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1.  Overexpression, purification, crystallization and crystallographic analysis of CopK of Cupriavidus metallidurans  
Overexpression, purification and crystallization of C. metallidurans CopK allowed the collection of a complete data set to 2.2 Å resolution.
CopK of Cupriavidus metallidurans is a 93-amino-acid protein whose mature form (73 amino acids) has been purified and crystallized by the hanging-drop vapour-diffusion method in 100 mM citrate pH 3.5, 200 mM Li2SO4, 20%(w/v) glycerol, 13%(w/v) PEG 8000. Crystals display orthorhombic symmetry, with unit-cell parameters a = 57.53, b = 128.65, c = 49.77 Å, and diffract to 2.2 Å resolution using synchrotron radiation.
doi:10.1107/S174430910502316X
PMCID: PMC1978110  PMID: 16511169
CopK; copper; Ralstonia; Cupriavidus metallidurans
2.  A primordial RNA modification enzyme: the case of tRNA (m1A) methyltransferase 
Nucleic Acids Research  2004;32(2):465-476.
The modified nucleoside 1-methyladenosine (m1A) is found in the T-loop of many tRNAs from organisms belonging to the three domains of life (Eukaryota, Bacteria, Archaea). In the T-loop of eukaryotic and bacterial tRNAs, m1A is present at position 58, whereas in archaeal tRNAs it is present at position(s) 58 and/or 57, m1A57 being the obligatory intermediate in the biosynthesis of 1-methylinosine (m1I57). In yeast, the formation of m1A58 is catalysed by the essential tRNA (m1A58) methyltransferase (MTase), a tetrameric enzyme that is composed of two types of subunits (Gcd14p and Gcd10p), whereas in the bacterium Thermus thermophilus the enzyme is a homotetramer of the TrmI polypeptide. Here, we report that the TrmI enzyme from the archaeon Pyrococcus abyssi is also a homotetramer. However, unlike the bacterial site-specific TrmI MTase, the P.abyssi enzyme is region-specific and catalyses the formation of m1A at two adjacent positions (57 and 58) in the T-loop of certain tRNAs. The stabilisation of P.abyssi TrmI at extreme temperatures involves intersubunit disulphide bridges that reinforce the tetrameric oligomerisation, as revealed by biochemical and crystallographic evidences. The origin and evolution of m1A MTases is discussed in the context of different hypotheses of the tree of life.
doi:10.1093/nar/gkh191
PMCID: PMC373318  PMID: 14739239
3.  The yggH Gene of Escherichia coli Encodes a tRNA (m7G46) Methyltransferase 
Journal of Bacteriology  2003;185(10):3238-3243.
We cloned, expressed, and purified the Escherichia coli YggH protein and show that it catalyzes the S-adenosyl-l-methionine-dependent formation of N7-methylguanosine at position 46 (m7G46) in tRNA. Additionally, we generated an E. coli strain with a disrupted yggH gene and show that the mutant strain lacks tRNA (m7G46) methyltransferase activity.
doi:10.1128/JB.185.10.3238-3243.2003
PMCID: PMC154064  PMID: 12730187
4.  Cloning and characterization of tRNA (m1A58) methyltransferase (TrmI) from Thermus thermophilus HB27, a protein required for cell growth at extreme temperatures 
Nucleic Acids Research  2003;31(8):2148-2156.
N1-methyladenosine (m1A) is found at position 58 in the T-loop of many tRNAs. In yeast, the formation of this modified nucleoside is catalyzed by the essential tRNA (m1A58) methyltransferase, a tetrameric enzyme that is composed of two types of subunits (Gcd14p and Gcd10p). In this report we describe the cloning, expression and characterization of a Gcd14p homolog from the hyperthermophilic bacterium Thermus thermophilus. The purified recombinant enzyme behaves as a homotetramer of ∼150 kDa by gel filtration and catalyzes the site- specific formation of m1A at position 58 of the T-loop of tRNA in the absence of any other complementary protein. S-adenosylmethionine is used as donor of the methyl group. Thus, we propose to name the bacterial enzyme TrmI and accordingly its structural gene trmI. These results provide a key evolutionary link between the functionally characterized two-component eukaryotic enzyme and the recently described crystal structure of an uncharacterized, putative homotetrameric methyltransferase Rv2118c from Mycobacterium tuberculosis. Interest ingly, inactivation of the T.thermophilus trmI gene results in a thermosensitive phenotype (growth defect at 80°C), which suggests a role of the N1-methylation of tRNA adenosine-58 in adaptation of life to extreme temperatures.
PMCID: PMC153742  PMID: 12682365
5.  The arcABDC Gene Cluster, Encoding the Arginine Deiminase Pathway of Bacillus licheniformis, and Its Activation by the Arginine Repressor ArgR 
Journal of Bacteriology  1998;180(24):6468-6475.
The arginine deiminase pathway enables Bacillus licheniformis to grow anaerobically on arginine. Both the presence of arginine and anaerobiosis are needed to trigger induction of the pathway. In this study we have cloned and sequenced the arc genes encoding the pathway. They appear clustered in an operon-like structure in the order arcA (arginine deiminase), arcB (ornithine carbamoyltransferase), arcD (putative arginine-ornithine antiporter), arcC (carbamate kinase). It was found that B. licheniformis has an arginine repressor, ArgR, homologous to the B. subtilis arginine repressor AhrC. Mutants affected in argR were isolated. These mutants have lost both repression by arginine of the anabolic ornithine carbamoyltransferase and induction of the arginine deiminase pathway. Electrophoretic band shift experiments and DNase I footprinting revealed that in the presence of arginine, ArgR binds to a site upstream from the arc promoter. The binding site is centered 108 nucleotides upstream from the transcription start point and contains a single Arg box.
PMCID: PMC107747  PMID: 9851988

Results 1-5 (5)