Structural insights into the second step of RNA-dependent cysteine biosynthesis in archaea: crystal structure of Sep-tRNA:Cys-tRNA synthase from Archaeoglobus fulgidus

In the ancient organisms, methanogenic archaea, lacking the canonical cysteinyl-tRNA synthetase, Cys-tRNA(Cys) is produced by an indirect pathway, in which O-phosphoseryl-tRNA synthetase ligates O-phosphoserine (Sep) to tRNA(Cys) and Sep-tRNA:Cys-tRNA synthase (SepCysS) converts Sep-tRNA(Cys) to Cys-tRNA(Cys). In this study, the crystal structure of SepCysS from Archaeoglobus fulgidus has been determined at 2.4 A resolution. SepCysS forms a dimer, composed of monomers bearing large and small domains. The large domain harbors the seven-stranded beta-sheet, which is typical of the pyridoxal 5'-phosphate (PLP)-dependent enzymes. In the active site, which is located near the dimer interface, PLP is covalently bound to the side-chain of the conserved Lys209. In the proximity of PLP, a sulfate ion is bound by the side-chains of the conserved Arg79, His103, and Tyr104 residues. The active site is located deep within the large, basic cleft to accommodate Sep-tRNA(Cys). On the basis of the surface electrostatic potential, the amino acid residue conservation mapping, the position of the bound sulfate ion, and the substrate amino acid binding manner in other PLP-dependent enzymes, a binding model of Sep-tRNA(Cys) to SepCysS was constructed. One of the three strictly conserved Cys residues (Cys39, Cys42, or Cys247), of one subunit may play a crucial role in the catalysis in the active site of the other subunit.     

Molecular basis of the antimutagenic activity of the house-cleaning inosine triphosphate pyrophosphatase RdgB from Escherichia coli

Inosine triphosphate pyrophosphatases, which are ubiquitous house-cleaning enzymes, hydrolyze noncanonical nucleoside triphosphates (inosine triphosphate (ITP) and xanthosine triphosphate (XTP)) and prevent the incorporation of hypoxanthine or xanthine into nascent DNA or RNA. Here we present the 1.5-Å-resolution crystal structure of the inosine triphosphate pyrophosphatase RdgB from Escherichia coli in a free state and in complex with a substrate (ITP + Ca^2 +) or a product (inosine monophosphate (IMP)). ITP binding to RdgB induced a large displacement of the α1 helix, closing the enzyme active site. This positions the conserved Lys13 close to the bridging oxygen between the α- and β-phosphates of the substrate, weakening the P_α-O bond. On the other side of the substrate, the conserved Asp69 is proposed to act as a base coordinating the catalytic water molecule. Our data provide insight into the molecular mechanisms of the substrate selectivity and catalysis of RdgB and other ITPases.     

Effect of inorganic and organic forms of selenium supplementation on development of larval Heliothis virescens

Selenium (Se) is an essential micronutrient for vertebrates though little is known about the effects on insects. Herbivorous insect larvae acquire Se from plant tissues in the inorganic form of sodium selenate and sodium selenite, and in the organic form of selenoamino acids, selenomethionine, and selenocystine. In this study, we document the effects of dietary supplementation with sodium selenite, sodium selenate, selenocystine, selenomethionine, and selenized yeast on the developmental rate of Heliothis virescens (Fabricius) (Lepidoptera: Noctuidae). Larvae tolerated high levels of Se (500 µg g^?1 Se) as sodium selenate and to a lesser extent as selenocystine. Lower levels of sodium selenite (>1 µg g^?1 Se) caused increased mortality, reduced rates of pupation, more pupal/adult intermediates, and reduced adult emergence. Selenomethionine proved toxic to larvae at levels above 25 µg g^?1 Se, significantly delaying pupation and raising mortality. Provision of Se as selenized yeast, which contains primarily selenomethionine, was also extremely detrimental to larval development and survival. The results indicate that the impact of dietary Se supplement for insects may differ from vertebrates.     

Crystal structure and mutational study of a unique SpoU family archaeal methylase that forms 2'-O-methylcytidine at position 56 of tRNA

The conserved cytidine residue at position 56 of tRNA contributes to the maintenance of the L-shaped tertiary structure. aTrm56 catalyzes the 2′-O-methylation of the cytidine residue in archaeal tRNA, using S-adenosyl-L-methionine. Based on the amino acid sequence, aTrm56 is the most distant member of the SpoU family. Here, we determined the crystal structure of Pyrococcus horikoshii aTrm56 complexed with S-adenosyl-L-methionine at 2.48 Å resolution. aTrm56 consists of the SPOUT domain, which contains the characteristic deep trefoil knot, and a unique C-terminal β-hairpin. aTrm56 forms a dimer. The S-adenosyl-L-methionine binding and dimerization of aTrm56 were similar to those of the other SpoU members. A structure-based sequence alignment revealed that aTrm56 conserves only motif II, among the four signature motifs. However, an essential Arg16 residue is located at a novel position within motif I. Biochemical assays showed that aTrm56 prefers the L-shaped tRNA to the λ form as its substrate.     

Structure of N-acetyl-beta-D-glucosaminidase (GcnA) from the endocarditis pathogen Streptococcus gordonii and its complex with the mechanism-based inhibitor NAG-thiazoline

The crystal structure of GcnA, an N-acetyl-β-d-glucosaminidase from Streptococcus gordonii, was solved by multiple wavelength anomalous dispersion phasing using crystals of selenomethionine-substituted protein. GcnA is a homodimer with subunits each comprised of three domains. The structure of the C-terminal α-helical domain has not been observed previously and forms a large dimerisation interface. The fold of the N-terminal domain is observed in all structurally related glycosidases although its function is unknown. The central domain has a canonical (β/α)₈ TIM-barrel fold which harbours the active site. The primary sequence and structure of this central domain identifies the enzyme as a family 20 glycosidase. Key residues implicated in catalysis have different conformations in two different crystal forms, which probably represent active and inactive conformations of the enzyme. The catalytic mechanism for this class of glycoside hydrolase, where the substrate rather than the enzyme provides the cleavage-inducing nucleophile, has been confirmed by the structure of GcnA complexed with a putative reaction intermediate analogue, N-acetyl-β-d-glucosamine-thiazoline. The catalytic mechanism is discussed in light of these and other family 20 structures.     

The crystal structure of DehI reveals a new alpha-haloacid dehalogenase fold and active-site mechanism

Haloacid dehalogenases catalyse the removal of halides from organic haloacids and are of interest for bioremediation and for their potential use in the synthesis of industrial chemicals. We present the crystal structure of the homodimer DehI from Pseudomonas putida strain PP3, the first structure of a group I α-haloacid dehalogenase that can process both l- and d-substrates. The structure shows that the DehI monomer consists of two domains of ∼ 130 amino acids that have ∼ 16% sequence identity yet adopt virtually identical and unique folds that form a pseudo-dimer. Analysis of the active site reveals the likely binding mode of both l- and d-substrates with respect to key catalytic residues. Asp189 is predicted to activate a water molecule for nucleophilic attack of the substrate chiral centre resulting in an inversion of configuration of either l- or d-substrates in contrast to d-only enzymes. These details will assist with future bioengineering of dehalogenases.     

Biochemical and structural analysis of substrate promiscuity in plant Mg2+-dependent O-methyltransferases

Plant S-adenosyl-l-methionine-dependent class I natural product O-methyltransferases (OMTs), related to animal catechol OMTs, are dependent on bivalent cations and strictly specific for the meta position of aromatic vicinal dihydroxy groups. While the primary activity of these class I enzymes is methylation of caffeoyl coenzyme A OMTs, a distinct subset is able to methylate a wider range of substrates, characterized by the promiscuous phenylpropanoid and flavonoid OMT. The observed broad substrate specificity resides in two regions: the N-terminus and a variable insertion loop near the C-terminus, which displays the lowest degree of sequence conservation between the two subfamilies. Structural and biochemical data, based on site-directed mutagenesis and domain exchange between the two enzyme types, present evidence that only small topological changes among otherwise highly conserved 3-D structures are sufficient to differentiate between an enzymatic generalist and an enzymatic specialist in plant natural product methylation.     

Crystal structures and mutagenesis of sucrose hydrolase from Xanthomonas axonopodis pv. glycines: insight into the exclusively hydrolytic amylosucrase fold

Neisseria polysaccharea amylosucrase (NpAS), a transglucosidase of glycoside hydrolase family 13, is a hydrolase and glucosyltransferase that catalyzes the synthesis of amylose-like polymer from a sucrose substrate. Recently, an NpAS homolog from Xanthomonas axonopodis pv. glycines was identified as a member of the newly defined carbohydrate utilization locus that regulates the utilization of plant sucrose in phytopathogenic bacteria. Interestingly, this enzyme is exclusively a hydrolase and not a glucosyltransferase; it is thus known as sucrose hydrolase (SUH). Here, we elucidated the novel functional features of SUH using X-ray crystallography and site-directed mutagenesis. Four different crystal structures of SUH, including the SUH-Tris and the SUH-sucrose and SUH-glucose complexes, represent structural snapshots along the catalytic reaction coordinate. These structures show that SUH is distinctly different from NpAS in that ligand-induced conformational changes in SUH cause the formation of a pocket-shaped active site and in that SUH lacks the three arginine residues found in the NpAS active site that appear to be crucial for NpAS glucosyltransferase activity. Mutation of SUH to insert these arginines failed to confer glucosyltransferase activity, providing evidence that its enzymatic activity is limited to sucrose hydrolysis by its pocket-shaped active site and the identity of residues in the vicinity of the active site.