Background: QueF is the only nitrile reductase known in biology.Results: x-ray crystallography and transient kinetics provide evidence for a thioimide adduct and its rate of formation. Conclusion: Formation of a thioimide adduct is a key step in the mechanism of reaction. Significance: This is the first structural observation of the nitrile reductase QueF with bound substrate.

Queuosine is a modified pyrrolopyrimidine nucleoside found in the anticodon loop of transfer RNA acceptors for the amino acids tyrosine, asparagine, aspartic acid, and histidine. Because it is exclusively synthesized by bacteria, higher eukaryotes must salvage queuosine or its nucleobase queuine from food and the gut microflora. Previously, animals made deficient in queuine died within 18 days of withdrawing tyrosine, a nonessential amino acid, from the diet (Marks, T., and Farkas, W. R. (1997) Biochem. Biophys. Res. Commun. 230, 233-237). Here, we show that human HepG2 cells deficient in queuine and mice made deficient in queuosine-modified transfer RNA, by disruption of the tRNA guanine transglycosylase enzyme, are compromised in their ability to produce tyrosine from phenylalanine. This has similarities to the disease phenylketonuria, which arises from mutation in the enzyme phenylalanine hydroxylase or from a decrease in the supply of its cofactor tetrahydrobiopterin (BH4). Immunoblot and kinetic analysis of liver from tRNA guanine transglycosylase-deficient animals indicates normal expression and activity of phenylalanine hydroxylase. By contrast, BH4 levels are significantly decreased in the plasma, and both plasma and urine show a clear elevation in dihydrobiopterin, an oxidation product of BH4, despite normal activity of the salvage enzyme dihydrofolate reductase. Our data suggest that queuosine modification limits BH4 oxidation in vivo and thereby potentially impacts on numerous physiological processes in eukaryotes.Bacteria and humans have co-evolved for millennia, and many examples exist of how various symbiotic and commensal partnerships contribute to human health and nutrition ranging from the metabolism of complex carbohydrates to the provision of vital micronutrients (1). Queuosine is an example of a micronutrient, synthesized exclusively by bacteria but which, for poorly defined reasons, is utilized by almost all eukaryotic species with the exception of the baker's yeast, Saccharomyces cerevisiae (2).Bacterial queuosine biosynthesis occurs in two stages. First, a series of five enzymatic steps convert guanosine triphosphate nucleoside (GTP) to the soluble 7-aminomethyl-7-deazaguanine molecule. Subsequently, 7-aminomethyl-7-deazaguanine is inserted into the wobble position of tRNA containing a GUN consensus sequence (Tyr, Asp, Asn, and His) by means of the single enzyme species, tRNA guanine transglycosylase (TGT), and is further remodeled in situ to queuosine (3). Eukaryotes must acquire queuosine or its free nucleobase, queuine, from food and the gut microflora. Curiously, both cytosolic and mitochondrial tRNA species are modified by queuosine (2). The eukaryotic enzyme that performs this reaction, queuine tRNA ribosyltransferase, has recently been identified as a heterodimeric complex, consisting of the eukaryotic homologue of the catalytic TGT subunit and a related protein called queuine tRNA ribosyltransferase domain containing 1 (QTRTD1), both of which localize to the mitochondria (4, 5).Stu...

The presence of the 7-deazaguanosine derivative archaeosine (G ؉ ) at position 15 in tRNA is one of the diagnostic molecular characteristics of the Archaea. The biosynthesis of this modified nucleoside is especially complex, involving the initial production of 7-cyano-7-deazaguanine (preQ 0 ), an advanced precursor that is produced in a tRNA-independent portion of the biosynthesis, followed by its insertion into the tRNA by the enzyme tRNA-guanine transglycosylase (arcTGT), which replaces the target guanine base yielding preQ 0 -tRNA. The enzymes responsible for the biosynthesis of preQ 0 were recently identified, but the enzyme(s) catalyzing the conversion of preQ 0 -tRNA to G ؉ -tRNA have remained elusive. Using a comparative genomics approach, we identified a protein family implicated in the late stages of archaeosine biosynthesis. Notably, this family is a paralog of arcTGT and is generally annotated as TgtA2. Structurebased alignments comparing arcTGT and TgtA2 reveal that TgtA2 lacks key arcTGT catalytic residues and contains an additional module. We constructed a Haloferax volcanii ⌬tgtA2 derivative and demonstrated that tRNA from this strain lacks G ؉ and instead accumulates preQ 0 . We also cloned the corresponding gene from Methanocaldococcus jannaschii (mj1022) and characterized the purified recombinant enzyme. Recombinant MjTgtA2 was shown to convert preQ 0 -tRNA to G ؉ -tRNA using several nitrogen sources and to do so in an ATP-independent process. This is the only example of the conversion of a nitrile to a formamidine known in biology and represents a new class of amidinotransferase chemistry.

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The overaccumulation of glycogen appears as a hallmark in various glycogen storage diseases (GSDs), including Pompe, Cori, Andersen, and Lafora disease. Accumulating evidence suggests that suppression of glycogen accumulation represents a potential therapeutic approach for treating these GSDs. Using a fluorescence polarization assay designed to screen for inhibitors of the key glycogen synthetic enzyme, glycogen synthase (GS), we identified a substituted imidazole, (rac)-2-methoxy-4-(1-(2-(1-methylpyrrolidin-2-yl)­ethyl)-4-phenyl-1H-imidazol-5-yl)­phenol (H23), as a first-in-class inhibitor for yeast GS 2 (yGsy2p). Data from X-ray crystallography at 2.85 Å, as well as kinetic data, revealed that H23 bound within the uridine diphosphate glucose binding pocket of yGsy2p. The high conservation of residues between human and yeast GS in direct contact with H23 informed the development of around 500 H23 analogs. These analogs produced a structure–activity relationship profile that led to the identification of a substituted pyrazole, 4-(4-(4-hydroxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-5-yl)­pyrogallol, with a 300-fold improved potency against human GS. These substituted pyrazoles possess a promising scaffold for drug development efforts targeting GS activity in GSDs associated with excess glycogen accumulation.

Glycogen synthase is a rate-limiting enzyme in the biosynthesis of glycogen and has an essential role in glucose homeostasis. The three-dimensional structures of yeast glycogen synthase (Gsy2p) complexed with maltooctaose identified four conserved maltodextrin-binding sites distributed across the surface of the enzyme. Site-1 is positioned on the N-terminal domain, site-2 and site-3 are present on the C-terminal domain, and site-4 is located in an interdomain cleft adjacent to the active site. Mutation of these surface sites decreased glycogen binding and catalytic efficiency toward glycogen. Mutations within site-1 and site-2 reduced the V max /S 0.5 for glycogen by 40-and 70-fold, respectively. Combined mutation of site-1 and site-2 decreased the V max /S 0.5 for glycogen by >3000-fold. Consistent with the in vitro data, glycogen accumulation in glycogen synthase-deficient yeast cells (⌬gsy1-gsy2) transformed with the site-1, site-2, combined site-1/site-2, or site-4 mutant form of Gsy2p was decreased by up to 40-fold. In contrast to the glycogen results, the ability to utilize maltooctaose as an in vitro substrate was unaffected in the site-2 mutant, moderately affected in the site-1 mutant, and almost completely abolished in the site-4 mutant. These data show that the ability to utilize maltooctaose as a substrate can be independent of the ability to utilize glycogen. Our data support the hypothesis that site-1 and site-2 provide a "toehold mechanism," keeping glycogen synthase tightly associated with the glycogen particle, whereas site-4 is more closely associated with positioning of the nonreducing end during catalysis.Glycogen synthase was the first reported intracellular target of insulin, and the enzyme catalyzes the linear polymerization of glucose residues from activated sugar donor molecules to the nonreducing end of the glycogen chain. Recent structural studies have shown that the enzyme folds into two Rossmann foldlike domains, with a deep cleft in between harboring the active site (1-3). Although the basic fold is conserved between the prokaryotic, archaeal, and eukaryotic enzymes, there are multiple sequence insertions in the eukaryotic enzymes. The largest of these insertions (a long coiled-coil insert in the C-terminal domain) gives rise to their unique tetrameric arrangement as well as the structural plasticity necessary for the complex regulation of glycogen synthase activity in eukaryotes (3). Furthermore, a conserved arginine cluster present in the C-terminal region of the eukaryotic enzymes mediates the sensitivity to inhibition by phosphorylation and activation by glucose 6-phosphate (4, 5). In our recent structural studies, we demonstrated that the middle two arginine residues 3 are necessary and sufficient to confer regulation by glucose 6-phosphate and that the first three arginine residues (Arg-580, Arg-581, and Arg-583) are required for full regulatory response to phosphorylation (3).The yeast Saccharomyces cerevisiae possesses two genes encoding glycogen synthase, GSY1 and GSY2, wh...

Significance Glycogen is a branched glucose polymer found in most animals, fungi, bacteria, and archaea as an osmotically neutral means of energy storage. Glycogen also contains minor amounts of phosphate which can be removed by a dual specificity phosphatase, laforin. Accumulation of phosphate results in highly insoluble glycogen deposits and underlies Lafora disease, a devastating form of myoclonus epilepsy. In this paper, we present structural and kinetic data that support a plausible mechanism by which phosphate is directly incorporated into glycogen by glycogen synthase.

BackgroundThe Cry6 family of proteins from Bacillus thuringiensis represents a group of powerful toxins with great potential for use in the control of coleopteran insects and of nematode parasites of importance to agriculture. These proteins are unrelated to other insecticidal toxins at the level of their primary sequences and the structure and function of these proteins has been poorly studied to date. This has inhibited our understanding of these toxins and their mode of action, along with our ability to manipulate the proteins to alter their activity to our advantage. To increase our understanding of their mode of action and to facilitate further development of these proteins we have determined the structure of Cry6Aa in protoxin and trypsin-activated forms and demonstrated a pore-forming mechanism of action.ResultsThe two forms of the toxin were resolved to 2.7 Å and 2.0 Å respectively and showed very similar structures. Cry6Aa shows structural homology to a known class of pore-forming toxins including hemolysin E from Escherichia coli and two Bacillus cereus proteins: the hemolytic toxin HblB and the NheA component of the non-hemolytic toxin (pfam05791). Cry6Aa also shows atypical features compared to other members of this family, including internal repeat sequences and small loop regions within major alpha helices. Trypsin processing was found to result in the loss of some internal sequences while the C-terminal region remains disulfide-linked to the main core of the toxin. Based on the structural similarity of Cry6Aa to other toxins, the mechanism of action of the toxin was probed and its ability to form pores in vivo in Caenorhabditis elegans was demonstrated. A non-toxic mutant was also produced, consistent with the proposed pore-forming mode of action.ConclusionsCry6 proteins are members of the alpha helical pore-forming toxins – a structural class not previously recognized among the Cry toxins of B. thuringiensis and representing a new paradigm for nematocidal and insecticidal proteins. Elucidation of both the structure and the pore-forming mechanism of action of Cry6Aa now opens the way to more detailed analysis of toxin specificity and the development of new toxin variants with novel activities.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0295-9) contains supplementary material, which is available to authorized users.