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Among all restriction endonucleases known to date, Bfil is unique in cleaving DNA in the absence of metal ions. Bfil represents a different evolutionary lineage of restriction enzymes, as shown by its crystal structure at 1.9-[Angstrom] resolution. The protein consists of two structural domains. The N-terminal catalytic domain is similar to Nuc, an EDTA-resistant nuclease from the phospholipase D superfamily. The C-terminal DNA-binding domain of Bfil exhibits a [beta]-barrel-like structure very similar to the effector DNA-binding domain of the [Mg.sup.2+]-dependent restriction enzyme EcoRII and to the B3-like DNA-binding domain of plant transcription factors. Bfil presumably evolved through domain fusion of a DNA-recognition element to a nonspecific nuclease akin to Nuc and elaborated a mechanism to limit DNA cleavage to a single double-strand break near the specific recognition sequence. The crystal structure suggests that the interdomain linker may act as an autoinhibitor controlling Bfil catalytic activity in the absence of a specific DNA sequence. A PSI-BLAST search identified a Bfil homologue in a Mesorhizobium sp. BNC1 bacteria strain, a plant symbiont isolated from an EDTA-rich environment. restriction endonuclease | x-ray crystallography

HslVU is a bacterial homolog of the proteasome, where HslV is the protease that is activated by HslU, an ATPase and chaperone. Structures of singly and doubly capped HslVU particles have been reported, and different binding modes have been observed. Even among HslVU structures with l-domains distal to HslV, no consensus mode of activation has emerged. A feature in the Haemophilus influenzae HslVU structure, insertion of the C termini of HslU into pockets in HslV, was not seen in all other structures of the enzyme. Here we report site-directed mutagenesis, peptide activation, and fluorescence experiments that strongly support the functional relevance of the C terminus insertion mechanism: we find that mutations in HslV that disrupt the interaction with the C termini of HslU invariably lead to inactive enzyme. Conversely, synthetic peptides derived from the C terminus of HslU bind to HslV with [10.sup.-5] M affinity and can functionally replace full HslU particles for both peptide and casein degradation but fail to support degradation of a folded substrate. Thus, the data can be taken as evidence for separate substrate unfoldase and protease stimulation activities in HslU. Enhanced HslV proteolysis could be due to the opening of a gated channel or allosteric activation of the active sites. To distinguish between these possibilities, we have mutated a series of residues that line the entrance channel into the HslV particle. Our mutational and fluorescence experiments demonstrate that allosteric activation of the catalytic sites is required in HslV, but they do not exclude the possibility of channel opening taking place as well. The present data support the conclusion that the H. influenzae structure with l-domains distal to HslV captures the active species and point to significant differences in the activation mechanism of HslV, ClpP, and the proteasome.

Heat shock locus V (HslV; also called ClpQ) is the proteolytic core of the ATP-dependent protease HslVU in Escherichia coli. It has sequence similarity with the [Beta]-type subunits of the eukaryotic and archaebacterial proteasomes. Unlike these particles, which display 72-point symmetry, it is a dimer of hexamers with 62-point symmetry. The crystal structure of HslV at 3.8-[Angstrom] resolution, determined by isomorphous replacement and symmetry averaging, shows that in spite of the different symmetry of the particle, the fold and the contacts between subunits are conserved. A tripeptide aide-hyde inhibitor, acetyl-Leu-Leu-norleucinal, binds to the N-terminal threonine residue of HslV, probably as a hemiacetal, relating HslV also functionally to the proteasomes of archaea and eukaryotes.

HslVU is an ATP-dependent prokaryotic protease complex. Despite detailed crystal and molecular structure determinations of free HslV and HslU, the mechanism of ATP-dependent peptide and protein hydrolysis remained unclear, mainly because the productive complex of HslV and HslU could not be unambiguously identified from the crystal data. In the crystalline complex, the I domains of HslU interact with HslV. Observations based on electron microscopy data were interpreted in the light of the crystal structure to indicate an alternative mode of association with the intermediate domains away from HslV. By generation and analysis of two dozen HslU mutants, we find that the amidolytic and caseinolytic activities of HslVU are quite robust to mutations on both alternative docking surfaces on HslU. In contrast, HslVU activity against the maltose-binding protein-SulA fusion protein depends on the presence of the I domain and is also sensitive to mutations in the N-terminal and C-terminal domains of HslU. Mutational studies around the hexameric pore of HslU seem to show that it is involved in the recognition/translocation of maltose-binding protein-SulA but not of chromogenic small substrates and casein. ATP-binding site mutations, among other things, confirm the essential role of the 'sensor arginine' (R393) and the 'arginine finger' (R325) in the ATPase action of HslU and demonstrate an important role for E321. Additionally, we report a better refined structure of the HslVU complex crystallized along with resorufin-labeled casein.

We present a biochemical and crystallographic characterization of active site mutants of the yeast 20S proteasome with the aim to characterize substrate cleavage specificity, subunit intermediate processing, and maturation. [Beta]1(Pre3), [Beta]2(Pup1), and [Beta]5(Pre2) are responsible for the postacidic, tryptic, and chymotryptic activity, respectively. The maturation of active subunits is independent of the presence of other active subunits and occurs by intrasubunit autolysis. The propeptides of [Beta]6(Pre7) and [Beta]7(Pre4) are intermediately processed to their final forms by [Beta]2(Pup1) in the wild-type enzyme and by [Beta]5(Pre2) and [Beta]1(Pre3) in the [Beta]2(Pup1) inactive mutants. A role of the propeptide of [Beta]1 (Pre3) is to prevent acetylation and thereby inactivation. A gallery of proteasome mutants that contain active site residues in the context of the inactive subunits [Beta]3(Pup3), [Beta]6(Pre7), and [Beta]7(Pre4) show that the presence of Gly-1, Thr1, Asp17, Lys33, Ser129, Asp166, and Ser169 is not sufficient to generate activity.

Cytosine methylation promotes deamination. In eukaryotes, CpG methylation is thought to account for CpG underrepresentation. Whether scarcity of CpGs in prokaryotic genomes is diagnostic for methylation is not clear. Here, we report that Mycoplasms tend to be CpG depleted and to harbor a family of constitutively expressed or phase variable CpG-specific DNA methyltransferases. The very CpG poor Mycoplasma penetrans and its constitutively active CpG-specific methyltransferase M.MpeI were chosen for further characterization. Genome-wide sequencing of bisulfite-converted DNA indicated that M.MpeI methylated CpG target sites both in vivo and in vitro in a locus-nonselective manner. A crystal structure of M.MpeI with DNA at 2.15-Å resolution showed that the substrate base was flipped and that its place in the DNA stack was taken by a glutamine residue. A phenylalanine residue was intercalated into the "weak" CpG step of the nonsubstrate strand, indicating mechanistic similarities in the recognition of the short CpG target sequence by prokaryotic and eukaryotic DNA methyltransferases. [ABSTRACT FROM AUTHOR]

The site-selective encoding of noncanonical amino acids (NAAs) is a powerful technique for the installation of novel chemical functional groups in proteins. This is often achieved by recoding a stop codon and requires two additional components: an evolved aminoacyl tRNA synthetase (AARS) and a cognate tRNA. Analysis of the most successful AARSs reveals common characteristics. The highest fidelity NAA systems derived from the Methanocaldococcus jannaschii tyrosyl AARS feature specific mutations to two residues reported to interact with the hydroxyl group of the substrate tyrosine. We demonstrate that the restoration of just one of these determinants for amino acid specificity results in the loss of fidelity as the evolved AARSs become noticeably promiscuous. These results offer a partial explanation of a recently retracted strategy for the synthesis of glycoproteins. Similarly, we reinvestigated a tryptophanyl AARS reported to allow the site-selective incorporation of 5-hydroxy tryptophan within mammalian cells. In multiple experiments, the enzyme displayed elements of promiscuity despite its previous characterization as a high fidelity enzyme. Given the many similarities of the TyrRSs and TrpRSs reevaluated here, our findings can be largely combined, and in doing so they reinforce the long-established central dogma regarding the molecular basis by which these enzymes contribute to the fidelity of translation. Thus, our view is that the central claims of fidelity reported in several NAA systems remain unproven and unprecedented. [ABSTRACT FROM AUTHOR]