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β Propellers: structural rigidity and functional diversity

Recently solved structures and proposed models have helped to reveal the structural characteristics of the beta-propeller fold, as well as the features that contribute to its high rigidity and stability. Possible strategies for identifying beta-propeller proteins in newly characterised sequences are helping to overcome the problems of predicting the beta-propeller fold from amino acid sequences.

Catalysis of serine oligopeptidases is controlled by a gating filter mechanism

Proteases have a variety of strategies for selecting substrates in order to prevent uncontrolled protein degradation. A recent crystal structure determination of prolyl oligopeptidase has suggested a way for substrate selection involving an unclosed seven‐bladed β‐propeller domain. We have engineered a disulfide bond between the first and seventh blades of the propeller, which resulted in the loss of enzymatic activity. These results provided direct evidence for a novel strategy of regulation in which oscillating propeller blades act as a gating filter during catalysis, letting small peptide substrates into the active site while excluding large proteins to prevent accidental proteolysis.

Catalytic and structural studies of RhI complexes of (−)-(2S,4S)-2,4-bis(diphenylphosphino)pentane. Asymmetric hydrogenation of acetophenonebenzylimine and acetophenone

Abstract Rhodium(I) complexes formed by (−)-(2 S ,4 S )-2,4-bis(diphenylphosphino)pentane (BDPP) are efficient catalysts for the hydrogenation of acetophenone and acetophenonebenzylimine. The composition of the solvent mixture and the reaction temperature have a marked influenced on the enantioselectivity. These effects are thought to be related to the enhanced conformational flexibility of six-membered rings when simple substrates without functional groups are coordinated to the rhodium. X-ray crystallographic studies reveal that in [Rh(( S , S )-BDPP)NBD] + ( 1 ) the ligand is in a chair conformation, and that in [Rh(( S , S )-BDPP)COD] + ( 2 ) the chelate ring is in a δ-skew conformation. Studies of Rh(( S , S )-BDPP)(NBD)Cl ( 3 ) in solution indicate a trigonal bipyramidal structure with a chair conformation of the ring in aromatic solvents and a conformationally labile ring in methanol.

Structure-function properties of prolyl oligopeptidase family enzymes

Prolyl oligopeptidase family enzymes regulate the activity of biologically active peptides and peptide hormones, and they are implicated in diseases, including amnesia, depression, diabetes, and trypanosomiasis. Distinctively, these enzymes hydrolyze only relatively short peptide substrates, while large structured peptides and proteins are not usually cleaved. Prolyl oligopeptidase has a C-terminal α/β-hydrolase catalytic domain that is similar to lipases and esterases. An N-terminal β-propeller domain regulates access to the buried active site, explaining the observed oligopeptidase activity. The catalytic and regulatory mechanisms have been investigated using a combination of X-ray crystallography, site-directed mutagenesis, and enzyme kinetic measurements. Crystal structures have now been determined for representative members of three of the four subfamilies and are facilitating a better understanding of the structure-function properties of these physiologically and pharmaceutically important enzymes.

Substrate Recognition Properties of Oligopeptidase B from Salmonella enterica Serovar Typhimurium

Oligopeptidase B (OpdB) is a serine peptidase broadly distributed among unicellular eukaryotes, gram-negative bacteria, and spirochetes which has emerged as an important virulence factor and potential therapeutic target in infectious diseases. We report here the cloning and expression of the opdB homologue from Salmonella enterica serovar Typhimurium and demonstrate that it exhibits amidolytic activity exclusively against substrates with basic residues in P(1). While similar to its eukaryotic homologues in terms of substrate specificity, Salmonella OpdB differs significantly in catalytic power and inhibition and activation properties. In addition to oligopeptide substrates, restricted proteolysis of histone proteins was observed, although no cleavage was seen at or near residues that had been posttranslationally modified or at defined secondary structures. This supports the idea that the catalytic site of OpdB may be accessible only to unstructured oligopeptides, similar to the closely related prolyl oligopeptidase (POP). Salmonella OpdB was employed as a model enzyme to define determinants of substrate specificity that distinguish OpdB from POP, which hydrolyzes substrates exclusively at proline residues. Using site-directed mutagenesis, nine acidic residues that are conserved in OpdBs but absent from POPs were converted to their corresponding residues in POP. In this manner, we identified a pair of glutamic acid residues, Glu(576) and Glu(578), that define P(1) specificity and direct OpdB cleavage C terminal to basic residues. We have also identified a second pair of residues, Asp(460) and Asp(462), that may be involved in defining P(2) specificity and thus direct preferential cleavage by OpdB after pairs of basic residues.

Purification, crystallization and preliminary X-ray crystallographic studies on acetolactate decarboxylase.

Acetolactate decarboxylase has the unique ability to decarboxylate both enantiomers of acetolactate to give a single enantiomer of the decarboxylation product, (R)-acetoin. A gene coding for alpha-acetolactate decarboxylase from Bacillus brevis (ATCC 11031) was cloned and overexpressed in B. subtilis. The enzyme was purified in two steps to homogeneity prior to crystallization. Three different diffraction-quality crystal forms were obtained by the hanging-drop vapour-diffusion method using a number of screening conditions. The best crystal form is suitable for structural studies and was grown from solutions containing 20% PEG 2000 MME, 10 mM cadmium chloride and 0.1 M Tris-HCl pH 7.0. They grew to a maximum dimension of approximately 0.4 mm and belong to the trigonal space group P3(1,2)21, with unit-cell parameters a = 47.0, c = 198.9 A. A complete data set was collected to 2 A from a single native crystal using synchrotron radiation.

Molecular dynamics, crystallography and mutagenesis studies on the substrate gating mechanism of prolyl oligopeptidase

Altered prolyl oligopeptidase (PREP) activity is found in many common neurological and other genetic disorders, and in some cases PREP inhibition may be a promising treatment. The active site of PREP resides in an internal cavity; in addition to the direct interaction between active site and substrate or inhibitor, the pathway to reach the active site (the gating mechanism) must be understood for more rational inhibitor design and understanding PREP function. The gating mechanism of PREP has been investigated through molecular dynamics (MD) simulation combined with crystallographic and mutagenesis studies. The MD results indicate the inter-domain loop structure, comprised of 3 loops at residues, 189-209 (loop A), 577-608 (loop B), and 636-646 (loop C) (porcine PREP numbering), are important components of the gating mechanism. The results from enzyme kinetics of PREP variants also support this hypothesis: When loop A is (1) locked to loop B through a disulphide bridge, all enzyme activity is halted, (2) nicked, enzyme activity is increased, and (3) removed, enzyme activity is only reduced. Limited proteolysis study also supports the hypothesis of a loop A driven gating mechanism. The MD results show a stable network of H-bonds that hold the two protein domains together. Crystallographic study indicates that a set of known PREP inhibitors inhabit a common binding conformation, and this H-bond network is not significantly altered. Thus the domain separation, seen to occur in lower taxa, is not involved in the gating mechanism for mammalian PREP. In two of the MD simulations we observed a conformational change that involved the breaking of the H-bond network holding loops A and B together. We also found that this network was more stable when the active site was occupied, thus decreasing the likelihood of this transition.

Structure of a Trypanosomatid Mitochondrial Cytochrome C with Heme Attached Via Only One Thioether Bond and Implications for the Substrate Recognition Requirements of Heme Lyase.

The principal physiological role of mitochondrial cytochrome c is electron transfer during oxidative phosphorylation. c‐Type cytochromes are almost always characterized by covalent attachment of heme to protein through two thioether bonds between the heme vinyl groups and the thiols of cysteine residues in a Cys‐Xxx‐Xxx‐Cys‐His motif. Uniquely, however, members of the evolutionarily divergent protist phylum Euglenozoa, which includes Trypanosoma and Leishmania species, have mitochondrial cytochromes c with heme attached through only one thioether bond [to an (A/F)XXCH motif]; the implications of this for the cytochrome structures are unclear. Here we present the 1.55 Å resolution X‐ray crystal structure of cytochrome c from the trypanosomatid Crithidia fasciculata. Despite the fundamental difference in heme attachment and in the cytochrome c biogenesis machinery of the Euglenozoa, the structure is remarkably similar to that of typical (CXXCH) mitochondrial cytochromes c, both in overall fold and, other than the missing thioether bond, in the details of the heme attachment. Notably, this similarity includes the stereochemistry of the covalent heme attachment to the protein. The structure has implications for the maturation of c‐type cytochromes in the Euglenozoa; it also hints at a distinctive redox environment in the mitochondrial intermembrane space of trypanosomes. Surprisingly, Saccharomyces cerevisiae cytochrome c heme lyase (the yeast cytochrome c biogenesis system) cannot efficiently mature Trypanosoma brucei cytochrome c or a CXXCH variant when expressed in the cytoplasm of Escherichia coli, despite their great structural similarity to yeast cytochrome c, suggesting that heme lyase requires specific recognition features in the apocytochrome.

Identification of the reactive cysteine residues in oligopeptidase B from Trypanosoma brucei

Oligopeptidase B (OpdB) from Trypanosoma brucei is a candidate therapeutic target in African trypanosomiasis. OpdB is an atypical serine peptidase, since activity is inhibited by thiol‐blocking reagents and enhanced by reducing agents. We have identified C256 as the reactive cysteine residue that mediates OpdB inhibition by N‐ethylmaleimide and iodoacetic acid. Modeling studies suggest that C256 adducts occlude the P1 substrate‐binding site, preventing substrate binding. We further demonstrate that C559 and C597 are responsible for the thiol‐enhancement of OpdB activity. These studies may facilitate the development of specific OpdB inhibitors with therapeutic potential, by exploiting these unique properties of this enzyme.

Assessing crystallization droplets using birefringence

In this paper, the detection of crystalline elements in protein crystallization droplets containing precipitate is illustrated using the rotating-polarizer microscope technique. The sensitivity of this automated birefringence technique enables the detection of microcrystals in a precipitate that appears to be amorphous using traditional methods of inspection. The technique is illustrated with lysozyme and glucose isomerase. Glucose isomerase microcrystals were used successfully for seeding experiments and the conditions of both of the systems were refined to produce crystals suitable for X-ray analysis. The results are relevant to the field of high-throughput crystallography as an automated crystal-detection method as well as being a useful tool for detailed precipitate analysis.