Still a Long Way to Fully Understanding the Molecular Mechanism of Escherichia coli Purine Nucleoside Phosphorylase

The results of several decades of studying the catalytic mechanism of Escherichia colt purine nucleoside phosphorylases (PNP) by solution studies and crystal structure determinations are presented. Potentially PNPs can be used for enzyme-activating prodrug gene therapy against solid tumours because of the differences in specificity between human and E. coli PNPs. Biologically active form of PNP from E. coli is a homohexamer that catalyses the phosphorolytic cleavage of the glycosidic bond of purine nucleosides. Two conformations of the active site are possible after substrate(s) binding: open and closed. A series of determined 3D-structures of PNP binary and ternary complexes facilitated the prediction of the main steps in the catalytic mechanism. For their validation the active site mutants: Arg24Ala, Asp204Ala, Arg217Ala, Asp204Asn and double mutant Asp204Ala/Arg217Ala were prepared, The activity tests confirm that catalysis involves protonation of the purine base at position N7 and give better insight into the cooperativity between subunits in this oligomeric enzyme

Synthesis, Crystal Structures and Properties of New Chloroniobium Cluster Hydroxides with Co-ordinated and Non-coordinated OH- Groups. The Presence of H3O2 Intercluster Bridges

The isostructural, trigonal clusters of the composition [Nb6Cl12(H20)6](0H)2 l/4CH30H • 6H20 1 and [Nb6Cl12(H20)6](OH)2 -6H20 2 have been isolated from alkaline solutions of [NbgClj^]2* cluster in alcohol/water (1:3) in the presence of NMe^H, while the presence of NEt^H yielded NEt^NbeCl^Hjjf^e^IS^O 3. Compound 1 crystallizes in trigonal space group R3m with a = 21.166(5) A and c = 23.812(4) Å, y = 120°, U = 9238.6(3) Å3, Z = 12. Two ciystal- lographically independent cluster octahedra (I and II) have been found in the unit cell of 1 in the ratio 3:1. The Nb-0 distances of 2.211(11) Å and 2.217(16) Å for I and 2.238(19) Å for II indicate six co-ordinated water molecules. The cluster units of I are connected by hydrogen bonds of 2.49(1) A into a two-dimensional framework. Compound 3 is monoclinic, space group P2j/n, a = 10.085(5) A, b = 11.446(4) Å, c = 14.971(6) Å, /? = 102.95(6)°, U = 1684.2(1) Å3, Z = 2. The short Nb-O distances at 2.167(6), 2.177(8) and 2.187(7) A and considerable reduction of the Nb-O stretching frequency in the IR spectra support the presence of H302 (co-ordinated water molecule from one cluster unit and co-ordinated OH~ group from the neighbouring cluster unit) bridges. Each cluster unit is joined to the six neighbouring units by intermolecular hydrogen bonds of distances 2.540(11) Å and 2.594(10) Å in a three-dimensional network

Preliminary Crystallographic Study of Streptomyces coelicolor Single-stranded DNA-binding Protein

Single-stranded DNA-binding proteins (SSBs) play a crucial role in DNA processing such as replication, repair and recombination in all organisms, from bacteria to human. Streptomyces coelicolor ssb gene was overexpressed in a heterologous host, Escherichia coli NM522. 15 mg of purified protein from 1 dm3 of culture was obtained in one-step procedure applying Ni2+ chelating chromatography. Among bacterial SSBs with the solved crystal structure, the S. coelicolor SSB displayed significant sequence similarity with those from Mycobacterium tuberculosis and Mycobacterium smegmatis, slow growing bacteria with a high GC content. Moreover, conserved amino acid region that forms additional ß strand in mycobacterial SSBs was also found in S. coelicolor SSB. The full-length protein readily crystallises in space group I222 or I212121 with unit-cell parameters a = 100.8, b = 102.1, c = 164.2 Å. The asymmetric unit is expected to contain four monomers with solvent content of 52–55 %

Crystal structure of dipeptidyl peptidase III from the human gut symbiont Bacteroides thetaiotaomicron

Bacteroides thetaiotaomicron is a dominant member of the human intestinal microbiome. The genome of this anaerobe encodes more than 100 proteolytic enzymes, the majority of which have not been characterized. In the present study, we have produced and purified recombinant dipeptidyl peptidase III (DPP III) from B. thetaiotaomicron for the purposes of biochemical and structural investigations. DPP III is a cytosolic zinc-metallopeptidase of the M49 family, involved in protein metabolism. The biochemical results for B. thetaiotaomicron DPP III from our research showed both some similarities to, as well as certain differences from, previously characterised yeast and human DPP III. The 3D-structure of B. thetaiotaomicron DPP III was determined by X-ray crystallography and revealed a two-domain protein. The ligand-free structure (refined to 2.4 Å) was in the open conformation, while in the presence of the hydroxamate inhibitor Tyr-Phe-NHOH, the closed form (refined to 3.3 Å) was observed. Compared to the closed form, the two domains of the open form are rotated away from each other by about 28 degrees. A comparison of the crystal structure of B. thetaiotaomicron DPP III with that of the human and yeast enzymes revealed a similar overall fold. However, a significant difference with functional implications was discovered in the upper domain, farther away from the catalytic centre. In addition, our data indicate that large protein flexibility might be conserved in the M49 family

Substrate Recognition by Novel Family of Amino Acid:[Carrier Protein] Ligases

Amino acid:[carrier protein] ligases (aa:CP ligases) are newly discovered homologs of aminoacyl- tRNA synthetases (aaRS) which aminoacylate carrier proteins instead of tRNA. Activated amino acids are attached to prosthetic group of carrier proteins by thioester bond. Weak thioacylation activity was observed for many conventional aaRS. Therefore, different thiols were investigated as substrate analogs for aa:CP ligases. Here we show that coenzyme A, dithiothreitol and cysteine are efficiently aminoacylated by aa:CP ligases. The crystal structure of aa:CP ligase from Bradyrhizobium japonicum in complex with coenzyme A was solved, and revealed that CoA, besides acting as the substrate analog of the carrier protein, also competes with ATP for binding to the active site. aa:CP ligases do not aminoacylate tRNA, although they remarkably resemble catalytic core of atypical seryl-tRNA synthetases (aSerRS) from methanogenic archaea. Since aa:CP ligases lack tRNA-binding domain, fusion proteins of aa:CP ligases and aSerRS tRNA-binding domain were prepared in attempt to restore tRNA aminoacylation activity. Although fusion proteins were able to bind tRNA through appended domain, tRNA could not substitute carrier proteins in aminoacylation reaction. (doi: 10.5562/cca1818

Crystallographic snapshots of ligand binding to hexameric purine nucleoside phosphorylase and kinetic studies give insight into the mechanism of catalysis

Purine nucleoside phosphorylase (PNP) catalyses the cleavage of the glycosidic bond of purine nucleosides using phosphate instead of water as a second substrate. PNP from Escherichia coli is a homohexamer, build as a trimer of dimers, and each subunit can be in two conformations, open or closed. This conformational change is induced by the presence of phosphate substrate, and very likely a required step for the catalysis. Closing one active site strongly affects the others, by a yet unclear mechanism and order of events. Kinetic and ligand binding studies show strong negative cooperativity between subunits. Here, for the first time, we managed to monitor the sequence of nucleoside binding to individual subunits in the crystal structures of the wild-type enzyme, showing that first the closed sites, not the open ones, are occupied by the nucleoside. However, two mutations within the active site, Asp204Ala/Arg217Ala, are enough not only to significantly reduce the effectiveness of the enzyme, but also reverse the sequence of the nucleoside binding. In the mutant the open sites, neighbours in a dimer of those in the closed conformation, are occupied as first. This demonstrates how important for the effective catalysis of Escherichia coli PNP is proper subunit cooperation

Cis and trans-4-Oxoazetidine-2-Sulphonic Acid Derivatives; Preparation and X-Ray Structure Determination

Cis and ćrans-4-oxoazetidine-2-sulphonic acid derivatives were prepared starting from penicillanate sulphoxides (1) and (4). The methylsulphonates (5), (7), (8) and (9) were formed by oxidation of 4-oxoazetidine-2-sulphinates (2), (3), and (6). Generally, 4-oxoazetidine-2-sulphonates were labile entities and hydrolyzed under mild conditions into sulphonic acids. These were isolated as acids (12) and salts (10), (11) and (14). The conformational isomers of the sulphonate (9a) were detected by \u27H NMR spectroscopy and confirmed by variable temperature experiments. X-Ray structure analyses of 9a were performed but there wasn’t any evidence for intramolecular hydrogen bonding