ABSTRACT
The complete genome sequence of Enterococcus faecalis V583, a vancomycin-resistant clinical isolate, revealed that more than a quarter of the genome consists of probable mobile or foreign DNA. One of the predicted mobile elements is a previously unknown vanB vancomycin-resistance conjugative transposon. Three plasmids were identified, including two pheromone-sensing conjugative plasmids, one encoding a previously undescribed pheromone inhibitor. The apparent propensity for the incorporation of mobile elements probably contributed to the rapid acquisition and dissemination of drug resistance in the enterococci.
Subject(s)
Biological Evolution , Enterococcus faecalis/genetics , Genome, Bacterial , Interspersed Repetitive Sequences , Sequence Analysis, DNA , Vancomycin Resistance/genetics , Adhesins, Bacterial/genetics , Bacterial Adhesion , Bacterial Proteins/genetics , Carrier Proteins/genetics , Carrier Proteins/metabolism , Chromosomes, Bacterial/genetics , Conjugation, Genetic , Conserved Sequence , DNA Transposable Elements , Digestive System/microbiology , Drug Resistance, Multiple, Bacterial , Enterococcus faecalis/drug effects , Enterococcus faecalis/pathogenicity , Enterococcus faecalis/physiology , Gene Transfer, Horizontal , Gram-Positive Bacterial Infections/microbiology , Humans , Lysogeny , Open Reading Frames , Oxidative Stress , Plasmids , Synteny , Virulence/genetics , Virulence Factors/geneticsABSTRACT
Structural genomics of proteins of unknown function most straightforwardly assists with assignment of biochemical activity when the new structure resembles that of proteins whose functions are known. When a new fold is revealed, the universe of known folds is enriched, and once the function is determined by other means, novel structure-function relationships are established. The previously unannotated protein HI1434 from H. influenzae provides a hybrid example of these two paradigms. It is a member of a microbial protein family, labeled in SwissProt as YbaK and ebsC. The crystal structure at 1.8 A resolution reported here reveals a fold that is only remotely related to the C-lectin fold, in particular to endostatin, and thus is not sufficiently similar to imply that YbaK proteins are saccharide binding proteins. However, a crevice that may accommodate a small ligand is evident. The putative binding site contains only one invariant residue, Lys46, which carries a functional group that could play a role in catalysis, indicating that YbaK is probably not an enzyme. Detailed sequence analysis, including a number of newly sequenced microbial organisms, highlights sequence homology to an insertion domain in prolyl-tRNA synthetases (proRS) from prokaryote, a domain whose function is unknown. A HI1434-based model of the insertion domain shows that it should also contain the putative binding site. Being part of a tRNA synthetases, the insertion domain is likely to be involved in oligonucleotide binding, with possible roles in recognition/discrimination or editing of prolyl-tRNA. By analogy, YbaK may also play a role in nucleotide or oligonucleotide binding, the nature of which is yet to be determined.
Subject(s)
Bacterial Proteins , Carrier Proteins/chemistry , Haemophilus influenzae/chemistry , Amino Acid Sequence , Amino Acyl-tRNA Synthetases/chemistry , Carbon-Oxygen Lyases , Carrier Proteins/isolation & purification , Crystallography, X-Ray , Lectins/chemistry , Models, Molecular , Molecular Sequence Data , Protein Folding , Protein Structure, Tertiary , Sequence Homology, Amino AcidABSTRACT
Many of the gene products of completely sequenced organisms are 'hypothetical' - they cannot be related to any previously characterized proteins - and so are of completely unknown function. Structural studies provide one means of obtaining functional information in these cases. A 'structural genomics' project has been initiated aimed at determining the structures of 50 hypothetical proteins from Haemophilus influenzae to gain an understanding of their function. Each stage of the project - target selection, protein production, crystallization, structure determination, and structure analysis - makes use of recent advances to streamline procedures. Early results from this and similar projects are encouraging in that some level of functional understanding can be deduced from experimentally solved structures.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Genome, Bacterial , Haemophilus influenzae/chemistry , Haemophilus influenzae/genetics , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Crystallization , Crystallography, X-Ray , Genes, Essential/genetics , Genes, Essential/physiology , Haemophilus influenzae/enzymology , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Conformation , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Structure-Activity RelationshipABSTRACT
Ser1406 of the allosteric region of the hamster CAD enzyme, carbamyl phosphate synthetase II (CPSase), is known to be phosphorylated in vitro by cAMP-dependent protein kinase (PKA). Metabolic labeling experiments described here demonstrate that CAD is phosphorylated in somatic cells in culture. Phosphorylation is stimulated by treating cells with 8-bromo-cAMP, a PKA activator. The stimulation is essentially prevented by pretreatment with H-89, a PKA specific inhibitor. Substitution of Ser1406 with alanine results in an enzyme with kinetics and allosteric regulation indistinguishable from unsubstituted CAD. However, substitution to glutamic acid increases CPSase activity by reducing the apparent Km (ATP). The UTP concentration required to give 50% inhibition is increased rendering this altered enzyme significantly less sensitive to feedback inhibition, but allosteric activation by PRPP is unaffected. While these data do not prove that Ser1406 is phosphorylated in vivo, they do indicate that a specific alteration at this residue can affect allosteric regulation.