Mapping the substrate binding site of phenylacetone monooxygenase from Thermobifida fusca by mutational analysis

Baeyer-Villiger monooxygenases catalyze oxidations that are of interest for biocatalytic applications. Among these enzymes, phenylacetone monooxygenase (PAMO) from Thermobifida fusca is the only protein showing remarkable stability. While related enzymes often present a broad substrate scope, PAMO accepts only a limited number of substrates. Due to the absence of a substrate in the elucidated crystal structure of PAMO, the substrate binding site of this protein has not yet been defined. In this study, a structural model of cyclopentanone monooxygenase, which acts on a broad range of compounds, has been prepared and compared with the structure of PAMO. This revealed 15 amino acid positions in the active site of PAMO that may account for its relatively narrow substrate specificity. We designed and analyzed 30 single and multiple mutants in order to verify the role of these positions. Extensive substrate screening revealed several mutants that displayed increased activity and altered regio- or enantioselectivity in Baeyer-Villiger reactions and sulfoxidations. Further substrate profiling resulted in the identification of mutants with improved catalytic properties toward synthetically attractive compounds. Moreover, the thermostability of the mutants was not compromised in comparison to that of the wild-type enzyme. Our data demonstrate that the positions identified within the active site of PAMO, namely, V54, I67, Q152, and A435, contribute to the substrate specificity of this enzyme. These findings will aid in more dedicated and effective redesign of PAMO and related monooxygenases toward an expanded substrate scope.     

DNA sequence specificity of a naphthylquinoline triple helix-binding ligand.

We have examined the effect of a naphthylquinoline triplex-binding ligand on the formation of intermolecular triplexes on DNA fragments containing the target sites A6G6xC6T6 and G6A6xT6C6. The ligand enhances the binding of T6C2, but not T2C6, to A6G6xC6T6 suggesting that it has a greater effect on TxAT than C+xGC triplets. The complex with T6C2 is only stable below pH 6.0, confirming the requirement for protonation of the third strand cytosines. Antiparallel triplexes with GT-containing oligonucleotides are also stabilised by the ligand. The complex between G5T5 and A6G6xC6T6 is stabilised by lower ligand concentrations than that between T5G5 and G6A6xC6T6. The ligand does not promote the interaction with GT-containing oligonucleotides which have been designed to bind in a parallel orientation. Although the formation of antiparallel triplexes is pH independent, we find that the ligand has a greater stabilising effect at lower pH, suggesting that the active species is protonated. The ligand does not promote the binding of antiparallel GA-containing oligonucleotides at pH 7.5 but induces the interaction between A5G5 and G6A6xT6C6 at pH 5.5. Ethidium bromide does not promote the formation of any of these triplexes and destabilises the interaction of acridine-linked pyrimidine-containing third strands with these target sites.     

Occurrence of melittoside in the seeds of Plantago media

The ¹³C NMR and 360 MHz ¹H NMR signals of melittoside and its acetate have been assigned.     

Selective catalysis of A.T base pair proton exchange in DNA complexes: imino proton NMR analysis.

Polyaromatic molecules with amino chain substituents, upon binding with DNA, selectively catalyze exchange of the A.T base pair protons with bulk water protons. The amine-catalyzed exchange is mediated by compounds which are A.T and G.C base sequence specific, intercalators, and outside binders. A mechanism for the selective exchange, involving transient opening and closing of individual A.T base pairs in the duplex, is discussed.     

Cooperative binding of the hnRNP K three KH domains to mRNA targets

The heterogeneous nuclear ribonucleoprotein (hnRNP) K homology (KH) domain is an evolutionarily conserved module that binds short ribonucleotide sequences. KH domains most often are present in multiple copies per protein. In vitro studies of hnRNP K and other KH domain bearing proteins have yielded conflicting results regarding the relative contribution of each KH domain to the binding of target RNAs. To assess this RNA-binding we used full-length hnRNP K, its fragments and the yeast ortholog as baits in the yeast three-hybrid system. The results demonstrate that in this heterologous in vivo system, the three KH domains bind RNA synergistically and that a single KH domain, in comparison, binds RNA weakly.     

Use of non-covalent labeling in illustrating ligand binding to human serum albumin via affinity capillary electrophoresis with near-infrared laser induced fluorescence detection

This paper demonstrates the use of a near-infrared (NIR) dye as a non-covalent label for human serum albumin (HSA). The dye is a water soluble, heptamethine cyanine dye. The utility of the dye as a tracer illustrating the binding of various drugs to HSA is demonstrated via affinity capillary electrophoresis with near-infrared laser-induced fluorescence detection (ACE-NIR-LIF). Additionally, the factors affecting the separation of relevant species were investigated. The change in quantum yield of the dye upon complexation with HSA was calculated. Spectrophotometric measurements were conducted to study the stoichiometry of the dye albumin complex.     

AutoMotif Server for prediction of phosphorylation sites in proteins using support vector machine: 2007 update

We present here the recent update of AutoMotif Server (AMS 2.0) that predicts post-translational modification sites in protein sequences. The support vector machine (SVM) algorithm was trained on data gathered in 2007 from various sets of proteins containing experimentally verified chemical modifications of proteins. Short sequence segments around a modification site were dissected from a parent protein, and represented in the training set as binary or profile vectors. The updated efficiency of the SVM classification for each type of modification and the predictive power of both representations were estimated using leave-one-out tests for model of general phosphorylation and for modifications catalyzed by several specific protein kinases. The accuracy of the method was improved in comparison to the previous version of the service (Plewczynski et al., “AutoMotif server: prediction of single residue post-translational modifications in proteins”, Bioinformatics 21: 2525–7, 2005). The precision of the updated version reached over 90% for selected types of phosphorylation and was optimized in trade of lower recall value of the classification model. The AutoMotif Server version 2007 is freely available at . Additionally, the reference dataset for optimization of prediction of phosphorylation sites, collected from the UniProtKB was also provided and can be accessed at .     

Support-vector-machine classification of linear functional motifs in proteins

Our algorithm predicts short linear functional motifs in proteins using only sequence information. Statistical models for short linear functional motifs in proteins are built using the database of short sequence fragments taken from proteins in the current release of the Swiss-Prot database. Those segments are confirmed by experiments to have single-residue post-translational modification. The sensitivities of the classification for various types of short linear motifs are in the range of 70%. The query protein sequence is dissected into short overlapping fragments. All segments are represented as vectors. Each vector is then classified by a machine learning algorithm (Support Vector Machine) as potentially modifiable or not. The resulting list of plausible post-translational sites in the query protein is returned to the user. We also present a study of the human protein kinase C family as a biological application of our method.     

Bis-4-aminoquinolines: novel triple-helix DNA intercalators and antagonists of immunostimulatory CpG-oligodeoxynucleotides

Six dimeric 2-(2-naphthyl)quinolin-4-amines with a linker between the amino groups and eight dimeric 2-(4-anilino)quinolin-4-amines linked between the anilino groups were synthesized and evaluated for their interaction with duplex/triplex DNA's and as antagonists of immunostimulatory oligodeoxynucleotides with a CpG-motif (CpG-ODN). The most powerful triple-helix DNA intercalator known to date, with high affinity toward T.A.T triplets and triplex/duplex selectivity, was found. The potent antagonism of immunostimulatory CpG-ODN by several bis-4-aminoquinolines is not related to their DNA interactions.