Takuo Osawa

Conserved Cysteine Residues of GidA Are Essential for Biogenesis of 5-Carboxymethylaminomethyluridine at tRNA Anticodon

The 5-carboxymethylaminomethyl modification of uridine (cmnm(5)U) at the anticodon first position occurs in tRNAs that read split codon boxes ending with purine. This modification is crucial for correct translation, by restricting codon-anticodon wobbling. Two conserved enzymes, GidA and MnmE, participate in the cmnm(5)U modification process. Here we determined the crystal structure of Aquifex aeolicus GidA at 2.3 A resolution. The structure revealed the tight interaction of GidA with FAD. Structure-based mutation analyses allowed us to identify two conserved Cys residues in the vicinity of the FAD-binding site that are essential for the cmnm(5)U modification in vivo. Together with mutational analysis of MnmE, we propose a mechanism for the cmnm(5)U modification process where GidA, but not MnmE, attacks the C6 atom of uridine by a mechanism analogous to that of thymidylate synthase. We also present a tRNA-docking model that provides structural insights into the tRNA recognition mechanism for efficient modification.

Crystal structure and chitin oligosaccharide-binding mode of a 'loopful' family GH19 chitinase from rye, Secale cereale, seeds

The substrate‐binding mode of a 26‐kDa GH19 chitinase from rye, Secale cereale, seeds (RSC‐c) was investigated by crystallography, site‐directed mutagenesis and NMR spectroscopy. The crystal structure of RSC‐c in a complex with an N‐acetylglucosamine tetramer, (GlcNAc)4, was successfully solved, and revealed the binding mode of the tetramer to be an aglycon‐binding site, subsites +1, +2, +3, and +4. These are the first crystallographic data showing the oligosaccharide‐binding mode of a family GH19 chitinase. From HPLC analysis of the enzymatic reaction products, mutation of Trp72 to alanine was found to affect the product distribution obtained from the substrate, p‐nitrophenyl penta‐N‐acetyl‐β‐chitopentaoside. Mutational experiments confirmed the crystallographic finding that the Trp72 side chain interacts with the +4 moiety of the bound substrate. To further confirm the crystallographic data, binding experiments were also conducted in solution using NMR spectroscopy. Several signals in the 1H–15N HSQC spectrum of the stable isotope‐labeled RSC‐c were affected upon addition of (GlcNAc)4. Signal assignments revealed that most signals responsive to the addition of (GlcNAc)4 are derived from amino acids located at the surface of the aglycon‐binding site. The binding mode deduced from NMR binding experiments in solution was consistent with that from the crystal structure.

Structural basis of tRNA agmatinylation essential for AUA codon decoding

The cytidine at the first position of the anticodon (C34) in the AUA codon-specific archaeal tRNAIle2 is modified to 2-agmatinylcytidine (agm2C or agmatidine), an agmatine-conjugated cytidine derivative, which is crucial for the precise decoding of the genetic code. Agm2C is synthesized by tRNAIle-agm2C synthetase (TiaS) in an ATP-dependent manner. Here we present the crystal structures of the Archaeoglobus fulgidus TiaS–tRNAIle2 complexed with ATP, or with AMPCPP and agmatine, revealing a previously unknown kinase module required for activating C34 by phosphorylation, and showing the molecular mechanism by which TiaS discriminates between tRNAIle2 and tRNAMet. In the TiaS–tRNAIle2–ATP complex, C34 is trapped within a pocket far away from the ATP-binding site. In the agmatine-containing crystals, C34 is located near the AMPCPP γ-phosphate in the kinase module, demonstrating that agmatine is essential for placing C34 in the active site. These observations also provide the structural dynamics for agm2C formation.

Biogenesis of 2-agmatinylcytidine catalyzed by the dual protein and RNA kinase TiaS

The archaeal AUA-codon specific tRNAIle contains 2-agmatinylcytidine (agm2C or agmatidine) at the anticodon wobble position (position 34). The formation of this essential modification is catalyzed by tRNAIle-agm2C synthetase (TiaS) using agmatine and ATP as substrates. TiaS has a previously unknown catalytic domain, which we have named the Thr18-Cyt34 kinase domain (TCKD). Biochemical analyses of Archaeoglobus fulgidus TiaS and its mutants revealed that the TCKD first hydrolyzes ATP into AMP and pyrophosphate, then phosphorylates the C2 position of C34 with the γ-phosphate. Next, the amino group of agmatine attacks this position to release the phosphate and form agm2C. Notably, the TCKD also autophosphorylates the Thr18 of TiaS, which may be involved in agm2C formation. Thus, the unique kinase domain of TiaS catalyzes dual phosphorylation of protein and RNA substrates.

Modulation of the transglycosylation activity of plant family GH18 chitinase by removing or introducing a tryptophan side chain

Transglycosylation (TG) activity of a family GH18 chitinase from the cycad, Cycas revoluta, (CrChiA) was modulated by removing or introducing a tryptophan side chain. The removal from subsite +3 through mutation of Trp168 to alanine suppressed TG activity, while introduction into subsite +1 through mutation of Gly77 to tryptophan (CrChiA‐G77W) enhanced TG activity. The crystal structures of an inactive double mutant of CrChiA (CrChiA‐G77W/E119Q) with one or two N‐acetylglucosamine residues occupying subsites +1 or +1/+2, respectively, revealed that the Trp77 side chain was oriented toward +1 GlcNAc to be stacked with it face‐to‐face, but rotated away from subsite +1 in the absence of GlcNAc at the subsite. Aromatic residues in the aglycon‐binding site are key determinants of TG activity of GH18 chitinases.

Crystal structures and inhibitor binding properties of plant class V chitinases: the cycad enzyme exhibits unique structural and functional features

A class V (glycoside hydrolase family 18) chitinase from the cycad Cycas revoluta (CrChiA) is a plant chitinase that has been reported to possess efficient transglycosylation (TG) activity. We solved the crystal structure of CrChiA, and compared it with those of class V chitinases from Nicotiana tabacum (NtChiV) and Arabidopsis thaliana (AtChiC), which do not efficiently catalyze the TG reaction. All three chitinases had a similar (α/β)8 barrel fold with an (α + β) insertion domain. In the acceptor binding site (+1, +2 and +3) of CrChiA, the Trp168 side chain was found to stack face-to-face with the +3 sugar. However, this interaction was not found in the identical regions of NtChiV and AtChiC. In the DxDxE motif, which is essential for catalysis, the carboxyl group of the middle Asp (Asp117) was always oriented toward the catalytic acid Glu119 in CrChiA, whereas the corresponding Asp in NtChiV and AtChiC was oriented toward the first Asp. These structural features of CrChiA appear to be responsible for the efficient TG activity. When binding of the inhibitor allosamidin was evaluated using isothermal titration calorimetry, the changes in binding free energy of the three chitinases were found to be similar to each other, i.e. between -9.5 and -9.8 kcal mol(-1) . However, solvation and conformational entropy changes in CrChiA were markedly different from those in NtChiV and AtChiC, but similar to those of chitinase A from Serratia marcescens (SmChiA), which also exhibits significant TG activity. These results provide insight into the molecular mechanism underlying the TG reaction and the molecular evolution from bacterial chitinases to plant class V chitinases.

Crystal Structure of the Csm3-Csm4 Subcomplex in the Type III-A CRISPR-Cas Interference Complex.

Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci play a pivotal role in the prokaryotic host defense system against invading genetic materials. The CRISPR loci are transcribed to produce CRISPR RNAs (crRNAs), which form interference complexes with CRISPR-associated (Cas) proteins to target the invading nucleic acid for degradation. The interference complex of the type III-A CRISPR-Cas system is composed of five Cas proteins (Csm1-Csm5) and a crRNA, and targets invading DNA. Here, we show that the Csm1, Csm3, and Csm4 proteins from Methanocaldococcus jannaschii form a stable subcomplex. We also report the crystal structure of the M. jannaschii Csm3-Csm4 subcomplex at 3.1Å resolution. The complex structure revealed the presence of a basic concave surface around their interface, suggesting the RNA and/or DNA binding ability of the complex. A gel retardation analysis showed that the Csm3-Csm4 complex binds single-stranded RNA in a non-sequence-specific manner. Csm4 structurally resembles Cmr3, a component of the type III-B CRISPR-Cas interference complex. Based on bioinformatics, we constructed a model structure of the Csm1-Csm4-Csm3 ternary complex, which provides insights into its role in the Csm interference complex.

Crystallization and preliminary X-ray diffraction analysis of the tRNA-modification enzyme GidA from Aquifex aeolicus.

The 5-carboxymethylaminomethyl modification of uridine at the first position of the tRNA anticodon is crucial for accurate protein synthesis by stabilizing the correct codon-anticodon pairing on the ribosome. Two conserved enzymes, GidA and MnmE, are involved in this modification process. Aquifex aeolicus GidA was crystallized in two different crystal forms: forms I and II. These crystals diffracted to 3.2 and 2.3 A resolution, respectively, using synchrotron radiation at the Photon Factory. These crystals belonged to space groups I2(1)2(1)2(1) and P2(1) with unit-cell parameters a = 101.6, b = 213.3, c = 231.7 A and a = 119.4, b = 98.0, c = 129.6 A, beta = 90.002 degrees , respectively. The asymmetric units of these crystals are expected to contain two and four molecules, respectively.

Crystallization and preliminary X-ray diffraction analysis of an archaeal tRNA-modification enzyme, TiaS, complexed with tRNAIle2 and ATP

The cytidine at the first anticodon position of archaeal tRNA(Ile2), which decodes the isoleucine AUA codon, is modified to 2-agmatinylcytidine (agm(2)C) to guarantee the fidelity of protein biosynthesis. This post-transcriptional modification is catalyzed by tRNA(Ile)-agm(2)C synthetase (TiaS) using ATP and agmatine as substrates. Archaeoglobus fulgidus TiaS was overexpressed in Escherichia coli cells and purified. tRNA(Ile2) was prepared by in vitro transcription with T7 RNA polymerase. TiaS was cocrystallized with both tRNA(Ile2) and ATP by the vapour-diffusion method. The crystals of the TiaS-tRNA(Ile2)-ATP complex diffracted to 2.9 Å resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the primitive hexagonal space group P3(2)21, with unit-cell parameters a = b = 131.1, c = 86.6 Å. The asymmetric unit is expected to contain one TiaS-tRNA(Ile2)-ATP complex, with a Matthews coefficient of 2.8 Å(3) Da(-1) and a solvent content of 61%.

Crystallization and preliminary X-ray diffraction analysis of a class V chitinase from Nicotiana tabacum.

The plant chitinases, which have been implicated in self-defence against pathogens, are divided into at least five classes (classes I, II, III, IV and V). Although the crystal structures of several plant chitinases have been solved, no crystal structure of a class V chitinase has been reported to date. Here, the crystallization of Nicotiana tabacum class V chitinase (NtChiV) using the vapour-diffusion method is reported. The NtChiV crystals diffracted to 1.2 Å resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a=62.4, b=120.3, c=51.9 Å. The asymmetric unit of the crystals is expected to contain one molecule.