complexes. The structures reveal unique features of the antibiotic interactions, both in terms of differences in their binding to MtbPBP3 and in comparison with structures of other PBPs and serine -lactamases, including the tautomerisation status of the carbapenem derived acyl-enzyme complexes. The results suggest that rather than hoping PBP inhibitors developed for other infections will work against TB, work should focus on developing PBP inhibitors specialized for treating TB.

Chemistry – A European Journal, 2020

This is the author manuscript accepted for publication and has undergone full peer review but has... more This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record.

Nature Catalysis, 2018

Commercial enzymatic processes require robust catalysts able to withstand elevated temperatures a... more Commercial enzymatic processes require robust catalysts able to withstand elevated temperatures and long incubations, conditions under which most native enzymes perform poorly. Incremental increases in thermostability can be achieved by repeated rounds of mutagenesis and screening, but general strategies are needed for designing highly thermostable enzymes a priori. Here we show that enzymes can be created that can withstand temperatures ~ 30 °C higher and incubations ≥ 100 times longer than extant forms in a single step using ancestral reconstruction. We exemplify the approach with the first ancestral resurrections of two unrelated enzyme families: cytochrome P450 monooxygenases, that stereo-and regioselectively functionalize un-activated C-H bonds in pharmaceutical, flavour, fragrance and other fine chemical syntheses; and ketol acid reductoisomerases, used to make butanol-based biofuels. This shows thermostability can be designed into proteins using sequence data alone, potentially enhancing the economic feasibility of any process or product requiring a highly stable protein.

Journal of Medicinal Chemistry, Apr 23, 2021

Scientific Reports, 2016

Human African Trypanosomiasis (HAT) is a life-threatening infectious disease caused by the protoz... more Human African Trypanosomiasis (HAT) is a life-threatening infectious disease caused by the protozoan parasite, Trypanosoma brucei (Tbr). Due to the debilitating side effects of the current therapeutics and the emergence of resistance to these drugs, new medications for this disease need to be developed. One potential new drug target is 6-oxopurine phosphoribosyltransferase (PRT), an enzyme central to the purine salvage pathway and whose activity is critical for the production of the nucleotides (GMP and IMP) required for DNA/RNA synthesis within this protozoan parasite. Here, the first crystal structures of this enzyme have been determined, these in complex with GMP and IMP and with three acyclic nucleoside phosphonate (ANP) inhibitors. The Ki values for GMP and IMP are 30.5 μM and 77 μM, respectively. Two of the ANPs have Ki values considerably lower than for the nucleotides, 2.3 μM (with guanine as base) and 15.8 μM (with hypoxanthine as base). The crystal structures show that whe...

PLoS ONE, 2013

Textilinin-1 is a Kunitz-type serine protease inhibitor from Australian brown snake venom. Its ab... more Textilinin-1 is a Kunitz-type serine protease inhibitor from Australian brown snake venom. Its ability to potently and specifically inhibit human plasmin (K i = 0.44 nM) makes it a potential therapeutic drug as a systemic anti-bleeding agent. The crystal structures of the human microplasmin-textilinin-1 and the trypsin-textilinin-1 complexes have been determined to 2.78 Å and 1.64 Å resolution respectively, and show that textilinin-1 binds to trypsin in a canonical mode but to microplasmin in an atypical mode with the catalytic histidine of microplasmin rotated out of the active site. The space vacated by the histidine side-chain in this complex is partially occupied by a water molecule. In the structure of microplasminogen the x 1 dihedral angle of the side-chain of the catalytic histidine is rotated by 67u from its ''active'' position in the catalytic triad, as exemplified by its location when microplasmin is bound to streptokinase. However, when textilinin-1 binds to microplasmin the x 1 dihedral angle of this amino acid residue changes by 2157u (i.e. in the opposite rotation direction compared to microplasminogen). The unusual mode of interaction between textilinin-1 and plasmin explains textilinin-19s selectivity for human plasmin over plasma kallikrein. This difference can be exploited in future drug design efforts.

Journal of Medicinal Chemistry, 2013

Keough et al.: Acyclic nucleoside phosphonates containing a second phosphonate group are potent i... more Keough et al.: Acyclic nucleoside phosphonates containing a second phosphonate group are potent inhibitors of the 6-oxopurine phosphoribosyltransferases and have antimalarial activity.

The Journal of Infectious Diseases, 2005

Acta Crystallographica Section D Biological Crystallography, 2004

Ketol-acid reductoisomerase (EC 1.1.1.86) catalyses the second reaction in the biosynthesis of br... more Ketol-acid reductoisomerase (EC 1.1.1.86) catalyses the second reaction in the biosynthesis of branched-chain amino acids. The reaction involves an Mg 2+-dependent alkyl migration followed by an NADPH-dependent reduction of the 2-keto group. Here, the crystallization of the Escherichia coli enzyme is reported. A form with a C-terminal hexahistidine tag could be crystallized under 18 different conditions in the absence of NADPH or Mg 2+ and a further six crystallization conditions were identi®ed with one or both ligands. With the hexahistidine tag on the N-terminus, 20 crystallization conditions were found, some of which required the presence of NADPH, NADP + , Mg 2+ or a combination of ligands. Finally, the selenomethionine-substituted enzyme with the N-terminal tag crystallized under 15 conditions. Thus, the enzyme is remarkably easy to crystallize. Most of the crystals diffract poorly but several data sets were collected at better than 3.2 A Ê resolution; attempts to phase them are currently in progress.

$Research paper thumbnail of Crystallization and preliminary diffraction studies of the ectodomain of the envelope glycoprotein D from herpes simplex virus 1 alone and in complex with the ectodomain of the human receptor HveA$

Acta Crystallographica Section D Biological Crystallography, 2002

ABSTRACT Gycoprotein D (gD) is a glycoprotein expressed on the surface of several human and anima... more ABSTRACT Gycoprotein D (gD) is a glycoprotein expressed on the surface of several human and animal alpha herpes viruses. Binding of gD to cell-surface receptors has been shown to be necessary for herpes simplex virus 1 and 2 (HSV-1 and HSV-2) cell entry. The gD ectodomain consists of 316 residues and has no sequence homology to any other proteins of known structure. Two fragments of the HSV-1 gD ectodomain (gD(22-260): residues 22-260 and gD(285): residues 1-285) have been crystallized in two crystal forms. The complex between gD(285) and the ectodomain of HveA, a gD cellular receptor member of the tumor necrosis factor (TNFR) superfamily, has also been crystallized. Moreover, insect-cell-expressed selenomethionine-substituted gD(285) has been purified and crystallized alone and in complex with HveA.

FEBS Journal, 2004

Ketol-acid reductoisomerase (EC 1.1.1.86; KARI; also known as acetohydroxy acid isomeroreductase;... more Ketol-acid reductoisomerase (EC 1.1.1.86; KARI; also known as acetohydroxy acid isomeroreductase; reviewed in [1]) is a bifunctional enzyme that catalyzes two quite different reactions, acting both as an isomerase and as a reductase (Fig. 1A). In the isomerase reaction, 2-hydroxy-2-methyl-3-ketobutyrate (better known as 2-acetolactate) is rearranged via an Mg 2+dependent methyl migration to produce 3-hydroxy-3-methyl-2-ketobutyrate (HMKB). In the reductase reaction, this 2-ketoacid undergoes an M 2+-dependent (Mg 2+ , Mn 2+ or Co 2+) reduction by NADPH to yield 2,3-dihydroxy-3-methylbutyrate. This product is the precursor of both valine and leucine. The third branched-chain amino acid, isoleucine, is produced in a pathway that parallels that of valine, employing the same series of enzymes, with KARI catalyzing the conversion of 2-hydroxy-2-ethyl-3-ketobutyrate to 2,3-dihydroxy-3-ethylbutyrate. KARI is the target of the experimental herbicides Hoe704 [2] and IpOHA [3] that are thought to be transition-state intermediates of the alkyl migration step. Both reactions occur at a common active site. One of the initial lines of evidence for a single active site was that the 2-ketoacid intermediate is not released and does not exchange with added HMKB [4]. However, the enzyme will catalyze the reduction of this intermediate if it is provided [4]. In addition to HMKB, KARI will catalyze the reduction of other

The FEBS journal, Jul 9, 2017

Acetohydroxyacid synthase (AHAS, E.C. 2.2.1.6) is the first enzyme in the branched-chain amino ac... more Acetohydroxyacid synthase (AHAS, E.C. 2.2.1.6) is the first enzyme in the branched-chain amino acid biosynthesis pathway. Five of the most widely used commercial herbicides (i.e. sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidinyl-benzoates and sulfonylamino-cabonyl-triazolinones) target this enzyme. Here we have determined the first crystal structure of a plantAHAS in the absence of any inhibitor (2.9 Å resolution) and itshows that the herbicide-binding site adopts a folded state even in the absence of an inhibitor. This is unexpected because the equivalent regions for herbicide bindingin uninhibited Saccharomyces cerevisiae AHAS crystal structures are either disordered,or adopt a different fold when the herbicide is not present. In addition, the structure provides anexplanation as to why some herbicides are more potent inhibitorsofArabidopsis thaliana AHAS compared to AHASs from other species (e.g.Saccharomyces cerevisiae). The elucidation of the native structure of pl...