Molecular Assembly of Clostridium botulinum progenitor M complex of type E.

Clostridium botulinum neurotoxin (BoNT) is released as a progenitor complex, in association with a non-toxic-non-hemagglutinin protein (NTNH) and other associated proteins. We have determined the crystal structure of M type Progenitor complex of botulinum neurotoxin E [PTC-E(M)], a heterodimer of BoNT and NTNH. The crystal structure reveals that the complex exists as a tight, interlocked heterodimer of BoNT and NTNH.

Discovery of a novel L-lyxonate degradation pathway in Pseudomonas aeruginosa PAO1.

The l-lyxonate dehydratase (LyxD) in vitro enzymatic activity and in vivo metabolic function were assigned to members of an isofunctional family within the mandelate racemase (MR) subgroup of the enolase superfamily. This study combined in vitro and in vivo data to confirm that the dehydration of l-lyxonate is the biological role of the members of this family. In vitro kinetic experiments revealed catalytic efficiencies of ∼10(4) M(-1) s(-1) as previously observed for members of other families in the MR subgroup.

Deamination of 6-aminodeoxyfutalosine in menaquinone biosynthesis by distantly related enzymes.

Proteins of unknown function belonging to cog1816 and cog0402 were characterized. Sav2595 from Steptomyces avermitilis MA-4680, Acel0264 from Acidothermus cellulolyticus 11B, Nis0429 from Nitratiruptor sp. SB155-2 and Dr0824 from Deinococcus radiodurans R1 were cloned, purified, and their substrate profiles determined.

Crystal structure of ChrR--a quinone reductase with the capacity to reduce chromate.

The Escherichia coli ChrR enzyme is an obligatory two-electron quinone reductase that has many applications, such as in chromate bioremediation. Its crystal structure, solved at 2.2 Å resolution, shows that it belongs to the flavodoxin superfamily in which flavin mononucleotide (FMN) is firmly anchored to the protein.

Unique and analogous functions of aquaporin 0 for fiber cell architecture and ocular lens transparency.

Aquaporin (AQP) 1 and AQP0 water channels are expressed in lens epithelial and fiber cells, respectively, facilitating fluid circulation for nourishing the avascular lens to maintain transparency. Even though AQP0 water permeability is 40-fold less than AQP1, AQP0 is selectively expressed in the fibers. Delimited AQP0 fiber expression is attributed to a unique structural role as an adhesion protein.

Structure of YqgQ protein from Bacillus subtilis, a conserved hypothetical protein.

The crystal structure of the hypothetical protein YqgQ from Bacillus subtilis has been determined to 2.1 A resolution. The crystals belonged to space group P2(1), with unit-cell parameters a = 51.

Domain organization in Clostridium botulinum neurotoxin type E is unique: its implication in faster translocation.

Clostridium botulinum produces seven antigenically distinct neurotoxins [C. botulinum neurotoxins (BoNTs) A-G] sharing a significant sequence homology. Based on sequence and functional similarity, it was believed that their three-dimensional structures will also be similar.

A common catalytic mechanism for proteins of the HutI family.

Imidazolonepropionase (HutI) (imidazolone-5-propanote hydrolase, EC 3.5.2.

X-ray crystal structure of the B component of Hemolysin BL from Bacillus cereus.

Bacillus cereus Hemolysin BL enterotoxin, a ternary complex of three proteins, is the causative agent of food poisoning and requires all three components for virulence. The X-ray structure of the binding domain of HBL suggests that it may form a pore similar to other soluble channel forming proteins. A putative pathway of pore formation is discussed.

Structural genomics of protein phosphatases.

The New York SGX Research Center for Structural Genomics (NYSGXRC) of the NIGMS Protein Structure Initiative (PSI) has applied its high-throughput X-ray crystallographic structure determination platform to systematic studies of all human protein phosphatases and protein phosphatases from biomedically-relevant pathogens. To date, the NYSGXRC has determined structures of 21 distinct protein phosphatases: 14 from human, 2 from mouse, 2 from the pathogen Toxoplasma gondii, 1 from Trypanosoma brucei, the parasite responsible for African sleeping sickness, and 2 from the principal mosquito vector of malaria in Africa, Anopheles gambiae. These structures provide insights into both normal and pathophysiologic processes, including transcriptional regulation, regulation of major signaling pathways, neural development, and type 1 diabetes.

Mechanism of action of a flavin-containing monooxygenase.

Elimination of nonnutritional and insoluble compounds is a critical task for any living organism. Flavin-containing monooxygenases (FMOs) attach an oxygen atom to the insoluble nucleophilic compounds to increase solubility and thereby increase excretion. Here we analyze the functional mechanism of FMO from Schizosaccharomyces pombe using the crystal structures of the wild type and protein-cofactor and protein-substrate complexes.

Common binding site for disialyllactose and tri-peptide in C-fragment of tetanus neurotoxin.

Clostridial neurotoxins are comprised of botulinum (BoNT) and tetanus (TeNT), which share significant structural and functional similarity. Crystal structures of the binding domain of TeNT complexed with disialyllactose (DiSia) and a tri-peptide Tyr-Glu-Trp (YEW) have been determined to 2.3 and 2.

N-terminal helix reorients in recombinant C-fragment of Clostridium botulinum type B.

Botulinum neurotoxins comprise seven distinct serotypes (A-G) produced by Clostridium botulinum. The crystal structure of the binding domain of the botulinum neurotoxin type B (BBHc) has been determined to 2A resolution. The overall structure of BBHc is well ordered and similar to that of the binding domain of the holotoxin.

Structure and mechanism of ADP-ribose-1''-monophosphatase (Appr-1''-pase), a ubiquitous cellular processing enzyme.

Appr-1''-pase, an important and ubiquitous cellular processing enzyme involved in the tRNA splicing pathway, catalyzes the conversion of ADP-ribose-1''monophosphate (Appr-1''-p) to ADP-ribose. The structures of the native enzyme from the yeast and its complex with ADP-ribose were determined to 1.9 A and 2.

Structural analysis of botulinum neurotoxin type E catalytic domain and its mutant Glu212-->Gln reveals the pivotal role of the Glu212 carboxylate in the catalytic pathway.

The seven serotypes of botulinum neurotoxins (A-G) produced by Clostridium botulinum share significant sequence homology and structural similarity. The functions of their individual domains and the modes of action are also similar. However, the substrate specificity and the peptide bond cleavage selectivity of their catalytic domains are different.

Role of metals in the biological activity of Clostridium botulinum neurotoxins.

Clostridium botulinum neurotoxins are the most potent toxins to humans and cause paralysis by blocking neurotransmitter release at the presynaptic nerve terminals. The toxicity involves four steps, viz., binding to neuronal cells, internalization, translocation, and catalytic activity.

Cloning, high level expression, purification, and crystallization of the full length Clostridium botulinum neurotoxin type E light chain.

The catalytic activity of the highly potent botulinum neurotoxins are confined to their N-terminal light chains ( approximately 50kDa). A full-length light chain for the type E neurotoxin with a C-terminal 6x His-tag, BoNT/E-LC, has been cloned in a pET-9c vector and over-expressed in BL21 (DE3) cells. BoNT/E-LC was purified to homogeneity by affinity chromatography on Ni-NTA agarose followed by exclusion chromatography using a Superdex-75 sizing column.

Structure of the dodecamer r(GAUCACUUCGGU) with four 5'-overhang nucleotides.

The crystal structure of an RNA dodecamer, r(GAUCACUUCGGU), was solved at 2.6 A resolution by the molecular-replacement method and refined to an R(work) of 18.8% (R(free) = 22.

Crystal structure of a putative CN hydrolase from yeast.

The crystal structure of a yeast hypothetical protein with sequence similarity to CN hydrolases has been determined to 2.4 A resolution by the multiwavelength anomalous dispersion (MAD) method. The protein folds as a four-layer alphabetabetaalpha sandwich and exists as a dimer in the crystal and in solution.

A novel mechanism for Clostridium botulinum neurotoxin inhibition.

Clostridium botulinum neurotoxins are zinc endopeptidase proteins responsible for cleaving specific peptide bonds of proteins of neuroexocytosis apparatus. The ability of drugs to interfere with toxin's catalytic activity is being evaluated with zinc chelators and metalloprotease inhibitors. It is important to develop effective pharmacological treatment for the intact holotoxin before the catalytic domain separates and enters the cytosol.