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Reversible Transfer (reversible + transfer)
Selected AbstractsORF6 from the clavulanic acid gene cluster of Streptomyces clavuligerus has ornithine acetyltransferase activityFEBS JOURNAL, Issue 8 2002Nadia J. Kershaw The clinically used beta-lactamase inhibitor clavulanic acid is produced by fermentation of Streptomyces clavuligerus. The orf6 gene of the clavulanic acid biosynthetic gene cluster in S. clavuligerus encodes a protein that shows sequence homology to ornithine acetyltransferase (OAT), the fifth enzyme of the arginine biosynthetic pathway. Orf6 was overexpressed in Escherichia coli (at ,,15% of total soluble protein by SDS/PAGE analysis) indicating it was not toxic to the host cells. The recombinant protein was purified (to >,95% purity) by a one-step technique. Like other OATs it was synthesized as a precursor protein which underwent autocatalytic internal cleavage in E. coli to generate , and , subunits. Cleavage was shown to occur between the alanine and threonine residues in a KGXGMXXPX--(M/L)AT (M/L)L motif conserved within all identified OAT sequences. Gel filtration and native electrophoresis analyses implied that the ORF6 protein was an ,2,2 heterotetramer and direct evidence for this came from mass spectrometric analyses. Although anomalous migration of the , subunit was observed by standard SDS/PAGE analysis, which indicated the presence of two bands (as previously observed for other OATs), mass spectrometric analyses did not reveal any evidence for post-translational modification of the , subunit. Extended denaturation with SDS before PAGE resulted in observation of a single major , subunit band. Purified ORF6 was able to catalyse the reversible transfer of an acetyl group from N -acetylornithine to glutamate, but not the formation of N -acetylglutamate from glutamate and acetyl-coenzyme A, nor (detectably) the hydrolysis of N -acetylornithine. Mass spectrometry also revealed the reaction proceeds via acetylation of the , subunit. [source] The three-dimensional structure of cytosolic bovine retinal creatine kinaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2001Dominic Tisi Creatine kinase (CK) catalyses the reversible transfer of the phosphate moiety from phosphocreatine (PCr) to ADP, generating creatine and ATP. The crystal structure of a cytosolic brain-type creatine kinase is reported at 2.3,Å. The biological dimer sits on a crystallographic twofold axis. The N-terminal residues of both subunits come very close to the crystallographic twofold at the dimer interface. The electron density observed is consistent with two alternative conformations for the N-termini, as previously found for chicken brain-type creatine kinase. [source] The structure of Staphylococcus aureus phosphopantetheine adenylyltransferase in complex with 3,-phosphoadenosine 5,-phosphosulfate reveals a new ligand-binding modeACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 10 2009Hyung Ho Lee Bacterial phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in the coenzyme A (CoA) biosynthetic pathway. It catalyzes the reversible transfer of an adenylyl group from ATP to 4,-phosphopantetheine (Ppant) to form dephospho-CoA (dPCoA) and pyrophosphate. Previous structural studies have revealed how several ligands are recognized by bacterial PPATs. ATP, ADP, Ppant and dPCoA bind to the same binding site in a highly similar manner, while CoA binds to a partially overlapping site in a different mode. To provide further structural insights into ligand binding, the crystal structure of Staphylococcus aureus PPAT was solved in a binary complex with 3,-phosphoadenosine 5,-phosphosulfate (PAPS). This study unexpectedly revealed a new mode of ligand binding to PPAT, thus providing potentially useful information for structure-based discovery of inhibitors of bacterial PPATs. [source] Cobalt-, zinc- and iron-bound forms of adenylate kinase (AK) from the sulfate-reducing bacterium Desulfovibrio gigas: purification, crystallization and preliminary X-ray diffraction analysisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2009A. V. Kladova Adenylate kinase (AK; ATP:AMP phosphotransferase; EC 2.7.4.3) is involved in the reversible transfer of the terminal phosphate group from ATP to AMP. AKs contribute to the maintenance of a constant level of cellular adenine nucleotides, which is necessary for the energetic metabolism of the cell. Three metal ions, cobalt, zinc and iron(II), have been reported to be present in AKs from some Gram-negative bacteria. Native zinc-containing AK from Desulfovibrio gigas was purified to homogeneity and crystallized. The crystals diffracted to beyond 1.8,Å resolution. Furthermore, cobalt- and iron-containing crystal forms of recombinant AK were also obtained and diffracted to 2.0 and 3.0,Å resolution, respectively. Zn2+,AK and Fe2+,AK crystallized in space group I222 with similar unit-cell parameters, whereas Co2+,AK crystallized in space group C2; a monomer was present in the asymmetric unit for both the Zn2+,AK and Fe2+,AK forms and a dimer was present for the Co2+,AK form. The structures of the three metal-bound forms of AK will provide new insights into the role and selectivity of the metal in these enzymes. [source] | ||||||||||