Structure of a Sir2 substrate, Alba, reveals a mechanism for deacetylation-induced enhancement of DNA binding.
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The targeted acetylation status of histones and several other transcriptional regulatory proteins plays an important role in gene expression, although the mechanism for this is not well understood. As a model to understand how targeted acetylation may effect transcription, we determined the x-ray crystal structure of the chromatin protein Alba from Archaeoglobus fulgidus, a substrate for the Sir2 protein that deacetylates it at lysine 11 to promote DNA binding by Alba. The structure reveals a dimer of dimers in which the dimer-dimer interface is stabilized by several conserved hydrophobic residues as well as the lysine 11 target of Sir2. We show that, in solution, the mutation of these hydrophobic residues or lysine 11 disrupts dimer-dimer formation and decreases DNA-binding affinity. We propose that the in vivo deacetylation of lysine 11 of archaeal Alba by Sir2 promotes protein oligomerization for optimal DNA binding. Implications for the mechanism by which histone acetylation modulates gene expression are discussed. (+info)
Molecular characterization of H2O2-forming NADH oxidases from Archaeoglobus fulgidus.
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Three NADH oxidase encoding genes noxA-1, noxB-1 and noxC were cloned from the genome of Archaeoglobus fulgidus, expressed in Escherichia coli, and the gene products were purified and characterized. Expression of noxA-1 and noxB-1 resulted in active gene products of the expected size. The noxC gene was expressed as well but the protein produced showed no activity in the standard Nox assay. NoxA-1 and NoxB-1 are both FAD-containing enzymes with subunit molecular masses of 48 and 69 kDa, respectively. NoxA-1 exists predominantly as homodimer, NoxB-1 as monomer. NoxA-1 and NoxB-1 showed pH optimum of 8.0 and 6.5, with specific NADH oxidase activities of 5.8 U.mg-1 and 4.1 U.mg-1, respectively. Both enzymes were specific for NADH as electron donor, but with different apparent Km values (NoxA-1, 0.13 mm; NoxB-1, 0.011 mm). The apparent Km values for oxygen differed significantly (NoxA-1, 0.06 mm; NoxB-1, 2.9 mm). In contrast with all mesophilic homologues, both enzymes were found to produce predominantly H2O2 instead of H2O. Despite apparent similarities, NoxB-1 is essentially different from NoxA-1. Whereas NoxA-1 resembles typical H2O-producing Nox enzymes that are expected to have a role in oxidative stress defence, NoxB-1 belongs to a small group of enzymes that is involved in catalysing the reduction of unsaturated acids and aldehydes, suggesting a role in fatty acid oxidation. Moreover, NoxB-1 contains a ferredoxin-like motif, which is absent in NoxA-1. (+info)
Archaeoglobus fulgidus CopB is a thermophilic Cu2+-ATPase: functional role of its histidine-rich-N-terminal metal binding domain.
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P1B-type ATPases transport heavy metal ions across cellular membranes. Archaeoglobus fulgidus CopB is a member of this subfamily. We have cloned, expressed in Escherichia coli, and functionally characterized this enzyme. CopB and its homologs are distinguished by a metal binding sequence Cys-Pro-His in their sixth transmembrane segment (H6) and a His-rich N-terminal metal binding domain (His-N-MBD). CopB is a thermophilic protein active at 75 degrees C and high ionic strength. It is activated by Cu2+ with high apparent affinity (K1/2 = 0.28 microm) and partially by Cu+ and Ag+ (22 and 55%, respectively). The higher turnover was associated with a faster phosphorylation rate in the presence of Cu2+. A truncated CopB lacking the first 54 amino acids was constructed to characterize the His-N-MBD. This enzyme showed reduced ATPase activity (50% of wild type) but no changes in metal selectivity, ATP dependence, or phosphorylation levels. However, a slower rate of dephosphorylation of the E2P(Cu2+) form was observed for truncated CopB. The data suggest that the presence of the His residue in the putative transmembrane metal binding site of CopB determines a selectivity for this enzyme that is different for that observed in Cu+/Ag+-ATPases carrying a Cys-Pro-Cys sequence. The His-NMBD appears to have a regulatory role affecting the metal transport rate by controlling the metal release/dephosphorylation rates. (+info)
Alkylation damage repair protein O6-alkylguanine-DNA alkyltransferase from the hyperthermophiles Aquifex aeolicus and Archaeoglobus fulgidus.
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AGT (O6-alkylguanine DNA alkyltransferase) is an important DNA-repair protein that protects cells from killing and mutagenesis by alkylating agents. The AGT genes from two extremely thermophilic organisms, the bacterium Aquifex aeolicus and the archaeon Archaeoglobus fulgidus were PCR-derived and cloned into an expression vector. The nucleotide sequence of the Aq. aeolicus AGT encodes a 201-amino-acid protein with a molecular mass of 23000 Da and Ar. fulgidus AGT codes for a 147-amino-acid protein with a molecular mass of 16718 Da. The Aq. aeolicus and Ar. fulgidus AGTs were expressed at high levels in Escherichia coli fused to an N-terminal polyhistidine tag that allowed single-step isolation and purification by metal-affinity chromatography. Both AGTs formed inclusion bodies and were not soluble under native purification conditions. Therefore AGT isolation was performed under protein-denaturation conditions in the presence of 8.0 M urea. Soluble AGT was obtained by refolding the AGT in the presence of calf thymus DNA. Both AGTs were active in repairing O6-methylguanine and, at a lower rate, O4-methylthymine in DNA. They exhibited thermostability and optimum activity at high temperature. The thermostable AGTs, particularly that from Aq. aeolicus, were readily inactivated by the low-molecular-mass inhibitor O6-benzylguanine, which is currently in clinical trials to enhance cancer chemotherapy. (+info)
Biochemical analysis of components of the pre-replication complex of Archaeoglobus fulgidus.
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The eukaryotic pre-replication complex is assembled at replication origins in a reaction called licensing. Licensing involves the interactions of a variety of proteins including the origin recognition complex (ORC), Cdc6 and the Mcm2-7 helicase, homologues of which are also found in archaea. The euryarchaeote Archaeoglobus fulgidus encodes two genes with homology to Orc/Cdc6 and a single Mcm homologue. The A.fulgidus Mcm protein and one Orc/Cdc6 homologue have been purified and investigated in vitro. The Mcm protein is an ATP-dependent, hexameric helicase that can unwind between 200 and 400 bp of duplex DNA. Deletion of 112 amino acids from the N-terminus of A.f Mcm produced a protein, which was still capable of forming a hexamer, was competent in DNA binding and was able to unwind at least 1 kb of duplex DNA. The purified Orc/Cdc6 homologue was also able to bind DNA. Both Mcm and Orc/Cdc6 show a preference for specific DNA structures, namely molecules containing a single stranded bubble that mimics early replication intermediates. Nuclease protection showed that the binding sites for Mcm and Orc/Cdc6 overlap. The Orc/Cdc6 protein bound more tightly to these substrates and was able to displace pre-bound Mcm hexamer. (+info)
Phenol hydroxylase from Bacillus thermoglucosidasius A7, a two-protein component monooxygenase with a dual role for FAD.
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A novel phenol hydroxylase (PheA) that catalyzes the first step in the degradation of phenol in Bacillus thermoglucosidasius A7 is described. The two-protein system, encoded by the pheA1 and pheA2 genes, consists of an oxygenase (PheA1) and a flavin reductase (PheA2) and is optimally active at 55 degrees C. PheA1 and PheA2 were separately expressed in recombinant Escherichia coli BL21(DE3) pLysS cells and purified to apparent homogeneity. The pheA1 gene codes for a protein of 504 amino acids with a predicted mass of 57.2 kDa. PheA1 exists as a homodimer in solution and has no enzyme activity on its own. PheA1 catalyzes the efficient ortho-hydroxylation of phenol to catechol when supplemented with PheA2 and FAD/NADH. The hydroxylase activity is strictly FAD-dependent, and neither FMN nor riboflavin can replace FAD in this reaction. The pheA2 gene codes for a protein of 161 amino acids with a predicted mass of 17.7 kDa. PheA2 is also a homodimer, with each subunit containing a highly fluorescent FAD prosthetic group. PheA2 catalyzes the NADH-dependent reduction of free flavins according to a Ping Pong Bi Bi mechanism. PheA2 is structurally related to ferric reductase, an NAD(P)H-dependent reductase from the hyperthermophilic Archaea Archaeoglobus fulgidus that catalyzes the flavin-mediated reduction of iron complexes. However, PheA2 displays no ferric reductase activity and is the first member of a newly recognized family of short-chain flavin reductases that use FAD both as a substrate and as a prosthetic group. (+info)
RSEARCH: finding homologs of single structured RNA sequences.
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BACKGROUND: For many RNA molecules, secondary structure rather than primary sequence is the evolutionarily conserved feature. No programs have yet been published that allow searching a sequence database for homologs of a single RNA molecule on the basis of secondary structure. RESULTS: We have developed a program, RSEARCH, that takes a single RNA sequence with its secondary structure and utilizes a local alignment algorithm to search a database for homologous RNAs. For this purpose, we have developed a series of base pair and single nucleotide substitution matrices for RNA sequences called RIBOSUM matrices. RSEARCH reports the statistical confidence for each hit as well as the structural alignment of the hit. We show several examples in which RSEARCH outperforms the primary sequence search programs BLAST and SSEARCH. The primary drawback of the program is that it is slow. The C code for RSEARCH is freely available from our lab's website. CONCLUSION: RSEARCH outperforms primary sequence programs in finding homologs of structured RNA sequences. (+info)
Divergent evolutions of trinucleotide polymerization revealed by an archaeal CCA-adding enzyme structure.
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CCA-adding enzyme [ATP(CTP):tRNA nucleotidyltransferase], a template-independent RNA polymerase, adds the defined 'cytidine-cytidine-adenosine' sequence onto the 3' end of tRNA. The archaeal CCA-adding enzyme (class I) and eubacterial/eukaryotic CCA-adding enzyme (class II) show little amino acid sequence homology, but catalyze the same reaction in a defined fashion. Here, we present the crystal structures of the class I archaeal CCA-adding enzyme from Archaeoglobus fulgidus, and its complexes with CTP and ATP at 2.0, 2.0 and 2.7 A resolutions, respectively. The geometry of the catalytic carboxylates and the relative positions of CTP and ATP to a single catalytic site are well conserved in both classes of CCA-adding enzymes, whereas the overall architectures, except for the catalytic core, of the class I and class II CCA-adding enzymes are fundamentally different. Furthermore, the recognition mechanisms of substrate nucleotides and tRNA molecules are distinct between these two classes, suggesting that the catalytic domains of class I and class II enzymes share a common origin, and distinct substrate recognition domains have been appended to form the two presently divergent classes. (+info)