Towards a general approach for tailoring the hydrophobic binding site of phenylalanine ammonia-lyases.

Unnatural substituted amino acids play an important role as chiral building blocks, especially for pharmaceutical industry, where the synthesis of chiral biologically active molecules still represents an open challenge. Recently, modification of the hydrophobic binding pocket of phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) resulted in specifically tailored PcPAL variants, contributing to a rational design template for PAL-activity enhancements towards the differently substituted substrate analogues. Within this study we tested the general applicability of this rational design model in case of PALs, of different sources, such as from Arabidopsis thaliana (AtPAL) and Rhodosporidium toruloides (RtPAL). With some exceptions, the results support that the positions of substrate specificity modulating residues are conserved among PALs, thus the mutation with beneficial effect for PAL-activity enhancement can be predicted using the established rational design model. Accordingly, the study supports that tailoring PALs of different origins and different substrate scope, can be performed through a general method. Moreover, the fact that AtPAL variants I461V, L133A and L257V, all outperformed in terms of catalytic efficiency the corresponding, previously reported, highly efficient PcPAL variants, of identical catalytic site, suggests that not only catalytic site differences influence the PAL-activity, thus for the selection of the optimal PAL-biocatalysts for a targeted process, screening of PALs from different origins, should be included.

Efficient Biodiesel Production Catalyzed by Nanobioconjugate of Lipase from Pseudomonas fluorescens.

The Amano lipase from Pseudomonas fluorescens (L-AK) was covalently immobilized on various carbon nanomaterials (functionalized single-walled carbon nanotubes and graphene oxide) and tested for biodiesel production. Using the most active lipase preparation (covalently immobilized L-AK on SwCNTNH2 derivatized with glycerol diglycidyl ether) under optimal conditions, quasi-complete conversion (>99%) of sunflower oil was obtained after only 4 h reaction time. Moreover, the biocatalyst maintained more than 99% of its initial activity in the batch system after multiple recycling experiments.

Efficient and Stable Magnetic Chitosan-Lipase B from Candida Antarctica Bioconjugates in the Enzymatic Kinetic Resolution of Racemic Heteroarylethanols.

Lipase B from Candida antarctica immobilized by covalent binding on sebacoyl-activated chitosan-coated magnetic nanoparticles proved to be an efficient biocatalyst (49.2-50% conversion in 3-16 h and >96% enantiomeric excess) for the enzymatic kinetic resolution of some racemic heteroarylethanols through transesterification with vinyl acetate. Under optimal conditions (vinyl acetate, n-hexane, 45 °C), the biocatalyst remains active after 10 cycles.

Tailored Mutants of Phenylalanine Ammonia-Lyase from Petroselinum crispum for the Synthesis of Bulky l- and d-Arylalanines.

Tailored mutants of phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) were created and tested in ammonia elimination from various sterically demanding, non-natural analogues of phenylalanine and in ammonia addition reactions into the corresponding (E)-arylacrylates. The wild-type PcPAL was inert or exhibited quite poor conversions in both reactions with all members of the substrate panel. Appropriate single mutations of residue F137 and the highly conserved residue I460 resulted in PcPAL variants that were active in ammonia elimination but still had a poor activity in ammonia addition onto bulky substrates. However, combined mutations that involve I460 besides the well-studied F137 led to mutants that exhibited activity in ammonia addition as well. The synergistic multiple mutations resulted in substantial substrate scope extension of PcPAL and opened up new biocatalytic routes for the synthesis of both enantiomers of valuable phenylalanine analogues, such as (4-methoxyphenyl)-, (napthalen-2-yl)-, ([1,1'-biphenyl]-4-yl)-, (4'-fluoro-[1,1'-biphenyl]-4-yl)-, and (5-phenylthiophene-2-yl)alanines.

Modern diversification of the amino acid repertoire driven by oxygen.

All extant life employs the same 20 amino acids for protein biosynthesis. Studies on the number of amino acids necessary to produce a foldable and catalytically active polypeptide have shown that a basis set of 7-13 amino acids is sufficient to build major structural elements of modern proteins. Hence, the reasons for the evolutionary selection of the current 20 amino acids out of a much larger available pool have remained elusive. Here, we have analyzed the quantum chemistry of all proteinogenic and various prebiotic amino acids. We find that the energetic HOMO-LUMO gap, a correlate of chemical reactivity, becomes incrementally closer in modern amino acids, reaching the level of specialized redox cofactors in the late amino acids tryptophan and selenocysteine. We show that the arising prediction of a higher reactivity of the more recently added amino acids is correct as regards various free radicals, particularly oxygen-derived peroxyl radicals. Moreover, we demonstrate an immediate survival benefit conferred by the enhanced redox reactivity of the modern amino acids tyrosine and tryptophan in oxidatively stressed cells. Our data indicate that in demanding building blocks with more versatile redox chemistry, biospheric molecular oxygen triggered the selective fixation of the last amino acids in the genetic code. Thus, functional rather than structural amino acid properties were decisive during the finalization of the universal genetic code.

Expanding the substrate scope of phenylalanine ammonia-lyase from Petroselinum crispum towards styrylalanines.

This study focuses on the expansion of the substrate scope of phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) towards the l-enantiomers of racemic styrylalanines rac-1a-d - which are less studied and synthetically challenging unnatural amino acids - by reshaping the aromatic binding pocket of the active site of PcPAL by point mutations. Ammonia elimination from l-styrylalanine (l-1a) catalyzed by non-mutated PcPAL (wt-PcPAL) took place with a 777-fold lower kcat/KM value than the deamination of the natural substrate, l-Phe. Computer modeling of the reactions catalyzed by wt-PcPAL indicated an unproductive and two major catalytically active conformations and detrimental interactions between the aromatic moiety of l-styrylalanine, l-1a, and the phenyl ring of the residue F137 in the aromatic binding region of the active site. Replacing the residue F137 by smaller hydrophobic residues resulted in a small mutant library (F137X-PcPAL, X being V, A, and G), from which F137V-PcPAL could transform l-styrylalanine with comparable activity to that of the wt-PcPAL with l-Phe. Furthermore, F137V-PcPAL showed superior catalytic efficiency in the ammonia elimination reaction of several racemic styrylalanine derivatives (rac-1a-d) providing access to d-1a-d by kinetic resolution, even though the d-enantiomers proved to be reversible inhibitors. The enhanced catalytic efficiency of F137V-PcPAL towards racemic styrylalanines rac-1a-d could be rationalized by molecular modeling, indicating the more relaxed enzyme-substrate complexes and the promotion of conformations with higher catalytic activities as the main reasons. Unfortunately, ammonia addition onto the corresponding styrylacrylates 2a-d failed with both wt-PcPAL and F137V-PcPAL. The low equilibrium constant of the ammonia addition, the poor ligand binding affinities of 2a-d, and the non-productive binding states of the unsaturated ligands 2a-d within the active sites of either wt-PcPAL or F137V-PcPAL - as indicated by molecular modeling - might be responsible for the inactivity of the PcPAL variants in the reverse reaction. Modeling predicted that the F137V mutation is beneficial for the KRs of 4-fluoro-, 4-cyano- and 4-bromostyrylalanines, but non-effective for the KR process of 4-trifluoromethylstyrylalanine.

Nanobioconjugates of Candida antarctica lipase B and single-walled carbon nanotubes in biodiesel production.

Carboxylated single-walled carbon nanotubes (SWCNTCOOH) were used as support for covalent immobilization of Candida antarctica lipase B (CaL-B) using linkers with different lengths. The obtained nanostructured biocatalysts with low diffusional limitation were tested in batch mode in the ethanolysis of the sunflower oil. SWCNTCOOH-CaL-B proved to be a highly efficient and stable biocatalyst in acetonitrile (83.4% conversion after 4h at 35°C, retaining >90% of original activity after 10 cycles).

Heterocycles 36. Single-Walled Carbon Nanotubes-Bound N,N-Diethyl Ethanolamine as Mild and Efficient Racemisation Agent in the Enzymatic DKR of 2-Arylthiazol-4-yl-alanines.

In this paper we describe the chemoenzymatic synthesis of enantiopure l-2-arylthiazol-4-yl alanines starting from their racemic N-acetyl derivatives; by combining the lipase-catalysed dynamic kinetic resolution of oxazol-5(4H)-ones with a chemical and an enzymatic enantioselective hydrolytic step affording the desired products in good yields (74%-78%) and high enantiopurities (ee > 99%). The developed procedure exploits the utility of the single-walled carbon nanotubes-bound diethylaminoethanol as mild and efficient racemisation agent for the dynamic kinetic resolution of the corresponding oxazolones.

Heterocycles 38. Biocatalytic Synthesis of New Heterocyclic Mannich Bases and Derivatives.

This paper describes the biocatalytic synthesis of new Mannich bases containing various heterocyclic rings (thiazole, furane, thiophene, pyridine) by applying the lipase catalyzed trimolecular condensation of the corresponding heterocyclic aldehydes with acetone and primary aromatic amines, in mild and eco-friendly reaction conditions. The obtained Mannich bases were acylated to their corresponding N-acetyl derivatives. All compounds were characterized by 1H-NMR, 13C-NMR and MS spectrometry.

Immobilization of Phenylalanine Ammonia-Lyase on Single-Walled Carbon Nanotubes for Stereoselective Biotransformations in Batch and Continuous-Flow Modes.

Carboxylated single-walled carbon nanotubes (SwCNTCOOH) were used as a support for the covalent immobilization of phenylalanine ammonia-lyase (PAL) from parsley by two different methods. The nanostructured biocatalysts (SwCNTCOOH-PALI and SwCNTCOOH-PALII) with low diffusional limitation were tested in the batch-mode kinetic resolution of racemic 2-amino-3-(thiophen-2-yl)propanoic acid (1) to yield a mixture of (R)-1 and (E)-3-(thiophen-2-yl)acrylic acid (2) and in ammonia addition to 2 to yield enantiopure (S)-1. SwCNTCOOH-PALII was a stable biocatalyst (>90 % of the original activity remained after six cycles with 1 and after three cycles in 6 m NH3 with 2). The study of ammonia addition to 2 in a continuous-flow microreactor filled with SwCNTCOOH-PALII (2 m NH3, pH 10.0, 15 bar) between 30-80 °C indicated no significant loss of activity over 72 h up to 60 °C. SwCNTCOOH-PALII in the continuous-flow system at 30 °C was more productive (specific reaction rate, rflow=2.39 µmol min-1 g-1) than in the batch reaction (rbatch=1.34 µmol min-1 g-1).

Composite aromatic boxes for enzymatic transformations of quaternary ammonium substrates.

Cation-π interactions to cognate ligands in enzymes have key roles in ligand binding and enzymatic catalysis. We have deciphered the key functional role of both charged and aromatic residues within the choline binding subsite of CTP:phosphocholine cytidylyltransferase and choline kinase from Plasmodium falciparum. Comparison of quaternary ammonium binding site structures revealed a general composite aromatic box pattern of enzyme recognition sites, well distinguished from the aromatic box recognition site of receptors.

Molecular cloning and characterization of a thermostable esterase/lipase produced by a novel Anoxybacillus flavithermus strain.

A thermophilic strain producing an extracellular esterase/lipase was isolated from a hot spring in Tasnad, Romania, and was identified phenotypically and by 16S rDNA sequencing as Anoxybacillus flavithermus (GenBank ID: JQ267733). The gene encoding the putative carboxyl esterase (GenBank ID: JX494348) was cloned by direct PCR amplification from genomic DNA. The protein, consisting of 246 amino acids and having a predicted molecular weight of 28.03 kDa, is encoded by an ORF of 741 bps. Expression was achieved in Escherichia coli and a recombinant protein with esterolytic activity and estimated molecular weight of 25 kDa was recovered and purified from the periplasmic fraction by IMAC. The purified enzyme, most active at 60-65°C and in the near-neutral range (pH 6.5-8), displayed a half-life at 60°C of about 5 h. Est/Lip displayed a relative tolerance to methanol, DMSO, acetonitrile, and low detergent concentrations (SDS, Triton) increased its thermostability. Highest activity was attained with p-nitrophenyl butyrate, but the enzyme was also able to hydrolyze long chain fatty acid esters, as well as triolein. The primary sequence and predicted tridimensional structure of the enzyme are very similar to those of other Anoxybacillus and Geobacillus carboxyl esterases in a distinct, recently described lipase family. Est/Lip was highly enantioselective, with preference for the (S)-enantiomer of substrates.

Early transcriptional pattern of angiogenesis induced by EGCG treatment in cervical tumour cells.

The major green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) has been shown to exhibit antitumour activities in several tumour models. One of the possible mechanisms by which EGCG can inhibit cancer progression is through the modulation of angiogenesis signalling cascade. The tumour cells' ability to tightly adhere to endothelium is a very important process in the metastatic process, because once disseminated into the bloodstream the tumour cells must re-establish adhesive connections to endothelium in order to extravasate into the target tissues. In this study, we investigated the anti-angiogenic effects of EGCG treatment (10 µM) on human cervical tumour cells (HeLa) by evaluating the changes in the expression pattern of 84 genes known to be involved in the angiogenesis process. Transcriptional analysis revealed 11 genes to be differentially expressed and was further validated by measuring the induced biological effects. Our results show that EGCG treatment not only leads to the down-regulation of genes involved in the stimulation of proliferation, adhesion and motility as well as invasion processes, but also to the up-regulation of several genes known to have antagonist effects. We observed reduced proliferation rates, adhesion and spreading ability as well as invasiveness of HeLa tumour cells upon treatment, which suggest that EGCG might be an important anti-angiogenic therapeutic approach in cervical cancers.

Preparation of unnatural amino acids with ammonia-lyases and 2,3-aminomutases.

Ammonia-lyases catalyze a wide range of processes leading to α,ß-unsaturated compounds by elimination of ammonia. In this chapter, ammonia-lyases are reviewed with major emphasis on their synthetic applications in stereoselective preparation of unnatural amino acids. Besides the synthesis of various unnatural α-amino acids with the aid of phenylalanine ammonia-lyases (PALs) utilizing the 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) prosthetic groups, the biotransformations leading to various unnatural ß-amino acids with phenylalanine 2,3-aminomutases using the same catalytic MIO prosthetic group are discussed. Cloning, production, purification, and biotransformation protocols for PAL are described in detail.

Role of chemical structure in molecular recognition by transferrin.

Studies of molecular recognition of chiral compounds by proteins are of importance from many points of view. The biological role of proteins in their interaction with small molecules is of fundamental interest and can be used in many different fields, for instance for in vitro analysis of optically active compounds. Studies in these areas need a detailed study of the interaction sites on the protein surface and the relationship between chemical structure and the complex formation ability of small molecules, such as drugs. The electrophoretic migration of charged compounds through a protein zone may provide information about the surface properties of the macromolecule in the interaction site. The interaction of human serum transferrin with tryptophan-methyl- (TME), ethyl- (TEE) and butyl-esters (TBE) has been investigated by capillary electrophoresis (CE) and model calculations. Differences in the separation of tryptophan derivatives were obtained by varying experimental parameters such as, pH, ionic strength of background electrolyte and the length of transferrin zone. Limited separation of the enantiomer pairs were observed at pH 5 and 7 with a maximum resolution at pH 6. The size of the ligands coupled to the chiral centre has importance in stereoselective recognition; however, a direct comparison of resolution different in same runs may lead to false conclusion if the experimental conditions are not comparable. With a careful evaluation of the data we obtained significant differences between the resolution of the smallest enantiomer pair compared to those of tryptophan derivatives with longer alkyl chains.

Experimental and quantum chemical study on the vibrational spectroscopy of N-methylphenothiazines: part 1.

In this work the authors deal with the vibrational spectroscopy of three derivatives of phenothiazine: the 10-methyl-10H-phenothiazine, the 10 methyl-10H-phenothiazine-3-carbaldehyde and the 10-methyl-10H-phenothiazine-3-yl-methanol. The authors investigated the vibrational spectroscopic behaviour of the phenothiazine skeleton and dealt with the aldehyde and the alcohol substituent effect on the vibrational spectroscopic and structural properties of these skeleton. The infrared and Raman spectra of the compounds have been recorded in condensed state. The Gaussian 98 program package was applied with the ab initio HF method since in this case beside the infrared also the Raman spectoroscopic properties appear in the output file. On the basis of the calculated force constants and geometric parameters, normal coordinate analysis was applied for the interpretation of the experimental vibrational spectra. Problems arose with the choice of the internal coordinates of the molecules. Full interpretations of the vibrational fundamentals of the compounds are presented. The relative mean deviations between the measured and calculated frequencies were below 1%.

Separation of N-alkyl phenothiazine sulfones by HPTLC using an optimum mobile phase.

In high performance thin layer chromatography some form of optimization is necessary if complete separation of all components is required. The selection of mobile phase composition is one of the most important components of an optimization strategies. The aim of this paper is the separation of the N-alkyl phenothiazine sulfones by high performance thin layer chromatography using an optimum mobile phase system. The optimum composition of mobile phase (toluene-ethyl ether-chloroform, 30:50:20, v/v) are given by the maximum of objective function (F(obj)=10.6110).