A small protein inhibits proliferating cell nuclear antigen by breaking the DNA clamp.

Proliferating cell nuclear antigen (PCNA) forms a trimeric ring that encircles duplex DNA and acts as an anchor for a number of proteins involved in DNA metabolic processes. PCNA has two structurally similar domains (I and II) linked by a long loop (inter-domain connector loop, IDCL) on the outside of each monomer of the trimeric structure that makes up the DNA clamp. All proteins that bind to PCNA do so via a PCNA-interacting peptide (PIP) motif that binds near the IDCL.

Thermococcus kodakarensis has two functional PCNA homologs but only one is required for viability.

Proliferating cell nuclear antigen (PCNA) monomers assemble to form a ring-shaped clamp complex that encircles duplex DNA. PCNA binding to other proteins tethers them to the DNA providing contacts and interactions for many other enzymes essential for DNA metabolic processes. Most eukarya and euryarchaea have only one PCNA homolog but Thermococcus kodakarensis uniquely has two, designated PCNA1 and PCNA2, encoded by TK0535 and TK0582, respectively.

Structure and activity of PA5508, a hexameric glutamine synthetase homologue.

The structure of PA5508 from Pseudomonas aeruginosa, a glutamine synthetase (GS) homologue, has been determined at 2.5 Å. Surprisingly, PA5508 forms single hexameric rings rather than the stacked double rings that are characteristic of GS.

Structure of aminodeoxychorismate synthase from Stenotrophomonas maltophilia.

PabB, aminodeoxychorismate synthase, is the chorismic acid binding component of the heterodimeric PabA-PabB complex that converts chorismic acid to 4-amino-4-deoxychorismate, a precursor of p-aminobenzoate and folic acid in microorganisms. The second component, a glutamine amidotransferase subunit, PabA, generates ammonia that is channeled to the PabB active site where it attacks C4 of a chorismate-derived intermediate that is covalently bound, through C2, to an active site lysine residue. The presence of a PIKGT motif was, until recently, believed to allow discrimination of PabB enzymes from the closely related enzyme anthranilate synthase, which typically contains a PIAGT active site motif and does not form a covalent enzyme-substrate intermediate with chorismate.

Crystal structures of two active proliferating cell nuclear antigens (PCNAs) encoded by Thermococcus kodakaraensis.

Proliferating cell nuclear antigen (PCNA) is a ring-shaped protein that encircles duplex DNA and plays an essential role in many DNA metabolic processes in archaea and eukarya. The eukaryotic and euryarchaea genomes contain a single gene encoding for PCNA. Interestingly, the genome of the euryarchaeon Thermococcus kodakaraensis contains two PCNA-encoding genes (TK0535 and TK0582), making it unique among the euryarchaea kingdom.

Structure and function of YghU, a nu-class glutathione transferase related to YfcG from Escherichia coli.

The crystal structure (1.50 Å resolution) and biochemical properties of the GSH transferase homologue, YghU, from Escherichia coli reveal that the protein is unusual in that it binds two molecules of GSH in each active site. The crystallographic observation is consistent with biphasic equilibrium binding data that indicate one tight (K(d1) = 0.

Analysis of the structure and function of YfcG from Escherichia coli reveals an efficient and unique disulfide bond reductase.

YfcG is one of eight glutathione (GSH) transferase homologues encoded in the Escherichia coli genome. The protein exhibits low or no GSH transferase activity toward a panel of electrophilic substrates. In contrast, it has a very robust disulfide-bond reductase activity toward 2-hydroxyethyldisulfide on par with mammalian and bacterial glutaredoxins.

A comparative biochemical and structural analysis of the intracellular chorismate mutase (Rv0948c) from Mycobacterium tuberculosis H(37)R(v) and the secreted chorismate mutase (y2828) from Yersinia pestis.

The Rv0948c gene from Mycobacterium tuberculosis H(37)R(v) encodes a 90 amino acid protein as the natural gene product with chorismate mutase (CM) activity. The protein, 90-MtCM, exhibits Michaelis-Menten kinetics with a k(cat) of 5.5+/-0.

Structural insights into membrane targeting by the flagellar calcium-binding protein (FCaBP), a myristoylated and palmitoylated calcium sensor in Trypanosoma cruzi.

The flagellar calcium-binding protein (FCaBP) of the protozoan Trypanosoma cruzi is targeted to the flagellar membrane where it regulates flagellar function and assembly. As a first step toward understanding the Ca(2+)-induced conformational changes important for membrane-targeting, we report here the x-ray crystal structure of FCaBP in the Ca(2+)-free state determined at 2.2A resolution.

Structure of isochorismate synthase in complex with magnesium.

The electron carrier menaquinone is one of many important bacterial metabolites that are derived from the key intermediate chorismic acid. MenF, the first enzyme in the menaquinone pathway, catalyzes the isomerization of chorismate to isochorismate. Here, an improved structure of MenF in a new crystal form is presented.

Crystal structure of the pyocyanin biosynthetic protein PhzS.

The human pathogen Pseudomonas aeruginosa produces pyocyanin, a blue-pigmented phenazine derivative, which is known to play a role in virulence. Pyocyanin is produced from chorismic acid via the phenazine pathway, nine proteins encoded by a gene cluster. Phenazine-1-carboxylic acid, the initial phenazine formed, is converted to pyocyanin in two steps that are catalyzed by the enzymes PhzM and PhzS.

2-Hydroxychromene-2-carboxylic acid isomerase: a kappa class glutathione transferase from Pseudomonas putida.

The enzyme 2-hydroxychromene-2-carboxylic acid (HCCA) isomerase catalyzes the glutathione (GSH)-dependent interconversion (Keq = 1.5) of HCCA and trans-o-hydroxybenzylidene pyruvic acid (tHBPA) in the naphthalene catabolic pathway of Pseudomonas putida. The dimeric protein binds one molecule of GSH very tightly (Kd approximately 5 nM) and a second molecule of GSH with much lower affinity (Kd approximately 2 to 11 microM).

Structural and functional analysis of the pyocyanin biosynthetic protein PhzM from Pseudomonas aeruginosa.

Pyocyanin is a biologically active phenazine produced by the human pathogen Pseudomonas aeruginosa. It is thought to endow P. aeruginosa with a competitive growth advantage in colonized tissue and is also thought to be a virulence factor in diseases such as cystic fibrosis and AIDS where patients are commonly infected by pathogenic Pseudomonads due to their immunocompromised state.

Biochemical and structural characterization of the secreted chorismate mutase (Rv1885c) from Mycobacterium tuberculosis H37Rv: an *AroQ enzyme not regulated by the aromatic amino acids.

The gene Rv1885c from the genome of Mycobacterium tuberculosis H37Rv encodes a monofunctional and secreted chorismate mutase (*MtCM) with a 33-amino-acid cleavable signal sequence; hence, it belongs to the *AroQ class of chorismate mutases. Consistent with the heterologously expressed *MtCM having periplasmic destination in Escherichia coli and the absence of a discrete periplasmic compartment in M. tuberculosis, we show here that *MtCM secretes into the culture filtrate of M.

Crystallization and X-ray diffraction analysis of salicylate synthase, a chorismate-utilizing enyme involved in siderophore biosynthesis.

Bacteria have evolved elaborate schemes that help them thrive in environments where free iron is severely limited. Siderophores such as yersiniabactin are small iron-scavenging molecules that are deployed by bacteria during iron starvation. Several studies have linked siderophore production and virulence.

Structure of the phenazine biosynthesis enzyme PhzG.

PhzG is a flavin-dependent oxidase that is believed to play a role in phenazine antibiotic synthesis in various bacteria, including Pseudomonas. Phenazines are chorismic acid derivatives that provide the producing organisms, including the opportunistic pathogen P. aeruginosa, with a competitive growth advantage.

Parallel evolutionary pathways for glutathione transferases: structure and mechanism of the mitochondrial class kappa enzyme rGSTK1-1.

The class kappa glutathione (GSH) transferase is an enzyme that resides in the mitochondrial matrix. Its relationship to members of the canonical GSH transferase superfamily has remained an enigma. The three-dimensional structure of the class kappa enzyme from rat (rGSTK1-1) in complex with GSH has been solved by single isomorphous replacement with anomalous scattering at a resolution of 2.

Crystal structure of Escherichia coli protein ybgI, a toroidal structure with a dinuclear metal site.

Background: The protein encoded by the gene ybgI was chosen as a target for a structural genomics project emphasizing the relation of protein structure to function.

Results: The structure of the ybgI protein is a toroid composed of six polypeptide chains forming a trimer of dimers. Each polypeptide chain binds two metal ions on the inside of the toroid.

Structure and mechanism of Pseudomonas aeruginosa PhzD, an isochorismatase from the phenazine biosynthetic pathway.

PhzD from Pseudomonas aeruginosa is an isochorismatase involved in phenazine biosynthesis. Phenazines are antimicrobial compounds that provide Pseudomonas with a competitive advantage in certain environments and may be partly responsible for the persistence of Pseudomonas infections. In vivo, PhzD catalyzes the hydrolysis of the vinyl ether functional group of 2-amino-2-deoxyisochorismate, yielding pyruvate and trans-2,3-dihydro-3-hydroxyanthranilic acid, which is then utilized in the phenazine biosynthetic pathway.

Assisting functional assignment for hypothetical Heamophilus influenzae gene products through structural genomics.

The three-dimensional structures of Haemophilus influenzae proteins whose biological functions are unknown are being determined as part of a structural genomics project to ask whether structural information can assist in assigning the functions of proteins. The structures of the hypothetical proteins are being used to guide further studies and narrow the field of such studies for ultimately determining protein function. An outline of the structural genomics methodological approach is provided along with summaries of a number of completed and in progress crystallographic and NMR structure determinations.

Local protein dynamics and catalysis: detection of segmental motion associated with rate-limiting product release by a glutathione transferase.

Glutathione transferase rGSTM1-1 catalyzes the addition of glutathione (GSH) to 1-chloro-2,4-dinitrobenzene, a reaction in which the chemical step is 60-fold faster than the physical step of product release. The hydroxyl group of Y115, located in the active site access channel, controls the egress of product from the active site. The Y115F mutant enzyme has a k(cat) (72 s(-)(1)) that is 3.

The Biological Macromolecule Crystallization Database: crystallization procedures and strategies.

The Biological Macromolecule Crystallization Database (BMCD) archives crystallization data from published reports for all forms of biological macromolecules that have produced crystals suitable for X-ray diffraction studies. The information includes the crystallization conditions, crystal data, comments about the crystallization procedure and information on the biological macromolecule or biological macromolecule complex. Crystallization procedures, including fast screens and more general procedures, can be developed effectively using this web-based resource (http://wwwbmcd.

Structural basis of thermostability. Analysis of stabilizing mutations in subtilisin BPN'.

The crystal structures of two thermally stabilized subtilisin BPN' variants, S63 and S88, are reported here at 1.8 and 1.9 A resolution, respectively.