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 <i>Pyrococcus horikoshii</i> 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

Pseudouridine Monophosphate Glycosidase: A New Glycosidase Mechanism

Pseudouridine (Ψ), the most abundant modification in RNA, is synthesized in situ using Ψ synthase. Recently, a pathway for the degradation of Ψ was described [Preumont, A., Snoussi, K., Stroobant, V., Collet, J. F., and Van Schaftingen, E. (2008) <i>J. Biol. Chem. 283</i>, 25238–25246]. In this pathway, Ψ is first converted to Ψ 5′-monophosphate (ΨMP) by Ψ kinase and then ΨMP is degraded by ΨMP glycosidase to uracil and ribose 5-phosphate. ΨMP glycosidase is the first example of a mechanistically characterized enzyme that cleaves a C–C glycosidic bond. Here we report X-ray crystal structures of <i>Escherichia coli</i> ΨMP glycosidase and a complex of the K166A mutant with ΨMP. We also report the structures of a ring-opened ribose 5-phosphate adduct and a ring-opened ribose ΨMP adduct. These structures provide four snapshots along the reaction coordinate. The structural studies suggested that the reaction utilizes a Lys166 adduct during catalysis. Biochemical and mass spectrometry data further confirmed the existence of a lysine adduct. We used site-directed mutagenesis combined with kinetic analysis to identify roles for specific active site residues. Together, these data suggest that ΨMP glycosidase catalyzes the cleavage of the C–C glycosidic bond through a novel ribose ring-opening mechanism

Formylglycinamide Ribonucleotide Amidotransferase from <i>Thermotoga maritima:</i> Structural Insights into Complex Formation

In the fourth step of the purine biosynthetic pathway, formyl glycinamide ribonucleotide (FGAR) amidotransferase, also known as PurL, catalyzes the conversion of FGAR, ATP, and glutamine to formyl glycinamidine ribonucleotide (FGAM), ADP, P_i, and glutamate. Two forms of PurL have been characterized, large and small. Large PurL, present in most Gram-negative bacteria and eukaryotes, consists of a single polypeptide chain and contains three major domains: the N-terminal domain, the FGAM synthetase domain, and the glutaminase domain, with a putative ammonia channel located between the active sites of the latter two. Small PurL, present in Gram-positive bacteria and archaea, is structurally homologous to the FGAM synthetase domain of large PurL, and forms a complex with two additional gene products, PurQ and PurS. The structure of the PurS dimer is homologous with the N-terminal domain of large PurL, while PurQ, whose structure has not been reported, contains the glutaminase activity. In <i>Bacillus subtilis</i>, the formation of the PurLQS complex is dependent on glutamine and ADP and has been demonstrated by size-exclusion chromatography. In this work, a structure of the PurLQS complex from <i>Thermotoga maritima</i> is described revealing a 2:1:1 stoichiometry of PurS:Q:L, respectively. The conformational changes observed in TmPurL upon complex formation elucidate the mechanism of metabolite-mediated recruitment of PurQ and PurS. The flexibility of the PurS dimer is proposed to play a role in the activation of the complex and the formation of the ammonia channel. A potential path for the ammonia channel is identified

<i>Burkholderia glumae</i> 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 <i>Burkholderia glumae</i>. The <i>tox</i> operon of <i>B. glumae</i> contains five putative toxoflavin biosynthetic genes <i>toxABCDE</i>. ToxA is a predicted <i>S</i>-adenosylmethionine-dependent methyltransferase, and <i>toxA</i> knockouts of <i>B. glumae</i> 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. <i>Methanococcus jannaschii</i> 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) <i>J. Bacteriol.</i>, <i>186</i>, 6360–6366]. Our structural studies show that the Thi4 orthologs from <i>M. jannaschii</i> and <i>Methanococcus igneus</i> are structurally similar to Thi4 from <i>Saccharomyces cerevisiae</i>. In addition, all active site residues are conserved except for a key cysteine residue, which in <i>S. cerevisiae</i> 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) <i>J. Am. Chem. Soc.</i>, DOI: 10.1021/jacs.6b00445]. Here we report X-ray crystal structures of Thi4 from <i>M. jannaschii</i> complexed with ADP-ribulose, the C205S variant of Thi4 from <i>S. cerevisiae</i> with a bound glycine imine intermediate, and Thi4 from <i>M. igneus</i> 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

Structure of a <i>Clostridium botulinum</i> C143S Thiaminase I/Thiamin Complex Reveals Active Site Architecture,

Thiaminases are responsible for the degradation of thiamin and its metabolites. Two classes of thiaminases have been identified based on their three-dimensional structures and their requirements for a nucleophilic second substrate. Although the reactions of several thiaminases have been characterized, the physiological role of thiamin degradation is not fully understood. We have determined the three-dimensional X-ray structure of an inactive C143S mutant of Clostridium botulinum (Cb) thiaminase I with bound thiamin at 2.2 Å resolution. The C143S/thiamin complex provides atomic level details of the orientation of thiamin upon binding to Cb-thiaminase I and the identity of active site residues involved in substrate binding and catalysis. The specific roles of active site residues were probed by using site directed mutagenesis and kinetic analyses, leading to a detailed mechanism for Cb-thiaminase I. The structure of Cb-thiaminase I is also compared to the functionally similar but structurally distinct thiaminase II

Reversal of the Substrate Specificity of CMP <i>N</i>‑Glycosidase to dCMP

MilB is a CMP hydrolase involved in the early steps of biosynthesis of the antifungal compound mildiomycin. An enzyme from the bacimethrin biosynthetic pathway, BcmB, is closely related to MilB in both sequence and function. These two enzymes belong to the nucleoside 2′-deoxyribosyltransferase (NDT) superfamily. NDTs catalyze <i>N</i>-glycosidic bond cleavage of 2′-deoxynucleosides via a covalent 2-deoxyribosyl-enzyme intermediate. Conservation of key active site residues suggests that members of the NDT superfamily share a common mechanism; however, the enzymes differ in their substrate preferences. Substrates vary in the type of nucleobase, the presence or absence of a 2′-hydroxyl group, and the presence or absence of a 5′-phosphate group. We have determined the structures of MilB and BcmB and compared them to previously determined structures of NDT superfamily members. The comparisons reveal how these enzymes differentiate between ribosyl and deoxyribosyl nucleotides or nucleosides and among different nucleobases. The 1.6 Å structure of the MilB–CMP complex reveals an active site feature that is not obvious from comparisons of sequence alone. MilB and BcmB that prefer substrates containing 2′-ribosyl groups have a phenylalanine positioned in the active site, whereas NDT family members with a preference for 2′-deoxyribosyl groups have a tyrosine residue. Further studies show that the phenylalanine is critical for the specificity of MilB and BcmB toward CMP, and mutation of this phenylalanine residue to tyrosine results in a 1000-fold reversal of substrate specificity from CMP to dCMP

Biochemical Characterization and Structural Basis of Reactivity and Regioselectivity Differences between <i>Burkholderia thailandensis</i> and <i>Burkholderia glumae</i> 1,6-Didesmethyltoxoflavin <i>N</i>‑Methyltransferase

<i>Burkholderia glumae</i> converts the guanine base of guanosine triphosphate into an azapteridine and methylates both the pyrimidine and triazine rings to make toxoflavin. Strains of <i>Burkholderia thailandensis</i> and <i>Burkholderia pseudomallei</i> have a gene cluster encoding seven putative biosynthetic enzymes that resembles the toxoflavin gene cluster. Four of the enzymes are similar in sequence to <i>Bg</i>ToxBCDE, which have been proposed to make 1,6-didesmethyltoxoflavin (1,6-DDMT). One of the remaining enzymes, <i>Bth</i>II1283 in <i>B. thailandensis</i> E264, is a predicted <i>S</i>-adenosylmethionine (SAM)-dependent <i>N</i>-methyltransferase that shows a low level of sequence identity to <i>Bg</i>ToxA, which sequentially methylates N6 and N1 of 1,6-DDMT to form toxoflavin. Here we show that, unlike <i>Bg</i>ToxA, <i>Bth</i>II1283 catalyzes a single methyl transfer to N1 of 1,6-DDMT <i>in vitro</i>. In addition, we investigated the differences in reactivity and regioselectivity by determining crystal structures of <i>Bth</i>II1283 with bound <i>S</i>-adenosylhomocysteine (SAH) or 1,6-DDMT and SAH. <i>Bth</i>II1283 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 <i>Bg</i>ToxA. In <i>Bth</i>II1283, the modeled SAM methyl group is directed toward the p orbital of N1, whereas in <i>Bg</i>ToxA, 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 <i>Bth</i>II1283, N1 is hydrogen bonded to a histidine residue whereas <i>Bg</i>ToxA does not supply an obvious basic residue for either N6 or N1 methylation

Identification of the Product of Toxoflavin Lyase: Degradation via a Baeyer–Villiger Oxidation

Toxoflavin (an azapteridine) is degraded to a single product by toxoflavin lyase (TflA) in a reaction dependent on reductant, Mn­(II), and oxygen. The isolated product was fully characterized by NMR and MS and was identified as a triazine in which the pyrimidine ring was oxidatively degraded. A mechanism for toxoflavin degradation based on the identification of the enzymatic product and the recently determined crystal structure of toxoflavin lyase is proposed