The use of Trichomonas vaginalis purine nucleoside phosphorylase to activate fludarabine in the treatment of solid tumors.

Treatment with fludarabine phosphate (9-ß-D-arabinofuranosyl-2-F-adenine 5'-phosphate, F-araAMP) leads to regressions and cures of human tumor xenografts that express Escherichia coli purine nucleoside phosphorylase (EcPNP). This occurs despite the fact that fludarabine (F-araA) is a relatively poor substrate for EcPNP, and is cleaved to liberate 2-fluoroadenine at a rate only 0.3% that of the natural E. coli PNP substrate, adenosine. In this study, we investigated a panel of naturally occurring PNPs to identify more efficient enzymes that may be suitable for metabolizing F-araA as part of experimental cancer therapy. We show that Trichomonas vaginalis PNP (TvPNP) cleaves F-araA with a catalytic efficiency 25-fold greater than the prototypic E. coli enzyme. Cellular extracts from human glioma cells (D54) transduced with lentivirus stably expressing TvPNP (D54/TvPNP) were found to cleave F-araA at a rate similar to extracts from D54 cells expressing EcPNP, although much less enzyme was expressed per cell in the TvPNP transduced condition. As a test of safety and efficacy using TvPNP, human head and neck squamous cell carcinoma (FaDu) xenografts expressing TvPNP were studied in nude mice and shown to exhibit robust tumor regressions, albeit with partial weight loss that resolved post-therapy. F-araAMP was also a very effective treatment for mice bearing D54/TvPNP xenografts in which approximately 10% of tumor cells expressed the enzyme, indicating pronounced ability to kill non-transduced tumor cells (high bystander activity). Moreover, F-araAMP demonstrated activity against D54 tumors injected with an E1, E3 deleted adenoviral vector encoding TvPNP. In that setting, despite higher F-araA cleavage activity using TvPNP, tumor responses were similar to those obtained with EcPNP, indicating factors other than F-Ade production may limit regressions of the D54 murine xenograft model. Our results establish that TvPNP is a favorable enzyme for activating F-araA, and support further studies in combination with F-araAMP for difficult-to-treat human cancers.

Structural Basis of the Substrate Selectivity of Viperin.

Viperin is a radical S-adenosylmethionine (SAM) enzyme that inhibits viral replication by converting cytidine triphosphate (CTP) into 3'-deoxy-3',4'-didehydro-CTP and by additional undefined mechanisms operating through its N- and C-terminal domains. Here, we describe crystal structures of viperin bound to a SAM analogue and CTP or uridine triphosphate (UTP) and report kinetic parameters for viperin-catalyzed reactions with CTP or UTP as substrates. Viperin orients the C4' hydrogen atom of CTP and UTP similarly for abstraction by a 5'-deoxyadenosyl radical, but the uracil moiety introduces unfavorable interactions that prevent tight binding of UTP. Consistently, kcat is similar for CTP and UTP whereas the Km for UTP is much greater. The structures also show that nucleotide binding results in ordering of the C-terminal tail and reveal that this region contains a P-loop that binds the γ-phosphate of the bound nucleotide. Collectively, the results explain the selectivity for CTP and reveal a structural role for the C-terminal tail in binding CTP and UTP.

Structural basis of elongation factor 2 switching.

Archaebacterial and eukaryotic elongation factor 2 (EF-2) and bacterial elongation factor G (EF-G) are five domain GTPases that catalyze the ribosomal translocation of tRNA and mRNA. In the classical mechanism of activation, GTPases are switched on through GDP/GTP exchange, which is accompanied by the ordering of two flexible segments called switch I and II. However, crystal structures of EF-2 and EF-G have thus far not revealed the conformations required by the classical mechanism. Here, we describe crystal structures of Methanoperedens nitroreducens EF-2 (MnEF-2) and MnEF-2-H595N bound to GMPPCP (GppCp) and magnesium displaying previously unreported compact conformations. Domain III forms interfaces with the other four domains and the overall conformations resemble that of SNU114, the eukaryotic spliceosomal GTPase. The gamma phosphate of GMPPCP is detected through interactions with switch I and a P-loop structural element. Switch II is highly ordered whereas switch I shows a variable degree of ordering. The ordered state results in a tight interdomain arrangement of domains I-III and the formation of a portion of a predicted monovalent cation site involving the P-loop and switch I. The side chain of an essential histidine residue in switch II is placed in the inactive conformation observed for the "on" state of elongation factor EF-Tu. The compact conformations of MnEF-2 and MnEF-2-H595N suggest an "on" ribosome-free conformational state.

The Crystal Structure of Dph2 in Complex with Elongation Factor 2 Reveals the Structural Basis for the First Step of Diphthamide Biosynthesis.

Elongation factor 2 (EF-2), a five-domain, GTP-dependent ribosomal translocase of archaebacteria and eukaryotes, undergoes post-translational modification to form diphthamide on a specific histidine residue in domain IV prior to binding the ribosome. The first step of diphthamide biosynthesis in archaebacteria is catalyzed by Dph2, a homodimeric radical S-adenosylmethionine (SAM) enzyme having a noncanonical architecture. Here, we describe a 3.5 Å resolution crystal structure of the Methanobrevibacter smithii (Ms) Dph2 homodimer bound to two molecules of MsEF-2, one of which is ordered and the other largely disordered. MsEF-2 is bound to both protomers of MsDph2, with domain IV bound to the active site of one protomer and domain III bound to a surface α-helix of an adjacent protomer. The histidine substrate of domain IV is inserted into the active site, which reveals for the first time the architecture of the Dph2 active site in complex with its target substrate. We also determined a high-resolution crystal structure of isolated MsDph2 bound to 5'-methylthioadenosine that shows a conserved arginine residue preoriented by conserved phenylalanine and aspartate residues for binding the carboxylate group of SAM. Mutagenesis experiments suggest that the arginine plays an important role in the first step of diphthamide biosynthesis.

Menaquinone Biosynthesis: Biochemical and Structural Studies of Chorismate Dehydratase.

Menaquinone (MK, vitamin K) is a lipid-soluble quinone that participates in the bacterial electron transport chain. In mammalian cells, vitamin K functions as an essential vitamin for the activation of several proteins involved in blood clotting and bone metabolism. MqnA is the first enzyme on the futalosine-dependent pathway to menaquinone and catalyzes the aromatization of chorismate by water loss. Here we report biochemical and structural studies of MqnA. These studies suggest that the dehydration reaction proceeds by a variant of the E1cb mechanism in which deprotonation is slower than water loss and that the enol carboxylate of the substrate is serving as the base.

Towards the structural characterization of the human methyltransferome.

Approximately 2000 structures of methyltransferases (MTases) are currently available, displaying fifteen different folds for binding a methyl donor and providing molecular level insight into nearly half the human methyltransferome. Several MTases involved in gene expression and regulation are catalytically inefficient when isolated, and their catalytic domains often show inhibitory active site architectures. Recently reported structures of complexes that more closely reflect biological context have begun to reveal the structural basis of activation. DNA and particular histone MTases are allosterically activated by binding histone modifications using reader domains or separate reader proteins, and some MTases operating beyond chromatin are activated by binding an activator protein. In this review, we describe the structural status of the human methyltransferome and then discuss newly revealed structural mechanisms of MTase activation.

Organometallic and radical intermediates reveal mechanism of diphthamide biosynthesis.

Diphthamide biosynthesis involves a carbon-carbon bond-forming reaction catalyzed by a radical S-adenosylmethionine (SAM) enzyme that cleaves a carbon-sulfur (C-S) bond in SAM to generate a 3-amino-3-carboxypropyl (ACP) radical. Using rapid freezing, we have captured an organometallic intermediate with an iron-carbon (Fe-C) bond between ACP and the enzyme's [4Fe-4S] cluster. In the presence of the substrate protein, elongation factor 2, this intermediate converts to an organic radical, formed by addition of the ACP radical to a histidine side chain. Crystal structures of archaeal diphthamide biosynthetic radical SAM enzymes reveal that the carbon of the SAM C-S bond being cleaved is positioned near the unique cluster Fe, able to react with the cluster. Our results explain how selective C-S bond cleavage is achieved in this radical SAM enzyme.

Biochemical Characterization and Structural Basis of Reactivity and Regioselectivity Differences between Burkholderia thailandensis and Burkholderia glumae 1,6-Didesmethyltoxoflavin N-Methyltransferase.

Burkholderia glumae converts the guanine base of guanosine triphosphate into an azapteridine and methylates both the pyrimidine and triazine rings to make toxoflavin. Strains of Burkholderia thailandensis and Burkholderia pseudomallei have a gene cluster encoding seven putative biosynthetic enzymes that resembles the toxoflavin gene cluster. Four of the enzymes are similar in sequence to BgToxBCDE, which have been proposed to make 1,6-didesmethyltoxoflavin (1,6-DDMT). One of the remaining enzymes, BthII1283 in B. thailandensis E264, is a predicted S-adenosylmethionine (SAM)-dependent N-methyltransferase that shows a low level of sequence identity to BgToxA, which sequentially methylates N6 and N1 of 1,6-DDMT to form toxoflavin. Here we show that, unlike BgToxA, BthII1283 catalyzes a single methyl transfer to N1 of 1,6-DDMT in vitro. In addition, we investigated the differences in reactivity and regioselectivity by determining crystal structures of BthII1283 with bound S-adenosylhomocysteine (SAH) or 1,6-DDMT and SAH. BthII1283 contains a class I methyltransferase fold and three unique extensions used for 1,6-DDMT recognition. The active site structure suggests that 1,6-DDMT is bound in a reduced form. The plane of the azapteridine ring system is orthogonal to its orientation in BgToxA. In BthII1283, the modeled SAM methyl group is directed toward the p orbital of N1, whereas in BgToxA, it is first directed toward an sp2 orbital of N6 and then toward an sp2 orbital of N1 after planar rotation of the azapteridine ring system. Furthermore, in BthII1283, N1 is hydrogen bonded to a histidine residue whereas BgToxA does not supply an obvious basic residue for either N6 or N1 methylation.

Structural studies of viperin, an antiviral radical SAM enzyme.

Viperin is an IFN-inducible radical S-adenosylmethionine (SAM) enzyme that inhibits viral replication. We determined crystal structures of an anaerobically prepared fragment of mouse viperin (residues 45-362) complexed with S-adenosylhomocysteine (SAH) or 5'-deoxyadenosine (5'-dAdo) and l-methionine (l-Met). Viperin contains a partial (ßα)6-barrel fold with a disordered N-terminal extension (residues 45-74) and a partially ordered C-terminal extension (residues 285-362) that bridges the partial barrel to form an overall closed barrel structure. Cys84, Cys88, and Cys91 located after the first ß-strand bind a [4Fe-4S] cluster. The active site architecture of viperin with bound SAH (a SAM analog) or 5'-dAdo and l-Met (SAM cleavage products) is consistent with the canonical mechanism of 5'-deoxyadenosyl radical generation. The viperin structure, together with sequence alignments, suggests that vertebrate viperins are highly conserved and that fungi contain a viperin-like ortholog. Many bacteria and archaebacteria also express viperin-like enzymes with conserved active site residues. Structural alignments show that viperin is similar to several other radical SAM enzymes, including the molybdenum cofactor biosynthetic enzyme MoaA and the RNA methyltransferase RlmN, which methylates specific nucleotides in rRNA and tRNA. The viperin putative active site contains several conserved positively charged residues, and a portion of the active site shows structural similarity to the GTP-binding site of MoaA, suggesting that the viperin substrate may be a nucleoside triphosphate of some type.

Substrate-Dependent Cleavage Site Selection by Unconventional Radical S-Adenosylmethionine Enzymes in Diphthamide Biosynthesis.

S-Adenosylmethionine (SAM) has a sulfonium ion with three distinct C-S bonds. Conventional radical SAM enzymes use a [4Fe-4S] cluster to cleave homolytically the C5',adenosine-S bond of SAM to generate a 5'-deoxyadenosyl radical, which catalyzes various downstream chemical reactions. Radical SAM enzymes involved in diphthamide biosynthesis, such as Pyrococcus horikoshii Dph2 (PhDph2) and yeast Dph1-Dph2 instead cleave the Cγ,Met-S bond of methionine to generate a 3-amino-3-carboxylpropyl radical. We here show radical SAM enzymes can be tuned to cleave the third C-S bond to the sulfonium sulfur by changing the structure of SAM. With a decarboxyl SAM analogue (dc-SAM), PhDph2 cleaves the Cmethyl-S bond, forming 5'-deoxy-5'-(3-aminopropylthio) adenosine (dAPTA, 1). The methyl cleavage activity, like the cleavage of the other two C-S bonds, is dependent on the presence of a [4Fe-4S]+ cluster. Electron-nuclear double resonance and mass spectroscopy data suggests that mechanistically one of the S atoms in the [4Fe-4S] cluster captures the methyl group from dc-SAM, forming a distinct EPR-active intermediate, which can transfer the methyl group to nucleophiles such as dithiothreitol. This reveals the [4Fe-4S] cluster in a radical SAM enzyme can be tuned to cleave any one of the three bonds to the sulfonium sulfur of SAM or analogues, and is the first demonstration a radical SAM enzyme could switch from an Fe-based one electron transfer reaction to a S-based two electron transfer reaction in a substrate-dependent manner. This study provides an illustration of the versatile reactivity of Fe-S clusters.

Lysine relay mechanism coordinates intermediate transfer in vitamin B6 biosynthesis.

Substrate channeling has emerged as a common mechanism for enzymatic intermediate transfer. A conspicuous gap in knowledge concerns the use of covalent lysine imines in the transfer of carbonyl-group-containing intermediates, despite their wideuse in enzymatic catalysis. Here we show how imine chemistry operates in the transfer of covalent intermediates in pyridoxal 5'-phosphate biosynthesis by the Arabidopsis thaliana enzyme Pdx1. An initial ribose 5-phosphate lysine imine is converted to the chromophoric I320 intermediate, simultaneously bound to two lysine residues and partially vacating the active site, which creates space for glyceraldehyde 3-phosphate to bind. Crystal structures show how substrate binding, catalysis and shuttling are coupled to conformational changes around strand ß6 of the Pdx1 (ßα)8-barrel. The dual-specificity active site and imine relay mechanism for migration of carbonyl intermediates provide elegant solutions to the challenge of coordinating a complex sequence of reactions that follow a path of over 20 Å between substrate- and product-binding sites.

Lyme disease spirochaete Borrelia burgdorferi does not require thiamin.

Thiamin pyrophosphate (ThDP), the active form of thiamin (vitamin B1), is believed to be an essential cofactor for all living organisms1,2. Here, we report the unprecedented result that thiamin is dispensable for the growth of the Lyme disease pathogen Borrelia burgdorferi (Bb)3. Bb lacks genes for thiamin biosynthesis and transport as well as known ThDP-dependent enzymes4, and we were unable to detect thiamin or its derivatives in Bb cells. We showed that eliminating thiamin in vitro and in vivo using BcmE, an enzyme that degrades thiamin, has no impact on Bb growth and survival during its enzootic infectious cycle. Finally, high-performance liquid chromatography analysis reveals that the level of thiamin and its derivatives in Ixodes scapularis ticks, the enzootic vector of Bb, is extremely low. These results suggest that by dispensing with use of thiamin, Borrelia, and perhaps other tick-transmitted bacterial pathogens, are uniquely adapted to survive in tick vectors before transmitting to mammalian hosts. To our knowledge, such a mechanism has not been reported previously in any living organisms.

Crystal Structures of the Iron-Sulfur Cluster-Dependent Quinolinate Synthase in Complex with Dihydroxyacetone Phosphate, Iminoaspartate Analogues, and Quinolinate.

The quinolinate synthase of prokaryotes and photosynthetic eukaryotes, NadA, contains a [4Fe-4S] cluster with unknown function. We report crystal structures of Pyrococcus horikoshii NadA in complex with dihydroxyacetone phosphate (DHAP), iminoaspartate analogues, and quinolinate. DHAP adopts a nearly planar conformation and chelates the [4Fe-4S] cluster via its keto and hydroxyl groups. The active site architecture suggests that the cluster acts as a Lewis acid in enediolate formation, like zinc in class II aldolases. The DHAP and putative iminoaspartate structures suggest a model for a condensed intermediate. The ensemble of structures suggests a two-state system, which may be exploited in early steps.

Biochemical and structural characterization of Klebsiella pneumoniae oxamate amidohydrolase in the uric acid degradation pathway.

HpxW from the ubiquitous pathogen Klebsiella pneumoniae is involved in a novel uric acid degradation pathway downstream from the formation of oxalurate. Specifically, HpxW is an oxamate amidohydrolase which catalyzes the conversion of oxamate to oxalate and is a member of the Ntn-hydrolase superfamily. HpxW is autoprocessed from an inactive precursor to form a heterodimer, resulting in a 35.5 kDa α subunit and a 20 kDa ß subunit. Here, the structure of HpxW is presented and the substrate complex is modeled. In addition, the steady-state kinetics of this enzyme and two active-site variants were characterized. These structural and biochemical studies provide further insight into this class of enzymes and allow a mechanism for catalysis consistent with other members of the Ntn-hydrolase superfamily to be proposed.

Burkholderia glumae ToxA Is a Dual-Specificity Methyltransferase That Catalyzes the Last Two Steps of Toxoflavin Biosynthesis.

Toxoflavin is a major virulence factor of the rice pathogen Burkholderia glumae. The tox operon of B. glumae contains five putative toxoflavin biosynthetic genes toxABCDE. ToxA is a predicted S-adenosylmethionine-dependent methyltransferase, and toxA knockouts of B. glumae are less virulent in plant infection models. In this study, we show that ToxA performs two consecutive methylations to convert the putative azapteridine intermediate, 1,6-didemethyltoxoflavin, to toxoflavin. In addition, we report a series of crystal structures of ToxA complexes that reveals the molecular basis of the dual methyltransferase activity. The results suggest sequential methylations with initial methylation at N6 of 1,6-didemethyltoxoflavin followed by methylation at N1. The two azapteridine orientations that position N6 or N1 for methylation are coplanar with a 140° rotation between them. The structure of ToxA contains a class I methyltransferase fold having an N-terminal extension that either closes over the active site or is largely disordered. The ordered conformation places Tyr7 at a position of a structurally conserved tyrosine site of unknown function in various methyltransferases. Crystal structures of ToxA-Y7F consistently show a closed active site, whereas structures of ToxA-Y7A consistently show an open active site, suggesting that the hydroxyl group of Tyr7 plays a role in opening and closing the active site during the multistep reaction.

From Suicide Enzyme to Catalyst: The Iron-Dependent Sulfide Transfer in Methanococcus jannaschii Thiamin Thiazole Biosynthesis.

Bacteria and yeast utilize different strategies for sulfur incorporation in the biosynthesis of the thiamin thiazole. Bacteria use thiocarboxylated proteins. In contrast, Saccharomyces cerevisiae thiazole synthase (THI4p) uses an active site cysteine as the sulfide source and is inactivated after a single turnover. Here, we demonstrate that the Thi4 ortholog from Methanococcus jannaschii uses exogenous sulfide and is catalytic. Structural and biochemical studies on this enzyme elucidate the mechanistic details of the sulfide transfer reactions.

Structural Basis for Iron-Mediated Sulfur Transfer in Archael and Yeast Thiazole Synthases.

Thiamin diphosphate is an essential cofactor in all forms of life and plays a key role in amino acid and carbohydrate metabolism. Its biosynthesis involves separate syntheses of the pyrimidine and thiazole moieties, which are then coupled to form thiamin monophosphate. A final phosphorylation produces the active form of the cofactor. In most bacteria, six gene products are required for biosynthesis of the thiamin thiazole. In yeast and fungi only one gene product, Thi4, is required for thiazole biosynthesis. Methanococcus jannaschii expresses a putative Thi4 ortholog that was previously reported to be a ribulose 1,5-bisphosphate synthase [Finn, M. W. and Tabita, F. R. (2004) J. Bacteriol., 186, 6360-6366]. Our structural studies show that the Thi4 orthologs from M. jannaschii and Methanococcus igneus are structurally similar to Thi4 from Saccharomyces cerevisiae. In addition, all active site residues are conserved except for a key cysteine residue, which in S. cerevisiae is the source of the thiazole sulfur atom. Our recent biochemical studies showed that the archael Thi4 orthologs use nicotinamide adenine dinucleotide, glycine, and free sulfide to form the thiamin thiazole in an iron-dependent reaction [Eser, B., Zhang, X., Chanani, P. K., Begley, T. P., and Ealick, S. E. (2016) J. Am. Chem. Soc. , DOI: 10.1021/jacs.6b00445]. Here we report X-ray crystal structures of Thi4 from M. jannaschii complexed with ADP-ribulose, the C205S variant of Thi4 from S. cerevisiae with a bound glycine imine intermediate, and Thi4 from M. igneus with bound glycine imine intermediate and iron. These studies reveal the structural basis for the iron-dependent mechanism of sulfur transfer in archael and yeast thiazole synthases.

Polyketide Ring Expansion Mediated by a Thioesterase, Chain Elongation and Cyclization Domain, in Azinomycin Biosynthesis: Characterization of AziB and AziG.

The azinomycins are a family of potent antitumor agents with the ability to form interstrand cross-links with DNA. This study reports on the unusual biosynthetic formation of the 5-methyl naphthoate moiety, which is essential for effective DNA association. While sequence analysis predicts that the polyketide synthase (AziB) catalyzes the formation of this naphthoate, 2-methylbenzoic acid, a truncated single-ring product, is formed instead. We demonstrate that the thioesterase (AziG) acts as a chain elongation and cyclization (CEC) domain and is required for the additional two rounds of chain extension to form the expected product.

Anaerobic 5-Hydroxybenzimidazole Formation from Aminoimidazole Ribotide: An Unanticipated Intersection of Thiamin and Vitamin B12 Biosynthesis.

Comparative genomics of the bacterial thiamin pyrimidine synthase (thiC) revealed a paralogue of thiC (bzaF) clustered with anaerobic vitamin B12 biosynthetic genes. Here we demonstrate that BzaF is a radical S-adenosylmethionine enzyme that catalyzes the remarkable conversion of aminoimidazole ribotide (AIR) to 5-hydroxybenzimidazole (5-HBI). We identify the origin of key product atoms and propose a reaction mechanism. These studies represent the first step in solving a long-standing problem in anaerobic vitamin B12 assembly and reveal an unanticipated intersection of thiamin and vitamin B12 biosynthesis.

Different polyamine pathways from bacteria have replaced eukaryotic spermidine biosynthesis in ciliates Tetrahymena thermophila and Paramecium tetaurelia.

The polyamine spermidine is absolutely required for growth and cell proliferation in eukaryotes, due to its role in post-translational modification of essential translation elongation factor eIF5A, mediated by deoxyhypusine synthase. We have found that free-living ciliates Tetrahymena and Paramecium lost the eukaryotic genes encoding spermidine biosynthesis: S-adenosylmethionine decarboxylase (AdoMetDC) and spermidine synthase (SpdSyn). In Tetrahymena, they were replaced by a gene encoding a fusion protein of bacterial AdoMetDC and SpdSyn, present as three copies. In Paramecium, a bacterial homospermidine synthase replaced the eukaryotic genes. Individual AdoMetDC-SpdSyn fusion protein paralogues from Tetrahymena exhibit undetectable AdoMetDC activity; however, when two paralogous fusion proteins are mixed, AdoMetDC activity is restored and spermidine is synthesized. Structural modelling indicates a functional active site is reconstituted by sharing critical residues from two defective protomers across the heteromer interface. Paramecium was found to accumulate homospermidine, suggesting it replaces spermidine for growth. To test this concept, a budding yeast spermidine auxotrophic strain was found to grow almost normally with homospermidine instead of spermidine. Biosynthesis of spermidine analogue aminopropylcadaverine, but not exogenously provided norspermidine, correlated with some growth. Finally, we found that diverse single-celled eukaryotic parasites and multicellular metazoan Schistosoma worms have lost the spermidine biosynthetic pathway but retain deoxyhypusine synthase.